antileishmanial, cytotoxic and genotoxic effects of
TRANSCRIPT
ANTILEISHMANIAL, CYTOTOXIC AND GENOTOXIC EFFECTS OF
ACTINOMYCIN D, Z3, Z5 AND HYDRAZINE DERIVATIVES OF
ISOSTEVIOL
Ph. D. Thesis
By
QAISAR JAMAL
(M. Sc., M. Phil)
DEPARTMENT OF ZOOLOGY
UNIVERSITY OF PESHAWAR
Session 2013-14
ANTILEISHMANIAL, CYTOTOXIC AND GENOTOXIC EFFECTS OF
ACTINOMYCIN D, Z3, Z5 AND HYDRAZINE DERIVATIVES OF
ISOSTEVIOL
Thesis submitted to the University of Peshawar in partial fulfilment of the requirements
for the degree of Doctor of Philosophy in Zoology under the Faculty of Life and
Environmental Sciences by
Qaisar Jamal
DEPARTMENT OF ZOOLOGY
UNIVERSITY OF PESHAWAR
Session 2013-14
AUTHOR’S DECLARATION
I, Qaisar Jamal Reg. No. 2005-I-2855, student of Doctor of Philosophy in the subject of
Zoology hereby solemnly declare that the subject matter printed in the thesis titled
“Antileishmanial, Cytotoxic and Genotoxic Effects of Actinomycin D, Z3, Z5 And Hydrazine
Derivatives of Isosteviol” is my own work, except wherever stated otherwise and has not been
printed, published and submitted as research work, thesis or publication in any form to any
university or research institution in Pakistan or abroad. If at any stage my declaration is found to
be incorrect even after my graduation, the University of Peshawar will have the right to withdraw
my PhD degree.
Qaisar Jamal
PLAGIARISM UNDERTAKING
I, Qaisar Jamal Reg. No. 2005-I-2855, student of Doctor of Philosophy in the subject of zoology
hereby solemnly declare that the research work presented in this thesis titled “Antileishmanial,
Cytotoxic and Genotoxic Effects of Actinomycin D, Z3, Z5 and Hydrazine Derivatives of
Isosteviol” is exclusively my own research work, except where stated otherwise. Moreover,
complete thesis has been written by me and wherever any minor contribution/help (if any) was
taken, has been duly acknowledged. I understand the zero-tolerance policy of Higher Education
Commission (HEC), Pakistan and the University of Peshawar towards plagiarism. Therefore, I as
an author of the thesis declare that no portion of my thesis has been plagiarized and any material
used as reference has been properly cited. I undertake that if I am found guilty of any formal
plagiarism in this thesis, the University reserves the right to withdraw/ revoke my PhD degree. I
also declare that HEC and University have the right to publish my name on the HEC/University
website under the list of students with plagiarised thesis.
Qaisar Jamal
CERTIFICATE OF APPROVAL
This is to certify that the thesis titled “Antileishmanial, Cytotoxic and Genotoxic Effects of
Actinomycin D, Z3, Z5 And Hydrazine Derivatives of Isosteviol” by Qaisar Jamal (Reg. No.
2005-I-2855) under the supervision of Professor Dr. Akram Shah submitted in partial fulfilment
of the requirements for the Doctor of Philosophy (PhD) in Zoology complies with the regulation
of the university to meet the accepted standards with respect to originality.
Student’s Name: Qaisar Jamal _________________________
Examination Committee
External Examiner: 1 _________________________
External Examiner: 2 _________________________
Internal Examiner: 1 _________________________
Supervisor: _________________________
Chairman/Head: _________________________
RESEARCH COMPLETION CERTIFICATE
It is certified that research work contained in this thesis titled “Antileishmanial, Cytotoxic
and Genotoxic Effects of Actinomycin D, Z3, Z5 And Hydrazine Derivatives of Isosteviol” has
been carried out and completed by Qaisar Jamal (Reg. No. 2005-I-2855) under my supervision. I
recommend it as it fulfills partial requirement for the award of PhD degree in Zoology.
Research Supervisor: _________________________
Professor Dr. Akram Shah
M.Sc. Zoology (Gold Medallist), PhD. (King’s College London)
Commonwealth Post-doctoral Fellowship (School of Medicine, King’s College London)
INSPIRE Postdoctoral Fellow (London School of Hygiene & Tropical Medicine)
Chairman: __________________________
Professor Dr. Akram Shah
Chairman, Department of Zoology
University of Peshawar, 25120 Pakistan
Dedication
This work is dedicated to Professor Dr. Muhammad Nasim Siddiqui (Late) who worked
sincerely and selflessly to glitter the Science of Zoology in Khyber Pakhtunkhwa Province
of Pakistan. May Allah Bless His Soul in Everlasting Peace and Rehmah, Aameen
Table of Contents
List of Table………………………………………………………………………………… i
List of Figures……………………………………………………………………………. ii-iv
List of Abbreviations and Symbols……………………………………………………... v-vi
Acknowledgements……………………………………………………………………. vii-viii
Abstract…………………………………………………………………………………... ix-x
1. Introduction………………………………………………………………………... 01
1.1.Cutaneous Leishmaniasis……………………………………………………….. 01
1.2.History and Discovery…………………………………………………………... 02
1.3.Taxonomy and Classification of Leishmania…………………………………… 03
1.4.Evolution of Leishmania………………………………………………………… 07
1.5.Life Cycle and Transmission……………………………………………………. 08
1.6.Epidemiology: Global Scenario………………………………………………… 10
1.7.Surface Molecules Crucial for Survival in Hostile Environments……………… 11
1.8. Genetics of Leishmania………………………………………………………… 12
1.9.Immunology of Leishmaniasis………………………………………………….. 13
1.9.1. Promastigotes in the Inoculum vs Innate Immunity…………………….. 13
1.9.2. Anti-Inflammatory and Immunomodulatory Effect of Sand Fly Saliva… 14
1.9.3. Host Immune Evasion…………………………………………………... 15
1.9.4. The oxidative Burst……………………………………………………... 17
1.9.5. Activation of Natural Killer Cells (NK) and Adaptive Immunity……… 18
1.9.6. Role of Natural Killer Cells…………………………………………….. 18
1.9.7. Th1 and Th2 Response-Mechanism and Outcome……………………... 20
1.10. Treatment Approaches for Leishmaniasis………………………………….. 21
1.10.1. Antimonials…………………………………………………………….. 23
1.10.2. Amphotericin B………………………………………………………… 24
1.10.3. Pentamidine…………………………………………………………….. 26
1.10.4. Miltefosine……………………………………………………………… 27
1.10.5. Paromomycin…………………………………………………………… 28
1.10.6. Phytomedicine in Experimental and Clinical Trials……………………. 28
1.10.7. Inorganic Agents in Experimental and Clinical trials…………………... 33
1.10.8. Diterpenoids……………………………………………………………. 35
1.10.9. Actinomycins…………………………………………………………... 36
1.11. Starain of Leishmania to be Used………………………………………….. 37
Aim and objectives of the Present Study………………………………………………… 37
2. Morphology and Culture Characterization of Leishmania tropica KWH23…… 38
2.1. Materials and Methods………………………………………………………… 38
2.1.1. Culture of Leishmania tropica KWH23 Promastigotes………………... 38
2.1.2. Evaluation of Morphological Variations of Promastigotes during
Culture………………………………………………………………….. 39
2.1.3. Cultivation of Axenic Amastigotes and Evaluation of Morphology…… 39
2.1.4. Acquisition of Axenic Amastigotes by Starvation……………………... 40
2.1.5. Viability during Transformation………………………………………. 40
2.2. Results…………………………………………………………………………. 41
2.2.1. Growth Curve of L. tropica KWH23 Promastigotes…………………… 41
2.2.2. Axenic Promastigote Morphotypes……………………………………... 42
2.2.3. Division of L. tropica Promastiogtes in Axenic Development…………. 43
2.2.4. Cultivation of Axenic Amastigotes……………………………………... 44
2.2.5. Changes in Outline and Flagella during Differentiation of Promastigotes to
Amastigotes…………………………………………………………….. 45
2.2.6. Viability during Transformation……………………………………….. 49
2.2.7. Natural Acquisition of Axenic Amastigotes……………………………. 50
2.2.8. Requirements for Counting, Fixation and Smearing of Leishmania tropica
KWH23………………………………………………………………….. 51
2.2.9. Revision of Strain Nomenclature………………………………………. 51
2.3. Discussion……………………………………………………………………... 52
3. In Vitro Cytotoxicity Assays of Actinomycins and Isosteviol Derivatives on Human
Peripeheral Blood Lymphocytes
……………………………………………………………………………………… 56
3.1. Materials……………………………………………………………………….. 57
3.1.1. Chemicals and Solutions……………………………………………….. 57
3.1.2. Disposables…………………………………………………………….. 57
3.1.3. Apparatus………………………………………………………………. 57
3.2. Methods……………………………………………………………………….. 58
3.2.1. Isolation of Human Peripheral Blood Lymphocytes…………………... 58
3.2.2. Inspecting Viability of Lymphocytes………………………………….. 58
3.2.3. Plating Lymphocytes and Adding Compounds……………………….. 59
3.2.4. Evaluation of Cytotoxicity and Cell Death In Vitro…………………… 59
3.2.5. Data Analysis…………………………………………………………... 59
3.3. Results………………………………………………………………………... 60
3.3.1. Cytotoxic Impact of Miltefosine on Lymphocytes……………………. 60
3.3.2. Cytotoxicity of 16 (2,4-Dinitrophenylhydrazine) Isosteviol…………...... 61
3.3.3. Cytotoxicity of 17 Hydroxy, 16 (2,4-dinitrophenylhydrazine)
Isosteviol………………………………………………………………. 62
3.3.4. Cytotoxicity of Benzyl ester, 16 (2,4-dinitrophenylhydrazine)
Isosteviol………………………………………………………………. 62
3.3.5. Cytotoxicity of Actinomycin D………………………………………… 63
3.3.6. Cytotoxicity of Actinomycin Z3………………………………………... 64
3.3.7. Cytotoxicity of Actinomycin Z5………………………………………... 65
3.4.Discussion………………………………………………………………………. 67
4. Antileishmanial Compounds Screening In Vitro……………………………….... 71
4.1.Materials and Methods………………………………………………………….. 72
4.1.1. Culture of the Leishmania tropica KWH23 Promastigotes…………….. 72
4.1.2. Generation of Axenic Amastigotes……………………………………... 72
4.1.3. Antileishmanial Activity………………………………………………... 73
4.1.4. Data Analysis…………………………………………………………… 73
4.2. Results………………………………………………………………………….. 74
4.2.1. Antileishmanial Activity of Miltefosine………………………………... 74
4.2.2. Antileishmanial Activity of 16 (2,4-dinitrophenylhydrazine)
Isosteviol………………………………………………………………... 75
4.2.3. Antileishmanial Activity of 17 hydroxy, 16 (2,4-dinitrophenylhydrazine)
Isosteviol ……………………………………………………………….. 75
4.2.4. Antileishmanial Activity of Benzyl ester 16 (2,4-dinitrophenylhydrazine)
Isosteviol………………………………………………………………... 76
4.2.5. Antileishmanial Activity of Actinomycin D……………………………. 77
4.2.6. Antileishmanial Activity of Actinomycin Z3………………………….... 78
4.2.7. Antileishmanial Activity of Actinomycin Z5…………………………... 79
4.3.Discussion………………………………………………………………………. 80
4.3.1. Mechanism of Action of Miltefosine…………………………………… 81
4.3.2. Antileishmanial Action of Isosteviol and Actinomycin D……………… 82
5. Evaluation of the Compounds Genotoxicity against Human Lymphocytes……. 86
5.1. Introduction…………………………………………………………………….. 86
5.2. Methodology………………………………………………………………….. 86
5.2.1. Application of Test Compounds to Lymphocytes for Genotoxicity….. 87
5.2.2. Slide Preparation and Mounting of Treated Lymphocytes…………… 87
5.2.3. Lysing of Lymphocyte Cells…………………………………………. 88
5.2.4. Single Cell (SC) Gel Electrophoresis………………………………… 88
5.2.5. Neutralization and Fixation…………………………………………… 88
5.2.6. Staining……………………………………………………………….. 88
5.2.7. Data Interpretation and Scoring………………………………………. 88
5.2.8. Statistical Analysis……………………………………………………. 89
5.3. Results………………………………………………………………………... 90
5.3.1. Genotoxicity of Miltefosine…………………………………………… 90
5.3.2. Genotoxicity of 16 (2, 4-dinitrophenylhydrazine) Isosteviol…………. 91
5.3.3. Genotoxicity of 17 hydroxy, 16 (2, 4-dinitrophenylhydrazine)
Isosteviol………………………………………………………………. 92
5.3.4. Genotoxicity of Benzyl ester 16 (2,4-dinitrophenylhydrazine)
Isosteviol………………………………………………………………. 93
5.3.5. Genotoxicity of Actinomycin D (M1) ………………………………… 94
5.3.6. Genotoxicity of Actinomycin Z3 (M2) ………………………………. 95
5.3.7. Genotoxicity of Actinomycin Z5 (M3) ………………………………. 96
5.4. Discussion……………………………………………………………………. 97
6. General Discussion………………………………………………………………. 99
Conclusion………………………………………………………………………………... 102
Recommendations………………………………………………………………………... 103
References…………………………………………………………………………….104-133
i
List of Tables
Table No. Title Page No.
1.1. Classification of Leishmania (modified from Adl et al., 2012; Lukeš et al., 2014) ……….06
1.2. Summary of the genome analysis for five different Leishmania species (Llanes et al., 2015).
……..........................................................................................................................................13
2.1.Promastigote to amastigote transformation ratio over 10-day duration based on Leishmania
morphology.………………………………………………………………………………….47
2.2. Size distribution of various differentiating and transforming amastigotes of Leishmania
tropica KWH23 in acidified medium……………………………………………………........48
2.3. Size distribution of the completely round, intermediates and oval forms of Leishmania
tropica amastigotes.………………………………………………………….........................48
ii
List of Figures
Figure No. Title Page No.
Figure 1.1. Classification of Leishmania (Adl et al., 2012; Lukeš et al., 2014) ……………05
Figure 1.2. Geographical distributions of various Leishmania spp.; sandflies and animal
reservoirs in the Old and New World. L: Leishmania (species), S: Sandfly (genus
or subgenus), R: Reservoir (genus or family) (Akhoundi et al., 2016)………….08
Figure 1.3. Life cycle of Leishmania showing different developmental stages of promastigote
in the sand fly midgut. The figures are from Ortiago et al., 2010 (A), Oliveira et
al., 2009 (B) and Dostálová and Volf, 2012 (C). ……………………………….10
Figure 1.4. Difference between the LPG of procyclics and metacyclics (A) and species-
specific variability in structure of LPG structure of other major glycoconjugates
(B). (A) from Spath et al., (2001) and (B) from Kamhavi, (2006) …………….12
Figure 1.5. Immunomodulation mechanism in macrophages (Bogdan and Röllinghoff, 1998)
…………………………………………………………………………………...17
Figure 1.6. Activation of natural killer cells in experimental visceral leishmaniasis (Bogdan et
al., 2012) ………………………………………………………………………...19
Figure 1.7. Immune response in cutaneous Leishmania infection (Abdossamadi et al., 2016)
…………………………………………………………………………………..20
Figure 2.1. Hemocytometer illustration representing set of 16 squares (marked in red colour)
used for counting promastigotes………………………………………………..38
Figure 2.2. Representative growth curve of Leishmania tropica KWH23 in vitro………… 41
Figure 2.3. Different stages of L. tropica KWH23 promastigotes representing cell cycle in
culture (see text for description). A naturally occurring amastigote (Arrow in “e”)
can be seen in a 13-day old promastigote culture………………………………42
Figure 2.4. Division of the L. tropica promastigotes in axenic development. Cells with 2
flagella single kinetoplast and single nucleus (A and B), Cells with single
flagellum, two kinetoplasts and single nucleus (C), Cells with Single flagellum,
two kinetoplasts and two nuclei (D and E) and Cells with two flagella, two
kinetoplasts and two nuclei near to the completion of cytokinesis (F). Solid
arrows show kinetoplasts and the others indicate flagella………………………44
iii
Figure 2.5. Promastigote to amastigote transformation. Change in the cell morphology and
loss of flagellum is seen progressively…………………………………………45
Figure 2.6. Viability assessment for Leishmania tropica amastigotes during transformation
and differentiation………………………………………………………………49
Figure 2.7. Unsurprisingly acquired amastigotes (A), Starved culture medium with pH 4.8-5.0
having naturally changed amastigotes (B). Standard RPMI 1640 growth medium
with pink colour and phenol red as a pH indicator (C)………………………….50
Figure 3.1. Concentration dependent cytotoxicity of Miltefosine against human peripheral
blood lymphocytes……………………………………………………………….61
Figure 3.2. Cytotoxicity of 16 (2,4-dinitrophenylhydrazine) Isosteviol against human
peripheral blood lymphocytes …………………………………………………...62
Figure 3.3. Cytotoxicity of 17 hydroxy, 16 (2,4-dinitrophenylhydrazine) Isosteviol against
human peripheral blood lymphocytes……………………………………………63
Figure 3.4. Cytotoxicity of Benzyl ester 16 (2,4-dinitrophenylhydrazine) Isosteviol against
human peripheral blood lymphocytes……………………………………………64
Figure 3.5. Cytotoxicity of Actinomycin D against human peripheral blood lymphocytes…65
Figure 3.6. Cytotoxicity of Actinomycin Z3 against human peripheral blood lymphocytes...66
Figure 3.7. Cytotoxicity of Actinomycin Z5 against human peripheral blood lymphocytes...67
Figure 4.1. Cutaneous leishmaniasis patients are treated in Peshawar Hospital with
intralesional injection of meglumine antimonate………………………………..71
Figure 4.2. Antileishmanial activity of miltefosine against promastigotes (A) and amastigotes
(B) of L. tropica KWH23……………………………………………………….74
Figure 4.3. Leishmanicidal activity of 16 (2,4-dinitrophenylhydrazine) Isosteviol against
promastigotes (A) and amastigotes (B) of L. tropica KWH23………………….75
Figure 4.4. Antileishmanial activity of 17 hydroxy, 16 (2,4-dinitrophenylhydrazine)
Isosteviol against promastigotes (A) and amastigotes (B) of L. tropica KWH23.76
Figure 4.5. Antileishmanial Activity of Benzyl ester 16 (2,4-dinitrophenylhydrazine)
Isosteviol against promastigotes (A) and amastigotes (B) of L. tropica KWH23.77
Figure 4.6. Antileishmanial activity of Actinomycin D (M1) against promastigotes (A) and
amastigotes (B) of L. tropica KWH23…………………………………………..78
iv
Figure 4.7. Antileishmanial activity of Actinomycin Z3 (M2) against promastigotes (A) and
amastigotes (B) of L. tropica KWH23…………………………………………..79
Figure 4.8. Antileishmanial activity of Actinomycin Z5 (M3) against the promastigotes (A)
and amastigotes (B) of L. tropica KWH23………………………………………80
Figure 4.9. Leishmania promastigotes (A) and amastigotes (B) showing acidocalcisomes
(https://www.sciencedirect.com/science/article/pii/S0020751907001038#fig1)..82
Figure 5.1. Genotoxicity of Miltefosine (Milt) at 100 uM, 25 uM and 1.25 uM (IC50 for
Leishmania tropica)…………………………………………………………….90
Figure 5.2. Genotoxicity of 16 (2,4-dinitrophenylhydrazine) isosteviol (ID) at 100 uM, 25 uM
and 4.447 uM (IC50 for Leishmania tropica)…………………………………...91
Figure 5.3. Genotoxicity of (ID7) at 100 uM, 25 uM and 5.033 uM (IC50 for Leishmania
tropica)………………………………………………………………………….92
Figure 5.4. Genotoxicity of Benzyl ester 16 (2,4-dinitrophenylhydrazine) isosteviol (IDB5) at
100 uM, 25 uM and (IC50 for Leishmania tropica)…………………………….94
Figure 5.5. Genotoxicity of Actinomycin D (M1) at 100 uM, 25 uM and 2.135 uM (IC50 for
Leishmania tropica)……………………………………………………………..95
Figure 5.6. Genotoxicity of Actinomycin Z3 (M2) at 100 uM, 25 uM and 1.76 μM IC50 for
Leishmania tropica……………………………………………………………...96
Figure 5.7. Genotoxicity of Actinomycin Z5 (M3) at 100 uM, 25 uM and 1.691 μM (IC50 for
Leishmania tropica)……………………………………………………………..97
v
List of Abbreviations and Symbols
ANOVA Analysis of variance
CR Complement receptor
DMSO Dimethyl Sulfoxide
EBVF Elongated barely visible flagellum
EDTA Ethylene diamine tetra-acetic acid
ELF Elongated long flagellum,
ENF Elongated no flagellum
ESF Elongated short flagellum
GP63 Metalloproteinase glycoprotein 63
GIPL Glycoionositolphospholipid
hi-FBS Heat-inactivated fetal bovine serum
HI-FCS Heat-inactivated fetal calf serum
ID 16 (2,4-Dinitrophenylhydrazine) Isosteviol
ID7 17 Hydroxy, 16 (2,4-dinitrophenylhydrazine) Isosteviol
IDB5 Benzyl ester 16 (2,4-dinitrophenylhydrazine) Isosteviol
IFN-γ Interferon Gamma
IL-10 Interleukin-10
IMS Industrial methylated spirit
IU International Unit
KWH23 Kuwait Teaching Hospital (Original ID of the strain of Leishmania
tropica)
LPG Liphophosphoglycan
M1 Actinomycin D
M2 Actinomycin Z3
M3 Actinomycin Z5
MIC Minimal inhibitory concentration
vi
Milt Miltefosine
mL Millilitre
Mɸ Macrophage
OBVF Ovoid barely visible flagellum
OLF Ovoid long flagellum
ONF Ovoid no flagellum
OSF Ovoid short flagellum
PBLs Peripheral blood lymphocytes
PBS Phosphate Buffer Saline
PCD Programmed cell death
Plain Negative control (Cells of parasites grown in medium only)
RBVF Rounded barely visible flagellum
RLF Rounded long flagellum
RNF Rounded no flagellum
RPMI-1640 Rosewell Park Memorial Institute (culture medium)
RSF Rounded short flagellum
SSG Sodium Stibogluconate
TGF-β Transforming Growth Factor-Beta
XTT 2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-
Carboxanilide
μM Micromolar
μm Micrometer
vii
Acknowledgements
I am greatly indebted to my research supervisor Professor Dr. Akram Shah whose support
accompanied me all the way long during my M.Sc., M.Phil and now Ph.D. Had he not been that
much generous to me, perhaps I won’t have made it possible to go through the worries of
research work. His sincere efforts and dedication did not let me, and many others, feel any dearth
of materials and equipment. All this was to make things easier for me and point me in the course
of the light at the end of the channel.
I am thankful to Dr. Syed Basit Rasheed, Assistant Professor Department of Zoology, University
of Peshawar for his wise and insightful suggestions during experimental work. His wholehearted
response was extremely encouraging and helpful during jammed moments of laboratory work.
He, being much busy investigator, never said a nope, when requested for assistance. His
guidance in writing the manuscript is worth salutation.
I will not forget enthusiastic help from Dr. Shumaila Noreen, Assistant Professor Department of
Zoology, University of Peshawar when she lent me the sum to pay my fees. Rather, it was this
onward that I could complete my official documentation necessary in academic degrees. Thanks
to her for being generous enough to cut out money from her expenditures during this growing
inflation to help me.
I really owe to acknowledge my hostel mate Mr. Ibadat Ur Rehman, then Lecturer Hakim Abdul
Jalil Nadvi University College for Boys, University of Peshawar and now Junior Scientist
Pakistan Atomic Energy Commission, for suggesting me to use ortho-phosphoric acid for the
acidification of medium for Leishmania amastigotes culture; that worked wonderfully.
My heartfelt thanks go to Dr. Sanaullah Khan, Associate Professor Department of Zoology,
University of Peshawar for his concern and provoking advices, on occasions, to pursue my Ph.D
work on priority.
I am also much grateful to Dr. Asad Ullah, Assistant Professor Department of Chemistry, Islamia
College Peshawar for providing me the test compounds. He has always been generous and
gracious enough to provide guidance and information on the compounds. His ready response will
always be remembered.
viii
I feel pleasure to express my gratitude to Dr. Muhammad Khisroon, Assistant Professor
Department od Zoology, University of Peshawar for his support during genotoxicity evaluation.
His efforts in establishing and optimizing the comet assay techniques in the Department of
Zoology, University of Peshawar are clear like broad day light. He has been generous enough in
providing material, technical and theoretical support during these assays. He also went through
that part of the manuscript and put in valuable suggestions.
Valuable suggestions and faithful advices from Dr. Sobia Wahid and Dr. Nazma Habib,
Assistant Professors Department of Zoology, are worth acknowledgment. We were lucky to use
Leishmania tropica KWH23 (MHOM/PK/2010/KWH23) collected and typed by Dr. Nazma
Habib during her PhD at London School of Hygiene & Tropical Medicine. Madam thanks for
opening this door of opportunity.
Thanks are also due to Mr. Zaigham Hasan and Dr. Farrah Zaidi, Dr. Mohammad Adnan,
Assistant Professors, Department of Zoology for advices, support and assistance.
I heartily acknowledge ministerial help from Mr. Farhat Ullah, Senior Assistant, Department of
Zoology, University of Peshawar and Ashfaq Ali, Junior Clerk Department of Zoology,
University of Peshawar.
Last, but not least, I feel privileged to thank my fellow PhD scholars Mr Ajmal Khan and Mr
Abid Ullah who helped me selflessly in running comet assay protocol. They provided me basic
understanding and practical handling of the technique.
Qaisar Jamal
ix
Abstract
In the present study in vitro culture of Leishmania tropica KWH23 (MHOM/PK/2010/KWH23),
was used for all the experiments. In axenic growth, Leishmania tropica promastigotes reached to
log, mid-log, late-log and stationary phases on day 4, 5, 6 and 7 in culture respectively. Among
stationary phase promastigotes higher density of metacyclic was reported. Nectomonad stage
promastigotes were found to be the longest and slender most individuals outnumbering any other
morphotype for the first three days in the culture. On day 3 onward, leptomonads appeared in the
culture to a ratio of 44%. They were distinguished from nectomondas by getting wider anteriorly.
Metacyclic promastigotes appeared in culture during log phase with metacyclic to leptomonad to
nectomonad ratio of 27, 43 and 29% respectively. During logarithmic growth, 17% of the
promastigotes were found to be dividing. Division normally proceeded from flagellum to
kinetoplast to nucleus. Amastigote stage was grown in vitro in axenic culture. Day 4 onward
most of the parasites in culture were represented by rounded and ovoid cells with no flagella.
Cell size decreased from 10.966μm of the promastigote to 3.138μm of round amastigote. During
the transformation process 96-98% viability was noted. When the promastigotes were left
without fresh medium change, they naturally changed to amastigotes due to pH drop. In a 10 day
follow up, the pH dropped from 7.4 to 4.8 and 91% of the parasites, at a density of 1.2x107,
changed to amastigotes having 97% viability. These amastigotes were successfully transformed
back to promastigotes in normal growth medium.
Antileishmanial, cytotoxic and genotoxic effects of Actinomycin D, Z3 and Z5 and Isosteviol
derivatives, 16 (2,4-dinitrophenylhydrazine) Isosteviol, 17-hydroxy 16 (2,4-
dinitrophenylhydrazine) Isosteviol and Benzyl ester 16 (2,4-dinitrophenylhydrazine) Isosteviol
were assessed. Miltefosine was used as standard positive control. Cytotoxicity was expressed i]n
terms of 50% inhibitory (IC50) values. The IC50 values of Miltefosine, 16 (2,4-
dinitrophenylhydrazine) Isosteviol, 17 hydroxy, 16 (2,4-dinitrophenylhydrazine) Isosteviol,
Benzyl ester 16 (2,4-dinitrophenylhydrazine) Isosteviol, actinomycin D, actinomycin Z3 and
actinomycin Z5 were 272.2μM (95 % CI= 143.6 to 515.7μM), 781.2μM (95 % CI= 240.8 to
2535.0μM), 294.1μM (95 % CI= 177.4 to 487.5μM), 421.8μM (95% CI= 211.3 to 842.1μM),
195.8μM (95% CI=135.3 to 283.2μM), 210.1μM (95% CI= 145.2 to 304.1μM), and 234.9μM
(95% CI= 155.5 to 354.9μM) respectively regarding cytotoxicity.
x
Regarding antileishmanial activity, Miltefosine, 16 (2,4-dinitrophenylhydrazine) Isosteviol, 17-
hydroxy 16 (2,4-dinitrophenylhydrazine) Isosteviol, Benzyl ester 16 (2,4-
dinitrophenylhydrazine) Isosteviol, Actinomycin D, Actinomycin Z3 and Actinomycin Z5 gave
IC50 values against L. tropica promastigotes and amastigotes as 10.80μM (95% CI=9.114 to
12.81μM), 1.245μM (95% CI=0.7250 to 2.138μM), 7.098μM (95% CI=5.328 to 9.455μM),
4.447μM (95% CI= 2.788 to 7.094μM), 5.603μM (95% CI= 4.628 to 6.784μM), 5.033μM (95%
CI= 3.189 to 7.945μM), 10.71μM (95% CI= 8.611 to 13.31), 6.794μM (95% CI= 4.248 to
10.87μM), 8.739μM (95% CI= 6.675 to 11.44μM), 2.135μM (95% CI= 1.419 to 3.211μM),
5.500μM (95% CI= 3.811 to 7.939μM), 1.760μM (1.136 to 2.728μM), 9.529μM (95% CI= 7.354
to 12.35μM) and 1.691μM (95% CI= 0.9559 to 2.991μM) respectively. In conclusion, L. tropica
KWH23 was extra sensitive to 16 (2,4-dinitrophenylhydrazine) Isosteviol.
Miltefosine gave least genotoxicity at 100, 25 and 1.25μM concentration having total comet
score (TCS) of 10, 8 and 8 respectively. Damage was non-significant (P>0.05) as compared to
1% DMSO and negative control. Compound 16 (2,4-dinitrophenylhydrazine) Isosteviol showed
concentration dependent genotoxicity. It gave TCS values of 207.33, 87.33 and 10.66
respectively at 100, 25 and 4.447μM concentration. The compound showed non-significant
genotoxic effects to the standard Miltefosine and 1% DMSO and negative control at all the
concentration (P>0.05). Compound 17 hydroxy, 16 (2,4-dinitrophenylhydrazine) Isosteviol
caused significant genotoxicity as compared to standard and negative control at 100 and 25μM
(P<0.05). At 5.033μM concentraton, however, the genotoxicity became non-significant (P>0.05).
Benzyl ester 16 (2,4-dinitrophenylhydrazine) Isosteviol was found to be non-genotoxic as
contrasted with the standard and negative control at all concentrations (P>0.05). The TCS values
calculated were 166.33, 85.33 and 15 respectively at 100, 25 and 6.794μM concentrations.
Actinomycin D showed highest degree of genotoxicity as compared to the standard, Isosteviol
derivatives and negative control at 100 and 25μM concentrations (P<0.05). But Genotoxicity
became non-significant at 2.135μM concentrations (P>0.05). Actinomycin Z3 was found to show
significant genotoxicity at all the concentration i.e., 100, 25 and 1.76μM in relation to standard
and negative control as well as Isosteviol derivatives (P<0.05). Actinomycin Z5 was also found
significantly genotoxic as compared to standard, negative control and Isosteviol derivatives at all
the concentration (P<0.05). In terms of TCS values the genotoxicity, however, greatly reduced
with decreasing concentration 100μM (337.666), 25μM (214.333), 1.691μM (23.666).
1
CHAPTER 1. INTRODUCTION
Leishmaniasis is a collective term for vector-borne diseases (Saunkratay et al., 2010) affecting
mainly the skin (cutaneous leishmaniasis), mucous membranes (muco-cutaneous leishmaniasis)
and reticulo-endothelial system (visceral leishmaniasis) of vertebrates including human (WHO,
2019). Obligate intracellular protozoans in the genus Leishmania (Order: Kinetoplatida; Family:
Trypanosomatidae) are the causative agents of the diseases (Gupta and Nishi, 2010; Hatam et al.,
2013; Llanes et al., 2015). Clinical manifestations of the infection show variability in relation to
the causative species and site of infection. Even same species may reflect different clinical
history in relation to different individuals, as well as hosts and geography (Auon and Bouratbine,
2014). Due to the fact, the categorization of Leishmania species to visceral and cutaneous in
humans is infrequently applicable in other mammalian hosts e.g., Leishmania infantum infection
in dogs has been found to be viscero-dermic and human cutaneous Leishmania species have been
recovered from rodent’s viscera (Roque and Jansen, 2014). Leishmaniasis is principally zoonotic
except Leishmania donovani and Leishmania tropica, though few reports suggest animal
reservoirs for these two Leishmania species across Asia and Africa (Burza et al., 2018).
1.1. Cutaneous Leishmaniasis
WHO estimates 0·7–1 million cutaneous leishmaniasis (CL) cases yearly in the world. Presently,
more than 90% of CL cases are presented in Afghanistan, Algeria, Brazil, Iran, Pakistan, Peru
Saudi Arabia and Syria. Due to cross-border migration of war refugees, the number of CL cases
has noticeably amplified in non-endemic republics. Leishmania species producing CL in Old
World are Leishmania major, L. tropica, and L. aethiopica, that are predominant round the Horn
of Africa, Middle East, Mediterranean Basin and the Indian Subcontinent. The New World CL
causing Leishmania species includes Leishmania amazonensis, L. braziliensis, L. guyanensis and
L. mexicana reported from Central and South America (Burza et al., 2018).
Cutaneous leishmaniasis shows variety in clinical presentation of signs and symptoms vacillating
from simple to diffuse forms. Simple form of the disease is characterized by single or multiple
localized, self- healing and painless skin lesions developed at the site of sandfly bite. In worse
cases, it can cause disfiguring scars leading to social stigmatization. Diffuse form demonstrates
nodular lesions dispersed over the body (Debrant et al., 2004; Bahrami et al., 2011; Corrêa et al.,
2
2005). With reference to CL distribution sandfly vector belonging to Genus Phlebotomus is
found in the Old World and Lutzomyia in the New World. Vectors other than phlebotomine
sandflies have recently been incriminated. Day feeding hematophagous midges in subgenus
Forcepomyia (Lasiohelea) Keifer have been found to transmit a Leishmania species (not
properly identified but closest to L. enrietii) in red kangaroo (Macropus rufus) in Australia
(Dougall et al., 2011). CL is either anthroponotic (L tropica) with human-to-human infection or
zoonotic (L aethiopica, L major and all species found in the New World) that can be transmitted
from animals to human (Burza et al., 2018; Soptin et al., 2015). Rodents (Order: Rodentia),
carnivores (Order: Carnivora), marsupials such as opossums (Order: Didelphimorphia), anteaters
and sloths (Order: Pilosa), armadillos (Order: Cingulata) and primates (Order: Primata) have
been incriminated major reservoirs in zoonotic CL reported in various parts of the world (Bari
and Rahman, 2008; Roque and Jansen, 2014).
1.2. History and Discovery
Leishmaniasis is an ancient disease which is presented as historical figures, statues, ceramics and
papyrus (Tuon et al., 2008). The presence of Leishmania-like species in prehistorical eras is
predicted in two fossil ambers. The first Leishmania-like fossil was reported from the proboscis
and alimentary region of a blood-packed vanished female sand fly Palaeomyia burmitis
preserved in a 100 million-year old Cretaceous Burmese amber. The second fossil was described
as Paleoleishmania neotropicum originated in the extinct sand fly Lutzomyia adiketis in a 20–30
million-year-old Dominican amber. Leishmania promastigotes and amastigotes were perceived
in the gut and proboscis of the sand fly; though, no human or animals blood cells were
recognized. Leishmania species has perhaps evolved in Mesozoic era (252–66 million years ago)
earlier to the disintegration of the supercontinent Pangaea. Though, the precise geographical
establishment of the Leishmania species is a substance of continuing discussion (Steverding,
2017).
Ancient history of leishmaniasis has been traced back from 1500-2500 BC based on explanation
of Old-World CL lesions on medications in the library of King Ashurbanipal from 7th century
BC and DNA studies from Egyptian mummies and other archaeological remains. Recent records
are, however, found with 10th century Persian Muslim physician, astronomer, thinker and writer
of the Islamic Golden Age Avicenna (Abu 'Ali al-Husayn ibni Sina) who gave a detailed
3
description of the then and now called Balkh sore from Northern Afghanistan. Later records of
cutaneous leishmaniasis from Middle East were given mostly with local names of the disease
referring to the cutaneous lesions. Ancient history of visceral leishmaniasis or “kala azar” is not
clearly recognized. It was first noticed in District Jessore in India in 1824 but was mistaken for a
virulent form of malaria even by Ronald Ross. Original explanation of kala-azar was by the army
doctor William Twining (1790–1835) when he published a paper in 1827 regarding patients in
Bengal, India, who seemed withered with splenomegaly, severe anemia and sporadic
temperature. The initial epidemic of kala-azar was documented in 1824-25 in village
Muhammedpore thirty miles east of Jessore with mortality rate of 30%. Its status as a different
disease remained hidden until the discovery of the causative agent of visceral form of the disease
(Leishmania donovani) independently by Scottish army doctor, William Leishman and a
Professor of physiology at Madras University, Charles Donovan in 1900 (Steverding, 2017).
Scottish doctor and naturalist Alexander Russell (1715–1768) in 1756 published a
comprehensive clinical explanation of both dry and wet forms of Oriental lesion when he was
working in Aleppo city of Syria. He assigned CL as wet zoonotic CL instigated by L. major and
dry anthroponotic CL produced by L. tropica with mercurial plaster as successful treatment for
both types (Steverding, 2017). A number of people such as American James Homer Wright,
however, had seen parasites of cutaneous form of the disease but could not have properly
described. It was in 1885 and 1898 when David Cunningham and a Russian military doctor,
Borosky respectively described the Leishmania parasite as what really it is (Cox, 2002).
1.3. Taxonomy and Classification of Leishmania
An accurate classification and systematic account that depicts a clear picture of the phylogeny
and evolutionary history is normally crucial in elucidating its biology, relationship with host and
identification of strains. Morphology has been the main aspect of classification of almost all
groups of parasites until very recent years. However, trustworthiness of morphological
characterization is mostly open to errors because of plasticity of morphological characteristics.
With the beginning of molecular techniques, the fields of systematics and phylogenetics have got
into new direction. Molecular approaches are now excessively used to revise the classifications
done using traditional methods. Molecular systematics is unraveling many curiosities regarding
4
host-parasite interactions by exploring various aspects of the biology of serious human and
animal parasites (Monis, 1999).
Although, after its first description in 1903, much work has been done regarding genus
Leishmania, however, the evolutionary history and taxonomic classification of its species
remains incomplete and debatable. Confusion in the hierarchic classification of the taxon exists
at the level of genus and lower categories. This conflicting situation and uncertainty regarding
the taxonomy of the taxon is mainly due to incompatibility of molecular and non-molecular data
(based on clinical manifestations, biology of the parasites, geographic distribution, epidemiology
and immunological and biochemical characteristics) as well as studies being inclined to mostly
human infecting species (Harkins et al., 2016; Schönian et al., 2010).
Currently, the 35 to 40 (Fraga et al., 2013; Lukeš et al., 2014) different species in genus
Leishmania (Family: Trypanosomatidae: Order: Kinetoplastea) have been divided into two
sections Euleishmania and Paraleishmania (Ramalho-Ortiago et al., 2010). The section
Euleishmania contains species in three sub-genera, Leishmania, Viannia and Sauroleishmania
(Momen and Cupolillo, 2000; Fraga et al., 2010) and a separate new subgenus described in
Ghana L. enrietti complex. Species infecting humans belong to the former two sub-genera and L.
enrietti complex. The division of human Leishmania species into two sub genera originally was
done by Lainson and Shaw (1987) and was based on the region-specific growth and development
in the gut of sand fly vector. Members of the former prefer to develop in the midgut and pyloric
region (suprapylarian development) while that of the latter in the hindgut (peripylarian
development). This division is now supported by molecular data especially study of Fraga et al.
(2010) using molecular marker heat-shock protein 70 gene. L enrietti complex consists of four
species L. enrietti itself, L. siamensis, L. martiquensis and an unnamed species from Australia
(Llanes et al., 2015; Kwakye-Nuako et al., 2015; Corrêa et al., 2005). Species that infect Old
World reptiles and have been isolated from several lizards are classified in a separate subgenus
of Leishmania called as Sauroleishmania. Status of Sauroleishmania has, however, remained
conflicting among the workers. Some of the workers consider it as a separate genus and do not
place it in either of the sections (Momen and Cupolillo, 2000; Cupolillo et al., 2000).
Sauroleishmania (in whatever as a separate genus or as a subgenus) comprises of four different
species L. (S.) hoogstraali, L. (S.) adleri, L. (S.) gymnodactyli and L. (S.) tarantolae (Kwakye-
5
Nuako et al., 2015). Paraleishmania includes Leishmania hertigi and L. deanei that infect
porcupines and L. colombensis, L. equatorensis and L. herreri of sloths and squirrels and an
apparently unclear group, usually considered a genus, Endotrypanum. Endotrypanum currently
has two species E. shaudini and E. monterogeii. L. colombensis has been isolated from many
different hosts including humans (Momen and Cupolillo, 2000; Cupolillo et al., 2000).
Figure 1.1. Classification of Leishmania (Adl et al., 2012; Lukeš et al., 2014).
6
Table 1.1. Classification of Leishmania (modified from Adl et al., 2012; Lukeš et al., 2014)
Section Complex Species Distribution
Euleishmania Leishmania donovani (OW) L. (L.) donovani OW
L. (L.) archibaldi OW
1. L. (L.) infantum 2. Syn. L. (L.) chagasi 1= OW, 2= NW
Leishmania tropica (OW) L. (L.) tropica OW
L. (L.) aethiopica OW
L. (L.) killicki OW
Leishmania major (OW) L. (L.) major OW
L. (L.) gerbilli OW
L. (L.) arabica OW
L. (L.) turanica OW
Leishmania amazonensis (NW) L. (L.) mexicana (syn. L. (L.) pifanoi) NW
L. (L.) amazonensis (Syn. L. (L.)
gamhami ) NW
L. (L.) aristidesi NW
L. (L.) venezuelensis NW
L. (L.) forattinii NW
Leishmania enrietti (OW) L. enrietti OW
L. siamensis OW
L. martiniquensis OW
Leishmania brasiliensis (NW) L. (V.) brasiliensis NW
L. (V.) peruviana NW
Leishmania guyanensis (NW) L. (V.) guyanensis NW
L. (V.) panamensis NW
L. (V.) shawi NW
L. (Vianna) sp not recognized to
complex L. (V.) naiffi NW
L. (V.) lainsoni NW
L. (V.) lindenbergi NW
L. (V.) utingensis NW
L. (S.) hoogstraali OW
(L. S.) adleri OW
L. (S.) gymnodactyli OW
L. (S.) tarantolae OW
Paraleishmania
L. colombiensis NW
L. equatorensis NW
L. herreri NW
L. hertigi NW
L. deanei NW
7
1.4. Evolution of Leishmania
The phylogeny of genus Leishmania has been established on both the traditional (vector
specificity, clinical picture, geographic distribution, reservoir) as well as molecular data but it
still remains incomplete and unanswered (Marcili et al., 2014). Based on eco-biological aspects
Leishmania evolution has been influnced by its reservoirs and vector hosts. Its has been
speculated to be evolving from an estimated one billion years (der Auwera et al., 2011). Fossil
record indicates that Excavata, a group to which euglinids and kinetoplastids belong, have
eveolved in the Ordovician epoch of Palaeozoic era. The primitive most ancestor of the current
Leishmania might not have evolved until 700 million years ago in Proterozoic eon, where the
kingdom animalia (metazoans) could have evolved to provide host (both vector and deinitive) for
the parasites. The primitive host could have been a water dwelling animal. The Ordovician
ancestors of the Leishmania might have been parasitizing fish and amphibians via leeches. The
evolution of winged insects may be 300 miilion years ago and appearance of primitive winged
insets in diptera (current vectors of Leishmania) might have facillitated divergence from
primitive to new forms of Leishmania. Recovery of fossil haematophagus winged insects from
cretaceous about 140 miilion years ago and ancestral Palaeoleishmania proteus from again
cretaceous some 100 miilion years ago reveals the ingestion of trypanosomatid like free living
flagellates by cretaceous sandfly larvae, propagating them to adults and transmiting to the
vertebrates; initiating the life cycle. The descendants for current Leishmania species might have
evolved in paleogene period about 50 million years ago (Tuon et al., 2008).
Several hypotheses have been proposed for the origin of Leishmania regarding geographic
regions but the conflict still exists. Based on the previously mentioned non-molecular data,
Leishmania is hypothesized to have evolved in palaearctic lizards with Sauroleishmania being a
sister clade. According to this criteria a subsequent spread of Leishmania to Nearctic and
Neotroic through land bridges is assumed. Recent molecular approaches, however, support a
Neotropical origin of the parasite and subsequent spread to the Palearctic and Nearctic reigions
of the world (Tuon et al., 2008).
8
Figure 1.2. Geographical distributions of various Leishmania spp.; sandflies and animal
reservoirs in the Old World and New World. L: Leishmania (species), S: Sandfly (genus or
subgenus), R: Reservoir (genus or family) (Akhoundi et al., 2016).
1.5. Life Cycle and Transmission
All species of Leishmania possess a digenetic life cycle in which they move between an
invertebrate vector (sand fly) and definitive vertebrate host. Females of about 30 different
species (Lukeš et al., 2007) of hematophagous sand flies belonging to Genus Phlebotomus in the
Old World and Lutzomyia in the New World, are primary vectors of all kinds of human
leishmaniasis around the world. Species in the subgenus Sauroleishmania are transmitted to
lizard by sand flies in genus Sergentomyia (Momen and Cupollilo, 2000). The life cycle begins
when a female fly takes blood meal from an infected mammalian or other vertebrate host. The
fly ingests infected macrophages containing the non-flagellated form of the parasite called
amastigote in their phagolysosomes. When the macrophages lyse in the gut of the sand fly, the
amastigote form emerges and is soon transformed to a flagellated or promastigote form. The
9
transformation from amastigote to promastigote takes about 4 to 7 days or sometimes 10 days
depending on species and is accompanied by several intermediate stages of the promastigote to
the final infective metacyclic form. Stimuli that triggers the amastigote to promastigote form
have been reported to be lower temperature (from 37oC to 24oC) and high pH (from 7.3 to 8.5) of
the sandfly gut. During early days after blood suckling, natural obstacles to Leishmania growth
comprise proteolytic enzymes, the peritrophic medium adjacent the swallowed blood meal and
sandfly’s immune response. During the transformation cycle, the parasite has to face several
challenges and barriers to reach the final stage. Having an appropriate adaptation to travel a safe
journey through this critical intermediary stage of transformation is necessary for the parasite to
avoid being perished otherwise. The early promastigotes, the weakly motile long procyclic stage
with short flagellum, remain for 2-3 days in the blood meal enclosed by the peritrophic matrix
(PM) secreted by the mucosa of the sand fly’s gut. At the emergence on the decomposition of
PM, the very first priority of the strongly motile long promastigote, now called nectomonads,
move toward the anterior midgut (in case of subgenus Leishmania (Leishmania) which is called
suprapylarian with reference to this anterior movement as against the peripylarian subgenus
Leishmania (Viannia) that enters the hindgut after emergence from PM and then come to
midgut). The nectomonad stage transform to short leptomonad stage (Figure 1.4) now in the
anterior midgut (Gossage et al., 2003; Oliveira et al., 2009; Ramalho-Ortiago et al., 2010).
10
Figure 1.3. Life cycle of Leishmania species showing different developmental stages* of
promastigote in the sandfly midgut (adopted from Ramalho-Ortiago et al., 2010 (A), Oliveira et
al., 2009 (B) and Dostálová and Volf, 2012 (C).
*The figures display amastigote to procyclic and nectomonad development which occurs in the blood meal enclosed
in peritrophic matrix (PM). Nondividing nectomonad emergence from PM is followed after its attachment to the
epithelium of the gut and its change to leptomonad stage. The leptomonad develops to haptomonads and then to the
infectious metacyclic stage.
1.6. Epidemiology: Global Scenario
Ninety-nine countries and three territories of the world in five different continents (Africa, South
America, Europe, Asia and Australia) embrace leishmaniasis (Fraga et al., 2013; Strekers et al.,
2014; Rodrigues et al., 2015). An estimated 350 million people are at risk of the disease and 12
million are currently affected. The annual incidence of the disease is estimated to be
approximately 2 million cases worldwide (Carriόn et al., 2011) with a break up of 0.2- 0.4
million and 0.7-1.2 million cases of visceral and cutaneous leishmaniasis respectively (Akhoundi
11
et al., 2016). The actual reported cases are however, 0.6 million (Rodrigues et al., 2015). Annual
death rate, due to visceral infection is 30,000-50,000 cases worldwide (WHO, 2018). Although
the disease is widely distributed around the world, majority of the disease incidence occurs in a
limited number of countries. Almost 90 % of the cases of visceral leishmaniasis are reported
from only six countries that include India, Sudan, South Sudan, Ethiopia, Bangladesh and Brazil.
For cutaneous leishmaniasis, 70-75 % of the cases come from ten countries including
Afghanistan, Algeria, Colombia, Ethiopia, Brazil, Iran, Syria, North Sudan, Costa Rica and Peru.
In most cases, leishmaniases are zoonoses mostly affecting poor people in the rural areas.
Domestic dogs have been proved as potential reservoir for human leishmaniasis and 13 of 21
human Leishmania species have been so far isolated from dogs (Beattie and Kaye, 2011;
Cantasessi et al., 2015; WHO, 2018).
1.7. Surface Molecules Crucial for Survival in Hostile Environments
Some of the surface molecules play important role in the parasite’s overall biology. Important
most of these are glycoconjugates phosphoglycans including liphophosphoglycan (LPG),
metalloproteinase glycoprotein 63 (gp63) also called leishmanolysin, secreted acid phosphatase
(SAP) and filamentous (fPPG) proteophosphoglycan (Kamhavi, 2006). All these are accounted
for adaptability of the Leishmania to avert and survive the adversities presented by the hostile
environments of both the vector and definitive host and are important virulence factors. LPG,
however, is at the peak of prestige in fighting against the hostilities of the hosts. This molecule is
involved in avoidance of being digested by sandfly gut enzymes, scavenging of free radicals of
oxygen in the blood meal, prevention of procyclic and nectomonad stages from being flushed out
with digested blood remnants (Vanier-Santos et al., 2002). Along with gp63, internalization of
metacyclics by macrophage occurs through complement receptors CR1, CR3, mannose receptors
(MR) and fibronectin (FnR) receptors (Teixeira et al., 2013).
Along with the morphological and many other biochemical alterations during metacyclogenesis,
one major change is the lengthening of LPG molecules on the surface of metacyclic promastigote
stage. It is the modified LPG that is not capable of binding to the epithelial wall of the midgut
and due to this forward migration becomes possible (Spath and Beverley, 2001; Vanier-Santos et
al., 2002). Figure 1.5 shows the structure and chemical composition of procyclic and metacyclic
12
LPG of L. major (A) and the species-specific variability in the LPG of L. major, L. donovani and
L. tropica and structure of other major glycoconjugates.
Figure 1.4. Difference between the LPG of procyclics and metacyclics (A) and species-specific
variability in structure of LPG structure of other major glycoconjugates (B). {(A) from Spath et
al., (2001) and (B) from Kamhavi, (2006)}.
1.8. Genetics of Leishmania
The haploid genome of Leishmania contains 32.4–34 Mb with 34-36 chromosomes respectively
in the L. (Leishmania) and L. (Viannia) genera. The reduction in the number of chromosomes of
the latter by 1 is due to the fusion of chromosomes 20 and 34 in the former. Recent genome
sequence analyses have shown ~8,000 protein forming genes arranged mostly in single promotor
operated polycistrons of about 200 genes each known as directional gene clusters (DGCs). As a
result, post-transcriptional modification is basic in the regulation of gene expression (Llanes et
al., 2015; Marques et al., 2015; Romano et al., 2014). Of the total identified genes, Leishmania
shares ~6,000 genes with the other trypanosomatids and 95 % of these are said to be conserved
(remained unchanged) among the species for which total genome sequences are available. So far
difference of only ~2-4 hundred genes have been found among the three species L. major, L.
infantum and L. braziliensis (Fiebig et al., 2015; Peacock et al., 2007).
13
Table 1.2. Summary of the genome analysis for five different Leishmania species (Llanes et
al., 2015).
1.9. Immunology of Leishmaniasis
The immune response to Leishmania infection is complex and not yet completely deciphered. It
is mediated by several different and inter-dependent players and pathways of both the innate and
adaptive or acquired prongs of the immune system. Murine models have been and are still the
main source for the in vivo investigation of immunity to Leishmania species-which, of course,
shows species-specificity on the part of both host as well as the pathogen (Lohoff et al., 1998).
1.9.1. Promastigotes in the Inoculum vs Innate Immunity
Not all the metacyclic promastigotes of the inoculum survive but rather a huge number, almost
90 % in case of L. amazonensis in murine model, suffer from complement lysis and phagocyte
killing through toll like receptors (TLRs) at the site of the inoculation. Although dendritic cells
(DC) play their part in the phagocytic killing, neutrophils are the first (30 seconds post
inoculation of L. major in C57BL/6 mice) players of innate defense that encounter the
promastigotes of the inoculum using TLRs. It has been proposed that infected neutrophils,
14
especially the apoptotic neutrophils, even help the parasite to evade being killed and are carried
harmlessly to the macrophages. In other words, the promastigotes stopover in the neutrophils
(Soong, 2008; Horta et. al., 2012; Zamora-Chimal et al., 2016). A small number of resistant
promastigotes are opsonized by C3b and are phagocytosed by DC through CR1 and CR3
receptors. Phagocytosis in this way causes the production of IL-2 in the DC and the inactivation
of protein kinase C and Mitogen activated protein kinase (MAPK) which is subsequently
preventive to oxidative burst and macrophage activation (Zamora-Chimal et al., 2016).
1.9.2. Anti-Inflammatory and Immunomodulatory Effect of Sand Fly Saliva
There have been several direct and indirect evidences that factors in the sand fly saliva pose anti-
inflammatory effects by modulation of the immune response. Saliva has been shown to contain a
variety of pharmacologically active substances including immunomodulatory proteins. These
modulators mostly cause impaired antigen presentation and suppression of enzymes that cause
prevention of oxidative burst by the macrophage (Andrade et al., 2007; Rogers et al., 2009;
Loría-Cervera and Andrade-Narváeza, 2014). It has been shown that factors in the saliva of both
Phlebotomus papatasi and Lutzomyia longipalpis inhibit Th-1 expressing cytokines from
macrophages and up regulate Th-2 expression. Dendritic cells are also inhibited by factors in the
saliva of P. papatasi and P. doboscqi, and probably Lutzomyia longipalpis, from antigen
presentation through autocrine signals in the form of prostaglandin E2 and IL-10 that act by
down regulating the MHC-II on cell surface. The same cytokines of the dendritic cells also
inhibit and diminish the infiltration to the site of inoculation through the inhibition of neutrophil
chemotactic factors MIP-1α, TNF-α and Leukotriene B4 (Abdeladhim et al., 2014;
Selvapandiyan et al., 2014). Sand fly saliva has recently got prominence in the development of
immune protection against leishmaniasis and the vaccine design. This has been inspired by
induction of antibodies against sand fly saliva and the development of protection in animal
model pre-exposed to the bite of uninfected sandflies or vaccinated with either whole saliva or
purified salivary proteins. Anti-saliva antibodies IgG1, IgG4 and IgE has been demonstrated in
the serum of both the experimental models as well as the human volunteers from non-endemic
foci exposed to experimental bite of non-infected Lutzomyia longipalpis. Patients from endemic
areas of visceral leishmaniasis when were investigated had higher levels of IgG (Bandrade et al.,
2007; Bezerril et al., 2012; Loría-Cervera and Andrade-Narváeza, 2014). Immune response to
15
sand fly saliva is, however, variable with respect to species. As evaluated in the mouse model,
mice pre-immunized with the saliva of Lutzomyia intermedia did not cause protection against L.
braziliensis infection while a protective response was achieved with the saliva of L. whatmani in
the form of INF-γ producing lymphocytes (Gomes et al., 2016).
1.9.3. Host Immune Evasion
One of the important properties of Leishmania parasite is its mechanism to evade the host
immune response by modulating it. The evasion starts from innate response which is the earliest
to encounter the parasite at the entry site and leads to the adaptive one. This is achieved by
altering the function of both dendritic cells (DCs) and macrophage. The functionally altered DCs
produce insufficient amount of pro-inflammatory cytokines including INF-γ, α and IL-12 and
produce anti-inflammatory molecules like TGF-β and IL-10. Due to this, macrophage on the
other hand is prevented to produce oxidative burst which help Leishmania parasite establishing
inside its phagolysosomes (Gannavaram et al., 2016).
Surface molecules of metacyclic, not procyclic, promastigotes, especially LPG, GP63, GIP and
GIPL, play important role in subversion of host immune response and ensuring parasite survival.
Apart from these surface molecules, a variety of secreted molecules and enzymes including
cysteine peptidases, β-mercaptoetahnol dependent metalloproteases, acid phosphatases and
proteophosphoglycans of amastigotes affect macrophage anti-parasite mechanism. In Leishmania
major metacyclic promastigotes, LPG prevents the membrane attack complex by inhibiting the
conjugation of C5 and C9 to subvert complement lysis. Similarly, in case of both L. major and L.
amazonensis, gp63 cleaves C3b to C3bi and opsonization with C3bi causes internalization
through complement receptor CR3 rather than CR1 through C3b. This prevents respiratory burst
as mentioned before. It is LPG that delays the phagosome-endosome fusion by suppression of
the endosomal markers rab7 and LAMP-1 and the accumulation of F-actin. LPG and gp63 also
provide protection against the hydrolytic enzymes of the lysosome. LPG provides strong
negative charge and galactose-manose repeating units that prevent the enzyme attack. GP63
activity at acidic pH of the phagolysosome shows its function against the proteolytic enzymes
but is not yet certain as mutants can also survive. In vitro studies reveal that macrophages
incubated with LPG of GIPL of Leishmania did not express inducible nitric oxide synthase
(iNOS) upon stimulation by INF-γ and/or lipopolysaccharides (LPS). This is a necessary enzyme
16
to produce nitric oxide, a component of the oxidative burst. Rather newly found molecules of
Leishmania the peroxidoxins LcPxn1 and 2 and the superoxide dismutase scavenge the nitrites
and reactive oxygen intermediates to prevent respiratory burst (Olivier et al., 2005). Toll like
receptors (TLRs) of the macrophage recognize the LPG ligand on metacyclic and amastigote-
specific antigens. This initiate a protective immune response by inducing IL-12, TNF-α and
reactive oxygen species. As investigated in Leishmania major and L. donovani, however, the
parasite has evolved mechanism to subvert the response. In the former, protein molecules SOCS-
1 and SOCS-3 signals down regulate the TLR2 using cytokines while in the latter host
deubiquitinating enzyme A20 is induced to impair the TLR2 mediated immune response. L.
amazonensis encounter the TLR2 response by producing the double stranded RNA dependent
protein kinase and IFN-β. TNF-β upregulate the production of superoxide dismutase that
subverts oxidative burst (Gupta et al., 2013)
Recently, role of the purinergic pathway in the immune evasion by the Leishmania parasite has
been described by various studies and has been comprehensively reviewed by de Figueiredo et
al. (2016). ATP release during cell injury causes inflammation by activating macrophages and
DCs through P2X7 receptors. To control inflammation and tissue damage, immune cells degrade
ATP to ADP and then AMP through ecto-nucleoside triphosphate diphosphohdrolase (E-
NTPDase) or CD39. AMP is then degraded to phosphate and adenosine through ecto-5´-
nucleotidase (CD73). Normally, through transporters, the adenosine concentration is kept
equimolar on both sides of the membrane. In case of infection or injury, however, extracellular
concentration of adenosine reaches to maximum of 10 μM from normally in nM. This
extraordinarily raised concentration is attributed to have role in upregulating IL-10 and
suppressing nitric oxide. Along with other trypanosomatids and Legionella, Leishmania species
especially L. tropic and L. amazonensis have been shown to express these enzymes and to
enhance IL-10 production and consequent NO suppression by the dendritic cells and
macrophage. Presence of AMP, adenosine, pyrases and 5′-nucleotidase in the sand fly saliva and
their role in anticoagulation by prevention of platelet aggregation and in immune modulation
have been confirmed in various studies. Figure 1.6 summarizes the immune evasion mechanism
and survival in the macrophage.
17
Figure 1.5. Immunomodulation mechanism in macrophages (Bogdan and Röllinghoff, 1998).
1.9.4. The Oxidative Burst
During oxidative burst, macrophage releases reactive oxygen and nitrogen species (ROS and
RNS). Most important of the former includes superoxide anion (O2ˉ) while nitric oxide (NO) and
NO2 in the latter. Super oxides are induced spontaneously upon internalization of the parasite but
to produce nitric oxide macrophage stimulation by INF-γ and tumor necrosis factor (TNF) is
necessary. Production of NO occurs due to the reduction of N-terminus of L-arginine by
inducible nitric oxide synthase (iNOS or NOS2) the synthesis is NADPH mediated and occurs
upon stimulation by INF-γ and TNF (Horta et al., 2014; Carneiro et al., 2016). ROS not only
kills the parasite but also cause apoptotic killing of host cell by targeting the enzymes of signal
transduction especially the protein-tyrosine phosphatases (PTPs) that control vital physiological
functions. Killing of the infected cells by the immune system enhance the chances of pathogens
18
clearance. Target site of ROSs in the PTPs is rich cystine residue. Leishmania parasite however
has evolved mechanism to counteract the cleavage of PTPs by ROS. L. donovani has been shown
in vitro in murine cell line RAW 264.7 to subvert the oxidative burst killing and apoptosis of
host cells as well as itself. The subversion has been found to be suppression of cytokine signaling
(SOCS) mediated thioredoxin induced healing of PTPs by reduction. In this way parasite can
maintain the environment that provide tenancy for it (Srivastav et al., 2014).
1.9.5. Activation of Natural Killer Cells (NK) and Adaptive Immunity
Activation of natural killer cells (NK) is initiated in the lymph nodes by the solubilized LPG
from the complement lysed promastigotes and LPG presented in association with the CD86, TLR
and MHC-II receptors by the DCs that have overcome and killed phagocytosis resistant
promastigotes. This activate the NK cells and T cells thereby initiating adaptive response. Thus,
the initial acquired or adaptive immune response to Leishmania, like many other intracellular
pathogens, is, as evaluated in murine models, through the cell mediated prong of the acquired
immunity. Thenceforth, the humoral response is initiated through the recruitment of specific
subtypes of helper T-cells. Th 1 response is inhibitory to the infection while Th 2 response
favours the establishment of infection (Schleicher et al., 2008; Zamora-Chimal et al., 2016;
Zamora-Chimal et al., 2017).
1.9.6. Role of Natural Killer Cells
Natural killer cells are lymphocytes that are from 5-20% of the total lymphocytes in the blood.
These are classified as CD3ˉ and CD16/56+. There are two functional subsets i.e., cytokine
producing ones and cytotoxic ones. Apart from the above-mentioned activation mechanism,
direct contact of the promastigotes with NK cells can also contribute to their activation (Leike et
al., 2011). NK cells are the primary source of INF-γ in the early days of infection which is
necessary for oxidative killing of the parasite by macrophage and for the priming of T-cell
especially the protective Th1 response (Glennie and Scott, 2016) that also produce INF-γ but
several weeks later (Scharton and Scott, 1993). Figure 1.6 shows the mechanism of how NK
cells are activated and what they do?
19
Existing interpretation of the stimulation and role of NK cells in experimental visceral leishmaniasis (i.v. injection
of mice with L. donovani). Metacyclic Leishmania promastigotes are internalized by CD11c+ myeloid DC, that
produce the NK cell- triggering cytokine IL-12 in a TLR 9-reliant way (Schleicher et al., 2007). Furthermore, to IL-
12 the cytokine IL-2 is vital for the initial instigation of NK cells, while IL-18 plays a contributing character and IL-
15 is totally expendable in this procedure. Acquaintance of plasmacytoid dendritic cells (pDC) to L. promastigotes
centrals to a huge release of type I interferons (IFN-α/β), which, is of slight significance for the start of splenic NK
cells. Infected macrophages (MΦ) are unaffected by NK cell lysis in vitro and in vivo, however, NK cells produce
IFN-γ, that triggers MΦ for killing amastigotes, a practice that needs TNF and iNOS articulated by the MΦ.
Figure 1.6. Activation and role of NK cells in Experimental Visceral leishmaniasis (Bogdan et
al., 2012).
20
1.9.7. Th1 and Th2 Response-Mechanism and Outcome
Susceptibility or resistance of host is strongly associated with the immune response that is
triggered by the Leishmania parasite. The response itself is influenced by a variety of host and
parasite related factors including species of the parasite, its genetic structure and host genetic,
biochemical and physiological standing. As investigated in the mouse and other animal models
of cutaneous and visceral leishmaniasis (Figure 1.7) and as described before Th1 response leads
to parasite clearance and healing while Th2 causes the establishment of parasite and its
persistence in the host cells. The two responses present different cytokine profiles and a
consequent variable recruitment of the macrophages. Dominant cytokine of the Th 1 response are
INF-γ, interleukins 1, 2 and 12 and TNF-α. During the Th 2 response interleukins 4,5,10 and 13
and TGF-β are produced that orchestrate a different mechanism of macrophage activation. Apart
from these, IL-27 and IL-17 are also important for launching an inflammatory response (Ajdary
et al., 2009; de Santana et al., 2017).
Figure 1.7. Immune response in cutaneous leishmaniasis infection (Abdossamadi et al., 2016).
21
Defensive character of IL-17 against intracellular amastigotes in visceral leishmaniasis has been
demonstrated by comparing its level in patients and controls. A higher level of IL-17 in the
patients before treatment is decreased but still higher than the control can be attributed to its role
in the orchestration of protective immune response. It has been demonstrated to synergize with
interferon gamma and elicit NO production by the MΦ (Savoia, 2015). Similarly, a mixed type of
Th1 and Th 2 immune response in chronic and none-healing patients infected with L. tropica has
been recognized. It is attributed to the production of cytokines of the two responses including
IFN-γ, IL-13 and IL-5 (Ajdary et al., 2009) as well as the less described cytokines CD26 and
CD30 (Ajdary et al., 2007).
1.10. Treatment Approaches for Leishmaniasis
Being a neglected disease and causing a 60,000 annual death toll and annual morbidity of 2,090
thousand disability-adjusted life years, leishmaniasis is one of the major tropical diseases that
needs a special concern regarding drug development. It is because till date no reliable vaccine is
available against the disease (Evans and Kedziersky, 2012; Brikholtz et al., 2011) and the
available chemotherapy, the mainstay to encounter the disease, has lost its effectivity against
several, if not all, strains of the Leishmania parasite. The main factor in the chemotherapeutic
failure is development of resistance by the parasite strains especially in the endemic foci for the
various forms of the disease e.g., visceral leishmaniasis in North Bihar (India) and Sudan (Ponte-
Sucre et al., 2017). In addition, the first line of defense against the parasite including pentavalent
antimonials, the polyene anti-fungal amphotericin B and its liposomal preparation (AmBisome)
and antibiotic pentamidine and drugs passing the clinical trials have a variety of other limitations
(Seifert and Croft, 2006). High cost, side effects and hospitalization for treatment are the main
categories of limitations of the drugs in market (Nagle et al., 2014). Research regarding drug
discovery is in progress for both visceral and cutaneous forms of leishmaniasis. Cutaneous
leishmaniasis is especially on the list of Drug for Neglected disease initiative (DNDi) that is a
non-profit organization for drug development and research for neglected diseases (Mears et al.,
2015). A variety of synthetic and natural organic molecules and inorganic compounds are being
tested for their bioactivity against leishmaniases. Active drug targets in the parasite proteome are
of special concern and are under extensive investigations. Immunomodulant are being searched
both in the natural and synthetic pool of chemical compounds. Phyto materials have, however,
22
been the primary source of not only folkloric medicine for two millennia but also provide us with
39% of the currently available therapies in the market (da Rosa et al., 2017).
The Genomic Institute of the Novartis Foundation (GNF) is one of the leading bodies aiming at
the drug research and development against kinetoplastids. After screening 700,000 small
molecules against the extracellular bloodstream stages of Trypanosoma cruzi and axenic
amastigotes of L. donovani, 2,000 hits (which are different molecules having revealed inhibitory
effects) have been identified. Out of these hits, 44 % have also shown antipathogen activity
against the intracellular stages of T. cruzi. While such attempts have not been as much
productive in case of Leishmania as for T. cruzi, high-throughput image-based screening assays
by automated confocal microscopy against intracellular form have dug out 350 hits out of
300,000 screened. Regarding promastigote stage, only 4% of the hits active were also found
active against intracellular amastigote stage. A more advanced and efficient approach to screen
molecules against intracellular pathogens might be ex vivo cultures of infected target organs with
genetically modified fluorescent strains. Such has been of significance in case of luciferase
containing L. donovani in murine splenic explants that could identify 200 active hits out of 4,035
compounds. The same is also under extensive investigation using L. major, another important
species of the pathogen. Alongside this, half a million different compounds have been passed
through preclinical trials in in vivo murine models using fluorescent and bioluminescent
protocols to monitor parasite load in relation to dose of candidate compounds (Ang et al., 2015).
Since the trend to develop new drugs is rather new one instigated by realization of resistance
acquired by pathogens to the existing drugs, it will take us time to find out new therapies
especially for eukaryotic parasites that are metabolically and biochemically more diverse and
complex. Rapid developments in the proteomics and genomics are certainly of importance in the
drug development and innovation. It is because they are unravelling hidden biochemical and
metabolic mysteries of pathogens and provide potential targets for compound screening. Certain
computational techniques (a multidisciplinary approach requiring contributions from
mathematicians, chemists and chemical engineers, parasitologists, pharmacists and computer
scientists) are playing magnificent role in predicting pharmaceutical potential of organic and
inorganic molecules (Sinha et al., 2017).
23
1.10.1. Antimonials
Antimonials, as mentioned before, have been first line of defense against leishmaniasis for about
7 decades and continue to be the primary treatment available in most of the countries where the
disease is endemic. As trivalent sodium antimonial tartrate, they were first used in 1905 against
trypanosomes by Plimmer and Thompson. As antileishmanial, trivalent antimonials were first
used against cutaneous leishmaniasis by Vianna and against visceral leishmaniasis by Di Cristina
and Caronia in Sicily and Rogers in India in 1915. At that time the drugs were, however, found
highly toxic due to which the use was almost abandoned. It was in 1925 when urea stibamine, a
pentavalent form, was synthesized and used to save many lives in India by Brahmachari. It took
another 20 years to develop one of the currently used antimonials pentavalent sodium
stibogluconate (Pentostam) in 1945 via an intermediate antimony gluconate (Solustibosan) in
1937 (Nagle et al., 2014).
Instead of known resistance developed by the parasite against the drug, high toxicity
(pancreatitis, hepatitis and liver enzyme dysfunction, arrhythmias, muscle and joint pains),
lengthy therapy and poor tolerance, we are compelled to still use it due to the unavailability of
affordable and safe alternate chemotherapy. Currently a 20-28-day therapy at 20mg/kg/day is
recommended by WHO for cutaneous leishmaniasis that has shown species specific and
geographic limitations. A 30 to 60 % failure rate has been noted in case of L. (V.) braziliensis
and 30 % treatment failure against L. (V.) guyanensis (Zauli-Nascimento et al., 2010). Due to
increased acquired resistance, the cure rate in India (the endemic focus of Northern Bihar) for
VL is less than 50% (Sundar and Singh, 2016). Although in use since 1905 in various forms, the
resistance mechanism as well as the mechanism of action of antimonials is still not completely
known. What we know in this respect, has also been inferred from experiments primarily done
with arsenate. To grasp the Leishmania amastigotes, a drug has to pass through diverse physical
barricades e.g., the plasma membrane of macrophage, parasitophorous vacuole and the parasite
membrane. This is the most immediate obstacle that affects the success of drugs against
intracellular parasites. Resistance to the antimonials might be due to the parasite-specific
mechanism of reduction of active form of drug in the intracellular environment. This might
probably be achieved by a variety of different mechanisms including low uptake, enzymatic
inactivation and metabolism of drug, efflux of drug and sequestration in cell organelles. Most of
24
the information regarding the resistance acquisition has, however, been inferred indirectly by
using pentavalent arsenate, a related compound, either in vitro or in murine models using limited
number of parasite species (Ashutosh et al., 2007; Yadav et al., 2015).
As such both forms of the drug are accumulated at pharmacological concentrations in both the
stages of various species of Leishmania, SbV shows negligible antileishmanial activity. It is now
almost a fact that the trivalent SbIII, and not the pentavalent prodrug SbV, is the active form
against the parasite. It has been suggested the pentavalent form is first reduced to the trivalent
form inside the parasite. The reduction has been speculated to be enzymatic through mediation of
both the host and parasite thiols or non-enzymatic where thiols themselves may bring
conversion. The parasite enzymes involved are thought to be thiol-dependent reductase or
antimoniate reductase or both. The parasite derived thiols that might mediate the conversion are
thought to be glutathione-spermine conjugate and trypanothione. Host derived thiols including
glutathione (macrophage cytosol), cysteine and cysteinyl-glycine (lysosome) are proposed to be
the main reducing agents in this process. Among these the latter two and trypanothione of the
parasite that work at lower pH have been found more efficient (Ashutosh et al., 2007; Frézard et
al., 2009).
The uptake of antimonials, especially the SbV, is not all clear. As evaluated in axenic
amastigotes, SbV uses protein receptors that resemble that of gluconate because gluconate
compete with SbV. The non-competition of pentavalent arsenic and phosphate for SbV rules out
the proposition that it uses transporter of these. Since both the forms of the drug accumulate
differentially in the axenic amastigote, it is logical to say that both the forms use different
mechanism to enter the parasite. As evaluated by the uptake experiments using both trivalent
arsenic and antimoniate, these use GlpF (an aquaglyceroporin that facilitate the entry of glycerol
in E. coli). These are also facilitated by aquaglyceroporins in the yeast and mammalian cells
(Brochu et al., 2003; Mandal et al., 2015).
1.10.2. Amphotericin B
Amphotericin B is a naturally occurring antimycotic that belongs to the polyene (heptaene)
subgroup of macrolides (Hamilton-Miller, 1973) obtained from Streptomyces nodosus. It is
strongly lipophilic and in conjugation with liposomes presents highly changed pharmacokinetic
properties than the conventional form; the amphotericin B deoxycholate (D-AMB). The
25
conventional form of the drug manifests severe infusion reactions like fever, chills and
thromboblephitis and cause renal toxicity, lowering of blood potassium level and myocarditis.
Although lipid formulations do not lack these complexities but present these with comparatively
less intensity and severity. Lipid based formulations being less toxic and more efficacious are of
great concern in the current scenario of drug development (Silva-Jardim et al., 2014; Yousuf et
al., 2016; Kip et al., 2017). Three main lipid formulations currently approved by FDA are
liposomal amphotericin B (LAMB), amphotericin B lipid complex (ABLC) and amphotericin B
colloidal dispersion (ABCD) (Hamil, 2013) respectively known by their trade names
AmBisome®, Abelcet ® and Amphocel TM or Amphotec ® (Kip et al., 2017). Amongst these,
AmBisome have been the most extensively used in clinical trials and in the treatment of visceral
leishmaniasis. In comparison to other liposomal formulations, AmBisome has been found more
effective and less toxic in mouse model. It was found more effective in reducing burden of
Aspergillus fumigatus in experimentally infected mice than Lambin®. Toxicity of AmBisone
was comparatively less than Lambin®. It has also been found less toxic and more effective than
another liposomal formulation Anfogrn in the treatment of experimental fungal infections in
mice (Azanza et al., 2015).
Normally used as second-line of therapy against VL and aggravated cases of CL and MCL,
amphotericin B is used as primary cure of VL in Bihar (India), where L. donovani has become
resistant to antimonials and have been abandoned (Croft and Oliaro, 2011; Singh et al., 2017).
FDA approved dose for LAMB against VL (Savovia, 2015) is 3 mg/kg/day for
immunocompetent patients on days 1-5, 14 and 21 while that for immunocompromised
individuals a dose of 3-5 mg/kg/day on days 1-5, 10, 17, 24, 31, and 38. More than 97% cure rate
has been achieved with this regimen (Georgiadous et al., 2015). However, for reasons apparently
unclear, variable degree of efficacy of liposomal formulations of AMB have been reported in
other studies. The variability in efficacy might be attributed to administration regimen, clinical
form of leishmaniasis as well as geography and ethnicity. Balasegaram et al. (2012) reviewed
and shared expert opinion regarding the use of LAMB (AmBisome) against various forms of
leishmaniasis. Their study revealed that the drug and its various formulations has mainly been
used against visceral leishmaniasis. They found scarce and limited record of amphotericin B use
against cutaneous and mucocutaneous leishmaniases. They have reported a cure rate of 95% and
98% with AmBisome at a single dose of 10 mg/kg in phase-III and 20 mg/kg over four doses in
26
phase-IV clinical trials respectively in South Asia, Latin America and Europe. This regimen has,
however, given poor results in East Africa. In combination with 100 mg/kg miltefosine for 8
days and 15 mg/kg parmomycin for 11 days, 5 mg/kg AmBisome has given a cure rate of 97.5%
each in South Asia.
Amphotericin B targets ergosterol, a lipid that is shared by fungi and Leishmania species in their
cell wall and cell membrane respectively. After binding to the ergosterol, the complex forms a
transmembrane channel that turns the membrane permeable to cations, water and glucose. The
impaired permeability leads to altered membrane potential due to the loss of potassium ion and
consequently cell death occurs (Saha et al., 1986; Ordóñez-Gutiérrez, 2007; Ayres et al., 2008).
Some of the recent studies have also investigated the interaction of amphotericin B with the host
macrophage cholesterol and impairment of promastigote-macrophage attachment and subsequent
internalization (Paila et al., 2010). While this is the case, we can suppose that loss of ions and
impairment of permeability of host cells, especially muscle cells, when AMB interacts with the
membrane cholesterol can be attributed to the toxicity of the drug on the host.
1.10.3. Pentamidine
It is an aromatic diamidine and primarily an antibacterial agent. It was first implied in the
treatment of visceral leishmaniasis as well as the various forms of cutaneous leishmaniasis in
1950s in India and Spain. It was however, in use for the treatment of trypanosomes since 1940s.
Due to higher degree of toxicity it is usually used as second-line of chemotherapy where the
antimonials fail to show efficacy (Loure and Yorke, 1939; Croft and Coombs, 2003; Nagle et al.,
2014; Griensven et al., 2016). In French Guiana and Suriname, it is used as first-line of treatment
for CL with dosage regimen of 3 mg/kg over 4 intramuscular (IM) injections administered every
second day or a single IM dose of 7 mg/kg. The latter might be repeated after 48 hours if needed.
With the former regimen, a cure rate of 35 % against cutaneous infection of L. braziliensis in
Peru and 96 % for CL in Colombia have been achieved as compared to the standard antimonial
therapy. With the latter dosage regimen, 78.8% and 83.6% of the CL patients could be cured.
Apart from this, other studies in Colombia has reported 95% cure rate in the case of CL. In case
of VL, however, a cure rate of 67-77% was acquired with a 15-injections course of 4 mg/kg/day
administered over 14 to 30 days in 1990s. Other studies have reported 98% cure rate with the
standard dose of 4 mg/kg/day three times a week in India. Mediterranean VL caused by L.
27
infantum have been treated successfully with 2-4 mg/kg/day over 3-5 weeks treatment. After
this, in concern of rapid acquisition of resistance, its use was abandoned due to switching to
amphotericin B deoxycholate as second line drug. When it comes to toxicity, its use is limited
because it causes insulin-dependent and irreversible diabetes mellites with an incidence rate of 4-
12%. Other complications associated to its use include nephrotoxicity, myocarditis, pyrexia,
hypoglycemia and hypotension (Balaña-Fouce et al., 1998; Mitropoulos et al., 2010; Nagle et
al., 2014). A recent study used pentamidine as prophylactic agent to prevent relapse in
immunocompromised patients over 12 months duration. For this purpose, they employed a single
dose of 3-4 mg/kg body weight once in 3-4 weeks. With this prophylactic regimen, relapse
prevention was 79% and 71% after 6 and 12 months respectively (Diro et al., 2015).
1.10.4. Miltefosine
The general class to which miltefosine belongs is alkyl phosphocholine which are basically
ether-alcohols. Its IUPAC name is hexadecyl 2-(trimethyl-azaniumyl) ethyl phosphate and is also
known as hexadecylophosphocholine. Its empirical formula is C21H46NO4P with molecular
weight of 407.57 g/mol. Other related compounds used as drugs are edelfosine, ilmofosine and
perifosine. Miltefosine was originally developed as anti-neoplastic agent and was used in topical
formulations for the treatment of tumors of breast by two different independent groups in
Germany and United Kingdom in 1980s. Later, its antileishmanial activity was found in vitro as
well as in vivo studies. Currently it is a licensed oral antileishmanial agent in India against L.
donovani (Dorlo et al., 2012). A comprehensive review of the chemistry and pharmacology of
the drug as anticancer and antileishmanial agent has been presented in the reference quoted
ahead. Miltefosine (Impavido) is administered orally for 28 days at 2.5 mg/kg body weight. With
this regimen a cure rate of 94-97% have been successfully achieved in India against Kala-azar
(Eissa et al., 2012; de Morais-Teixeira et al., 2011). This has, however, dropped to 84 % in India
and Bangladesh in recent years. The cure rate against cutaneous infection caused by L.
panamensisis and L. braziliensis in Colombia was once more than 90 % but has gradually
reduced to 69.8 % in case of the former and to 49 % in the latter case. Studies in Brazil have
reported 75 % and 71% cure rate against the cutaneous disease respectively caused by L.
braziliensis and L. guyanensis. In Bolivia against the mucocutaneous infection by L. braziliensis
in Guatemala against the cutaneous leishmaniasis caused by L. braziliensis and L. mexicana and
28
in Afghanistan against L. tropica, a cure rate of 70 %, 53 % and 63 % have been achieved
respectively (Verla et al., 2012).
The miltefosine family of drugs cause apoptosis of Leishmania parasite, most expectedly by
direct degradation of the parasite DNA (Khademvatan et al., 2011; Verla et al., 2012).
1.10.5. Paromomycin
Paromomycin or aminosidine is an aminoglycoside antibiotic metabolite of Streptomyces
rimosus (Pujol-Burgués et al., 2014) that has been known and used as antileishmanial over the
last 60 years. However, its use as antileishmanial agent has been scattered and seemingly less
frequent than antimonials in the past. While it was found more efficacious in antimony resistant
cases during various clinical trials of both the VL and CL, it has been approved for parenteral
administration especially in the endemic state of Bihar in India where antimony resistant L.
donovani is prevalent. Reluctance in the use of paromomycin through parenteral route is due to it
being hepatotoxic, ototoxic and causing pain at the site of administration (Croft et al., 2006;
Banerjee et al., 2011). It is a WHO recommended first-line drug in combination with sodium
stibogluconate (SSG) against VL in Sudan. It is usually administered parenterally over 17 days
as compared to the 30 days regimen of SSG alone. The regimen is followed for both the freshly
infected and SSG treated relapse cases (Atia et al., 2015). Experimental topical ointments of the
aminosidine over two weeks treatment have been found to effectively reduce the parasite burden
and achieve clinical cure of CL. A more prolonged four-week topical application have been
effective in curing 2/3 of the patients in Iran leaving 1/3 to get antimonial therapy (Asilian et al.,
2003). Liposomal formulations for both the topical and parenteral use have almost traveled a
successful journey through the lab dish and animal trials to reach the clinical use (Bavarsad et
al., 2012). In conjugation to the liposomes, paromomycin is better absorbed, well tolerated and is
with improved therapeutic index (Gaspar et al., 2015).
1.10.6. Phytomedicine in Experimental and Clinical Trials
As mentioned before, plants have been, are and most certainly will be the primary source to
serve human with remedies for his illnesses. Many unexplored compounds in plants can act as
effective, harmless remedies and as replacement of the existing medications (Kaur et al., 2014).
The bulk of recent literature presenting experimental and trial-based use of thousands of plant-
29
derived agents against various species of Leishmania in vitro and in vivo in animal models is, of
course, hard to review completely. However, it is an attempt to review at least a fraction of the
scattered wealth of knowledge regarding phytomedicine in various infections including CL.
Plant derived compounds act against pathogens either directly by physically damaging them,
targeting their biosynthetic pathways and by modulating their immune systems. Withania
somnifera or winter cherry also known as Indian Ginseng, Asgand and Koti Laal in Pushto
language have been a candidate in folk remedies throughout the Sub-continent. It has been
extensively investigated for bioactivities and have revealed antioxidant, anticancer, anti-
inflammatory, anti-rheumatic, anti-tuberculosis, antianxiety, senescence preventive and
immunomodulatory properties. Withaferin-A and Sitoindosides VII-X isolated from this plant
have been associated with the above-mentioned activities and proliferative effects on superoxide
dismutase and catalase. Its constituents have been shown to positively affect leukopoiesis. W.
somnifera extract is hepato and nephro-protective and has anti-leishmanial activity exerted
through immunomodulation has recently been explored. A dose of 200 mg/kg in combination
with Aspharagus racemosus extract significantly reduced burden of L. donovani in liver and
spleen of BALB/c mice and induced strong Th 1 response. As evaluated by liver and renal
function tests, animals treated with the herbal extract had normal values as compared to the
control and SSG treated groups. At a dose of 350 mg/kg along with 5 mg/kg cisplatin, having
known hepatic and renal toxicity, significantly minimized the toxicity and enhanced parasite
clearance. However, when administered alone showed less potency against the pathogen. This
substantiated its role in immunomodulation. At 200 mg/kg along with A. racemosus extract and
at 350 mg/kg alone, it positively regulated the Th 1 cytokines (IFN-γ, IL-2, IG 2a) and antibodies
and downregulated those of the Th 2 cytokines including IL-4, IL-10 and IG1 (Sachdeva et al.,
2013; Alali et al., 2014; Kaur et al., 2014). W. somnfera constituents withanolides especially
chemotypes NMITLI-118, NMITLI-101R and withaferin-A have caused strong protective
immune response against L. donovani in hamster. In the treated groups, mRNAs protective
immune mechanism including inducible nitric oxide synthase, IFN-γ, IL-2 and TNF-α
heightened significantly but a reduction was seen in the anti-inflammatory cytokines like IL-4,
IL-10 and TGF-β and enhanced Leishmania-specific LTT response and elevated IgG2, ROS and
NO were seen. Intensity of these activities was more in NMITLI-10 followed by NMITLI-118
and withaferin-A (Tripathi et al., 2014). When administered to experimentally infected hamsters
30
in combination with the ED50 doses of miltefosine (10 mg/kg), paromomycin (30 mg/kg) and
amphotericin B (0.5 mg/kg) for 5 days each, the chemotype NMITLI-101R caused 98%, 94%
and 93% clearance respectively. This also exerted some effects on immunomodulation. It had a
strong effect on the up-regulation of inducible nitric oxide synthase and Th1 cytokines like IL-12
and IFN-γ while the Th2 cytokines IL-4, IL-10 and TGF-β were down regulated. This therapy
was also effective in generation of reactive oxygen species, nitric oxide, Leishmania-specific
IgG2 and significantly enhanced the delayed type hypersensitivity (Tripathi et al., 2017). In vitro
assays of crude extracts of the W. somnifera against the promastigotes and amastigotes of L.
donovani have been found effective with IC50 of 78 and 63 μg/mL. Withaferin-A, a constituent
isolated from the crude extract was found highly active against both promastigotes and
amastigotes with the IC50 values of 12.5 and 9.5 μg/mL respectively (Sharma et al., 2009).
Essential oils from a variety of different plants have been the most used plant derived bioactive
agents showing a wide range of activity against bacteria, fungi, viruses, and protozoa.
Commercial use of 300 of the currently known 3000 plant derived essential oils in the
pharmaceutical, agronomy, food, cosmetic and sanitary industries is on the record. Main
constituents of many of these oils are terpenes and terpenoids and other aromatic compounds.
Terpenes in these oils are mono-, di- and triterpenes. Monoterpenes are, however, in the highest
quantity (up to 90 %). Most of such oils contain a few components in higher concentrations
while the rest are present only in fractions (Akhtar et al., 2014; Le et al., 2017). Chenopodium
ambrosioides has been used over centuries as anti-parasitic remedy in South American folk
medicine. Main component of the plant that have been used and have potency against helminths
and protozoans is its essential oil. The essential oil is further constituted of three principle
components, carvacrol (62%), ascaridole (22%), and caryophyllene oxide (5%). These oils have
been commercially extracted and used as anti-parasitic agents but at a stage were abandoned to
use due to toxicities and sometime fatalities both in human and experimental animals. It is
genotoxic and causes gastroenteritis characterized by immediate hyperemia, leading to nervous
symptoms like headache, facial flushing, impaired vision, vertigo, incoordination and paresthesia
(Mnzote et al., 2009). Antimicrobial and anti-parasitic activity of the oil is extensively being
investigated. It has shown good in vitro activity against a variety of Gram positive and Gram
negative bacteria and different strains of Candida albicans (Brahim et al., 2015). The oil has
31
shown promising results, both in vitro and in vivo in mouse model, against L. amazonensis. In
vitro 50% effective dose of 3.7 and 4.6 μL/mL have been achieved against the promastigotes and
amastigotes respectively. When administered at 30 mg/kg orally, intraperitonially and
intralesionally, this essential oil showed variable degree of antileishmanial activity against L.
amazonensis. It was more effective in reducing parasite burden intraperitonially followed by oral
and intralesional treatment. Intraperitoneal administration was, however, more toxic than the
others (Monzote et al., 2006; Monzote et al., 2007). In another experiment, when administered at
a dose of 30 mg/kg every day for 14 days intralesionally brought significant healing and parasite
clearance in the footpad lesions of experimentally infected BALB/c mice with L. amazonensis.
As against this, an artificial mixture of main constituents i.e., ascarodole, carvacrol and
caryophyllene oxide at the same dose regimen in 30:70 DMSO-saline solution was not found
effective and caused mortality. Lesion morphology and morphometry in this case was
comparable to that treated with the mixture of DMSO-saline and the untreated control. Positive
control treated with 28 mg/kg glucantime caused less healing than the essential oils (Monzote et
al., 2014). Hydroethanolic extract of C. ambrosioides administered intralesionally caused a
significant reduction in the parasite load of L. amazonensis in footpad lesions, draining lymph
nodes and spleen in relation to standard antimony treatment and negative control. Treatment
regimen followed was 5 mg/kg extract and 28 mg/kg meglumine antimoniate every 4th day for 5
treatments. Oral treatment did not show satisfactory results in the parasite reduction. Heightened
production of nitric oxide was observed in the culture of peritoneal and lymph node macrophages
showing immunomodulatory effect of the plant (Patrício et al., 2008). Different combination of
the three major constituents of C. ambrosioides essential oil gave variable results against L.
amazonensis both in vitro and in vivo in BALB/c mice. A 1:4 ascaridol-carvacrol combination
gave excellent results in vitro against both the promastigotes and intracellular amastigotes and
was not cytotoxic. The same combination in a 20:80 mg/kg ascaridol-carvacrol was found far
better than the control in reducing the lesion size and the parasite burden (Pastor et al., 2015).
The action mechanism of ascaridol has been evaluated in the promastigotes of Leishmania
tarentolae. Once inside the parasite, it is cleaved in the presence of ferrous ion to release radicals
that are toxic to the parasite (Geroldinger et al., 2017).
32
Syzigium cumini (Jamun) essential oil with 87.7% monoterpenes has shown in vitro
antileishmanial activity against L. amazonensis promastigotes. An IC50 of 36 mg/L was obtained
at 24 hours incubation of promastigotes with the oils (Dias et al., 2013). A purified constituent of
S. cumini essential oil α-pinene, has shown promising results against both the stages of L.
amazonensis. IC50 values of 19.7, 16.1 and 15.6 μg/mL have been respectively achieved against
the promastigotes, axenic amastigotes and intramacrophage amastigotes. It was found to exert
the antileishmanial effect through enhancing phagocytic and lysosomal activity of the
macrophage and raising production of nitric oxide. They presented negligible cytotoxicity as
evaluated by haemolysis and exposure to the murine macrophages. The half minimal cytotoxic
concentration (CC50) and half minimal haemolytic concentration (HC50) of the essential oil and
α-pinene were 614.1 and 425.2 μg/mL and 874.3 and 233.3 μg/mL respectively (Rodrigues et al.,
2015). When tested against the same species of the parasite, its hexanoic extract showed
considerable leishmanicidal activity against the promastigote giving an IC50 of 31.64 μg/mL
(Ribeiro et al., 2014).
Demarchi et al. (2015) extracted and used essential oils and 6,7-dehydroroyleanone from
African plant Tetradenia riparia (ginger bush) against promastigotes and intramacrophage
amastigotes of L. amazonensis. Incubation of the promastigotes with the compounds for 24, 48
and 72 hours gave the LD50 values of 0.5, 0.3 and 0.8 μg/mL and 16.9, 14.9 and 3 μg/mL for the
essential oil and 6,7-dehydroroyleanone respectively. Morphological alterations were also
induced in the treated promastigotes. Both the agents caused a significant reduction in the
infection index in the murine macrophages infected with amastigotes and caused little
haemolysis in human erythrocytes. The compound 6,7-dehydroroyleanone was, however, toxic
to murine macrophages.
Thirty-seven essential oils from Vietnamese plants were tested against promastigotes of L.
mexicana at 25 and 50 nL/mL by Le et al., (2017). Of 37 oils they found 5 oils with considerable
leishmanicidal activity with 99% promastigote killing at the 25 nL/mL and an IC50 value of less
than 10 nL/mL. These 5 oils were from Amomum aromaticum (IC50=9.25 nL/mL),
Cinnamomum cassia (IC50=2.92 nL/mL), Elsholtzia ciliate (IC50=8.49 nL/mL), Ocimum
gratissimum (IC50=4.85 nL/mL) and Zingiber zerumbet (IC50=3.34 nL/mL). When evaluated
33
for cytotoxicity against WI-38 fibroblasts and J774 macrophages, no toxicity was reported. This
study provided valuable preliminary information about the bioactivity and cytotoxicity of the 5
plants. Further studies were suggested to work out the lowering of cytotoxicity and efficacy in
the animal models and beyond.
Currently, literature regarding use of phytochemicals against microorganism in general and
Leishmania in specific is growing like weeds, therefore bulk of literature available on the use of
plant extracts, fractions, and isolated compounds against Leishmania is practically not possible to
review here. Over the last three decades, extensive work has been done throughout the world
both in developed and developing countries and it would need a book of several volumes to
completely accommodate the data. Although most, if not all of the phytoproducts are used in the
lab dish and against animal model stages of Leishmania. The hugeness of literature can be
illustrated with a simple example of a review published by Soosaraei et al. (2017), where7500
articles were found from 1999 to 2015 on electronic databases like EBSCO, ScienceDirect,
PubMed, Google Scholar, Scopus and four Persian databases Magiran, Iran doc, Iran Medex and
the Scientific Information Database (SID). Out of these articles 1750 were found irrelevant by
title. Since they were interested in the articles published in Iran, so they included only 68 in their
review. Interestingly, in these 68 articles a total of 188 experiments using 98 plants were
performed of which 140 were in vitro and 48 were in vivo.
1.10.7. Inorganic Agents in Experimental and Clinical Trials
Over the last three decades, like phytomedicinal chemists, the inorganic and organometallic
chemists have been equally busy in developing metal-based therapies against different parasites
including Leishmania. The primary design strategies they have been focusing on are; enhancing
of the antiparasitic efficacy by the incorporation of metal centre to existing agents, improvement
of bioavailability of the existing agents by bioactive ligand coordination, targeting DNA with
metal coordinated intercalating agents, and description of metal inhibitors of parasite specific
enzymes.
Discovering group-specific common targets in Leishmania and their cousins like
trypanosomatids and subsequent development of therapeutic agents against common targets is
34
like killing two birds with one stone (Gambino and Otero, 2017). Organometallic complexes of
various transition metals like zinc, copper, lead, osmium, rubidium, ruthenium, platinum,
bismuth, gold, silver, tin, nickel, boron, and cobalt have been employed for their antiparasitic
potential. Copper and zinc complexes derived from dipyridophenazine, a DNA intercalator, have
shown excellent leishmanicidal activity against both the promastigote and intracellular
amastigote form of L. infantum. The in vitro assays have, however, found them cytotoxic to
human macrophages (Madureira et al., 2013; Tahghighi, 2014). Cytoplasmic disorganization,
vacuolation, and induction of binucleated cells have been the effects of these compounds in
promastigotes (Novarro et al., 2003). Copper conjugates with β-diketonates have been evaluated
against the promastigotes of L. amazonensis. Cu(acetylacetone)2, Cu(trifluoroacetylacetone)2,
Cu(hexaluoroacetylacetone)2 and of Cu(nitrilotriacetic acid)2 gave ED50 values after 24, 48, and
72 hours of incubation as 31,84 and 94 μM, 68, 57 and 43 μM, 135, 124 and 68 μM and 263, 293
and 122 μM respectively. With the same incubation regimen, however, the ED50 values were 83,
19, 15 μM, 74, 31, 24 μM, 137, 39, 12 μM and 342, 137,13 μM respectively (Portas et al., 2012).
Lead complexes derived from 2,5-anhydro-D-manitol are potential antitrypanosomatid enzyme
inhibitors. Out of the 55 derivatives, 13 and 16 caused more than 50% inhibition of
Trypanosoma brucei phosphofructokinase and Leishmania mexicana pyruvate kinase
respectively (Nowicki et al., 2008). Three different complexes of ruthenium showed promising
activity against L. amazonensis, L. braziliensis and L. mexicana. Moreover, murine macrophages
RAW 246.7 treated with the complexes at the same concentration showed 90% viability (Costa
et al., 2017). Silver complexes of polypyridyl have been found effective against the
promastigotes of L. mexicana at 10 μM/L in vitro. [Ag (1,10-phenantroline-5,6-dione)2] NO3 and
[Ag (dipyrido[3,2-a: 2’,3’-c]phenazine)2] NO3 were shown as DNA metallo-intercalators and
were proposed to exert their antileishmanial activity through DNA inhibition (Navarro et al.,
2006). Carboxylate derivatives of tributyltin (IV) complexes have shown promising activity
against promastigotes of Leishmania tropica KWH23 at a concentration of 20 μg/mL. All the
tested compounds caused 95-97% lethality and gave 50% inhibitory concentration from 0.008-
0.954 μg/mL. Moreover, they gave much higher IC50 values when tested against the HepG2 and
THP1 cell lines at the same concentration (Waseem et al., 2017).
35
Organometallic complexes of the platinum-II and ruthenium-I have been found to cause growth
inhibition of both the flagellated and amastigote stages of L. donovani. RhI(CO)2 Cl (2-
Aminobenzothiazole) complex interacted with the nucleic acid as well as protein biosynthetic
pathways of the parasite and was most potent against the parasite. It was, however, toxic to J774
murine cell line. Complexes [(Cis-PtII (DDH) (2,5-Dihidroxibenzensulfonic)2 and RhI (CO)2
Cl(5-Cl-2-Methilbenzothiazole)] gave rather unsatisfactory results causing morphological
alterations only but were found safe and nonaggressive to the J774 cells (Mesa-Valle et al.,
1996). Some complexes derived from (2,2′:6′,2′′-terpyridine) platinum-(II) have been able to
cause effective inhibition of the intracellular forms of both the L. donovani and Trypanosoma
cruzi at 1μM concentration and the extracellular form of T. brucei at 0.03 μM (Lowe et al.,
1999). Although it got fame much latter in the last quarter of 20th century with the discovery of
an organometallic complex of Platinum II, the cisplatin, an anticancer agent, scattered use of the
inorganic metals and their complexes has been known since ancient times. Important step in the
use of inorganic therapeutics was the discovery of arsenophenyglycine and arshphenamine by
Paul Elhrich for sleeping sickness and syphilis respectively (Caballero et al., 2015). Cisplatin in
vitro antileishmanial outcome has earlier been established. Kaur et al. (2010) studied
antileishmanial action of 0.5mg/kg body weight and 1mg/kg body weight of the cisplatin drug L.
donovani infected BALB/c mice. Significant reduction in the parasite burden was noted
by1mg/kg body weight. Akhtari et al. (2019) reported effective in vitro antileishmanial action of
a cisplatin nano-formulation against promastigotes and amastigotes of Leishmania major. The
IC50 of cisplatin nano-formulation was 0.11±0.09μM for amastigotes of Leishmania major i.e.,
41-fold lesser than that of Glucantime (4.52±1.31μM), signifying that a minor quantity of
cisplatin nano-formulation in contrast with Glucantime is needed to execute 50% of intracellular
amastigotes.
A complete review of the ongoing extensive investigational research on the metal-based
remedies for the parasitic diseases and physiological disorders is beyond the scope of this
document.
1.10.8. Diterpenoids
Diterpenoids is a class of natural metabolites of bacterial, fungal and plant origin widely used in
industry and medicine. They are all derived through enzymatic cyclization of (E,E,E)-
36
geranylgeranyl diphosphate. The enzyme used in diterpene production is diterpene synthase
(Smanski et al., 2012). Two important sub-groups of the diterpenoids are ent-kauranes (e.g.,
steviol) and ent-beyeranes (e.g., isosteviol). Interconversion and chemical modification of these
to modify their biological and biochemical properties can be done. These have been found to
possess bactericidal, malaricidal, antidiabetic, anti-inflammatory and anti-tumour activities
(Cherney et al., 2013; Wang et al., 2018). Ent-kaurane diterpenoids ent-kaur-16-en-19-oic acid
and ent-kauran-16α-ol (Da Costa et al., 1996) and pimarane diterpenoid pimaric acid (Rubio et
al., 2005) have been investigated to effectively inhibit the growth of Trypanosoma cruzi at 500
μg/mL, 200 μg/mL and 150 μg/mL respectively. When tested against different pathogenic
protozoa, ent-3-a-hydroxy-kaur-16-en-18-ol (ent-kaurane) and ent-7-oxo-pimara-8,15-diene-18-
ol (ent-pimarane) were found much effective. The former showed significant activity against
Plasmodium falciparum (IC50=3.5 μM) and Leishmania donovani (IC50=2.5 μM) while the
latter was found effective against P. falciparum only (IC50=3.8 μM) (Nogueira et al., 2016). Iso-
kaurenoate, an esterified derivative of kaurenoic acid, showed high effectivity against P.
falciparum strain W2 (IC50=8.8 μM) (Batista et al., 2013). Members of thiosemicarbazones,
derivatives of kaurenoic acid which is a diterpenoid, have been found to possess trypanocide
activity against the Trypanosoma cruzi with IC50 ranging from 2-24 μM (Haraguchi et al.,
2011). Flavonoids from Stevia satureiifolia var. satureiifolia, also producing steviosides, steviols
and isosteviols, have been found to inhibit the epimatigotes, amastigotes and trypomastigotes of
T. cruzi and promatigotes of Leishmania braziliensis. Compounds eupatorin and 5-
desmethylsinensetin caused considerable inhibition on the epimatigotes (IC50s 0.2 and 0.4
μg/mL respectively) and trypomastigote (IC50s 61.8 and 71.1 μg/mL respectively). Compound
5-desmethylsinensetin showed IC50 of 87.7 and 37 μg/mL respectively against the amastigotes
of T. cuzi and promatigotes of L. braziliensis (Beer et al., 2016). 4-nitro- and 2,4-
dinitrophenylhydrazone, isosteviol derivative, have been found to show activity against T. cruzi
with IC50 value of 17.5 to 22.58 μg/mL (Ullah et al., 2016). Ullah et al. (2019) have
comprehensively reviewed the biological activities and synthesis of isosteviol from Stevia
rebaudiana.
1.10.9. Actinomycins
Actinomycins are the secondary metabolites of actimomycetes, a hallmark group of bacteria
regarding antibiotic production. Genus Streptomyces is of special concern and produces 35-50
37
kinds of actinomycins (Katz and Pugh, 1960). Actinomycin D has been found to hamper DNA
synthesis (Cooper and Braverman, 1977), cause cytostasis and enzymogenesis (Grand et al.,
1972; Nsi-Emvo and Raul, 1984) and up to 2005 about 3300 publications it has been a subject of
(Koba and Konopa, 2005). This inhibition on DNA synthesis attributes the anti-tumor effects of
actinomycicn D and C3 (Hollstein, 1974).
1.11. Leishmania Strain to be Used
Although there had been scattered studies and reports about leishmaniasis (Rowland et al., 1999)
in Khyber Pakhtunkhwa (KP) after its emergence due to post Russian invasion and refugees’
influx to Pakistan, it was not until 2010 that a serious and detailed study about genetic
characterization of the causative agent was undertaken. Dr Nazma Habib Khab, Assistant
Professor Department of Zoology, University of Peshawar Pakistan, then PHD student at London
School of Hygiene and Tropical Medicine worked out the molecular characterization and
phylogeny of isolates from KP, Pakistan (Khan et al., 2016). Being a strain of local origin and
easily available in local repositories, Leishmania strain KWH23 was used in this tudy.
Aim and Objectives of the Present Study
This study aimed to explore antileishmanial properties of some plant and microbial metabolites.
Main objectives of this study were to;
1. Study growth characteristics and progression of Leishmania tropica KWH23 in vitro
2. Explore antileishmanial effects of Actinomycin D, Z3, Z5 and Hydrazine derivatives of
Isosteviol
3. Assess cytotoxic effects of the mentioned test compounds on human lymphocytes.
4. Evaluate genototoxic effects of the test compounds through Comet Assay.
38
Chapter 2. Morphology and Culture Characterization of
Leishmania tropica KWH23
2.1. Materials and Methods
2.1.1. Culture of Leishmania tropica KWH23 Promastigotes
Leishmania tropica KWH23 promastigotes culture was initiated in RPMI-1640 (with 20 mM
HEPES and L-glutamine without NaHCO3) medium (Sigma-Aldrich Europe) at a densiy of
1×105/mL in tissue culture flask with surface area of 25 cm2, canted neck, and non-vented cap.
The RPMI-1640 medium was supplimented with 10 % heat-inactivated fetal bovine serum (hi-
FBS, Sigma-Aldrich Europe), 100 µg/mL streptomycin and 100 IU penicillin (Sigma-Aldrich
Europe). The growth was monitored daily by counting in improved Newbaur haemocytometer
over a week. The coverslip was moisturized with clean water and coverslip was affixed to
the hemocytometer. The occurrence of Newton's refraction rings below the coverslip specified
appropriate adhesion. Promastigotes were kept in in a Petri dish containing formaldehyde
impregnated tissue and all 4 sets of 16 corners were counted in hemocytometer (Figure 2.1).
These cells were averaged and multiplied by 104 to get number of promastigotes/mL. The growth
curve for Leishmania tropica KWH23 promastigotes was then made as shown in Figure 2.2.
Figure 2.1. Hemocytometer illustration representing set of 16 squares (marked in red colour)
used for counting promastigotes.
39
2.1.2. Evaluation of Morphological Variations of Promastigotes during Culture
Morphologic variability was monitored daily through Giemsa stained smear over a period of one
week. About 20µL of culture was smeared on albuminized slides and fixed in methanol for 1-3
minutes after air drying at room temperature. Various concentrations of the methanol were tested
to fix the Leishmania tropica KWH23 promastigotes. Fixed smears were stained for 14-19
minutes with 1 in 10 Gibco-KaryoMAX Giemsa stain (Thermo-Fisher, Germany) stock solution
in water (WHO, 2016). Smears were observed under the powerful oil immersion objective
(1000x magnification) of the light microscope (Leitz Dialux 22 EB, Germany) and photographs
were obtained by using USB microscope camera or sometime mobile phone camera. Though
comparable in appearance and aroma to (drinking) ethanol, methanol is poisonous to the nervous
system. Contact to methanol through ingestion, inhalation, or absorption can cause impaired
vision, unconsciousness and death. Personal protective equipment (PPE) like covers for eyes
(safety glasses), face (mask) and skin (gloves) were used during fixation process.
Temporal variation in the length, width, and shape of the promastigotes and length of the flagella
were qualitatively accessed to determine different Leishmania tropica promastigote morphotypes
in the axenic culture. Metacyclic promastigotes were also assesed through scanning electron
microscopy. For the morphological examination, Leishmania tropica promastigotes were fixed in
2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer for one to three hours. The coverslips
were washed in 1% HCL in industrial methylated spirit (IMS) at 50oC, washed thoroughly under
tap water, dried and stored. Afterward, promastigotes were stick to poly-L-lysine-covered
coverslips and dehydrated in ascending concentration (20%, 30%, 50%, 70% and 90%) of
ethanol, with each step of 5-10 minutes duration.
2.1.3. Cultivation of Axenic Amastigotes and Evaluation of Morphology
Axenically grown Leishmania tropica amastigotes has become an interesting life cycle stage for
drugs screening and for investigating biochemical as well as molecular variability between the
promastigote and amastigote stages. A variety of different protocols have been followed to
axenically transform promastigotes to amastigotes for different Leishmania species. In the
present study, 1x106/mL Leishmania tropica promastigotes in the stationary phase were grown at
37oC in humidified conditions lacking CO2 in RPMI 1640 medium fortified with 20% heat-
inactivated fetal bovine serum (hi-FBS, Sigma-Aldrich Europe), 100 µg/mL streptomycin, 100
40
IU penicillin and L-glutamine. The pH of the medium was adjusted at 4.4 using phosphoric acid
(85% aqueous solution BDH Chemicals, UK). The amastigotes were stained with Giemsa stain,
and phase contrast microscopy was used to monitor the transformation on daily basis and fully
transformed amastigotes were also confirmed through scanning electron microscopy. During the
promastigote to amastigote transformation, the change in cell shape was observed daily by smear
fixation and staining and expressed in terms of percentage from elongated promastigote form in
the initial inoculum to the fully formed amastigotes with a variety of oval forms in between.
Presence, length and absence of the flagellum was also recorded. Reduction in the cell size was
verified through stage and ocular micrometry. The size measurement was mostly based on the
fully round and oval amastigotes. The diameter was confirmed after taking mean for 20
randomly selected individual amastigotes cell measurements.
2.1.4. Acquisition of Axenic Amastigotes by Starvation
Amastigote stages were also acquired by Leishmania tropica in the in vitro culture, when the
promastigote culture was kept starved without medium changes, accidentally for the first time,
and then several times when repeated. This was a total accidental happening, which occurred due
to a week vacation when, to be on the safe side, the culture was sub-passaged in 25 mL complete
RPMI1640 medium in 25 cm2 non-vented flasks at 26°C, the normal temperature for
promastigotes. After opening, to my surprise, the culture medium was plasma-yellow and on
observation revealed a bulk of amastigotes. These were processed as mentioned before. This
practice was then repeated several times deliberately to confirm the conversion.
2.1.5. Viability during Transformation
Viability of cells is a necessary aspect in the drug sensitivity and biological studies. The viability
of Leishmania tropica during promastigote to amastigote transformation was checked daily over
a week using trypan blue exclusion and was expressed as percentage of viable cells. After
complete transformation, the amastigotes were cultured in the normal RPMI1640 as mentioned
above for promastigotes to check the viability.
41
2.2. Results
2.2.1. Growth Curve of L. tropica KWH23 Promastigotes
Flagellum, kinetoplast and nucleus are distinct morphological indicators of Kinetoplastida and L.
tropica owns a single flagellum, nucleus and kinetoplast and each duplicate once during the
entire cell cycle (Wheeler et al., 2011). The flagellum growth inducts first, trailed by mitosis
then kinetoplast division that twitches after the commencement of nuclear anaphase. The growth
of L. tropica KWH23 promastigotes through growth curve over one week period in the culture is
shown in figure 2.1. For the first three days of the culture, a stunted growth was observed. On
day 4, the culture reached log phase of the growth. On day 5th to 6th the mid and late log phases
were achieved. This means peaked growth was achieved on day 6. On day 7, the culture
inwarded stationary phase with a promastigote density of 1×107/mL. The promastigotes became
sluggish with decreased motility and were slithering around. Cells were looking stumpy with
long flagella. The promastigote body looked like a carrot with swollen anterior and tapered
posterior (Figure 2.3-b).
Figure 2.2. Representative growth curve of Leishmania tropica KWH23 in vitro.
-2000000
0
2000000
4000000
6000000
8000000
10000000
12000000
14000000
16000000
1 2 3 4 5 6 7 8
Nu
mb
er o
f P
rom
asti
gote
s/m
L
Days
42
2.2.2. Axenic Promastigote Morphotypes
Various morphotypes were determined through one week developmental cycle for L. tropica
KWH23 promastigotes. Culture was initiated from stationary phase promastigotes. It was infinite
passage number of the L. tropica promastigotes strain KWH23. Promastigotes of L. tropica at
different developmental forms were observed in these 8 days, some promastigote cells were in
the course of division i.e., in mitotic (M) stage of the cell cycle, whereas the rest were at other
stages of interphase including G1, S and G2 phases. In present study a promastigote cell in M
phase was demarcated as having two nuclei and/or kinetoplasts having incomplete cytokinesis,
as noted by inspection of Giemsa-stained slides (Figure 2.3).
Figure 2.3. Different stages of L. tropica KWH23 promastigotes representing cell cycle in
culture (see text for description). A naturally occurring amastigote (Arrow in “e”) can be seen in
a 13-day old promastigote culture.
43
On day one almost all of the promastigotes were nectomonads (Figure 3.2-b) having long,
slender and progressively tapering bodies with long flagella about one and a half to twice as long
as the promastigote body. On day 2 only 5% of the promastigotes were leptomonads (Fig 2.3-c)
with still slender and long cells and longer flagella almost indistinguishable from those of
nectomonads. In leptomonads, the cell body tapered posteriorly and cell to flagellum ratio
decreased. On day three, the ratio of leptomonads rose to 44 %. During the log phase on day 4,
metacyclics appeared in the culture. The matacyclics to leptomonads to nectomonads were 27%,
43% and 29% respectively. Metacyclics had long flagella and short stumpy cell bodies with
anterior end much broader than the posterior as compared to that in leptomonads and
nectomonads. The posterior end of the body was bearing a slender tail like extension (Figure 2.3
d-f). Promastigotes number increased till day 6 ( late log phase), where onward the stationary
phase reached and the number of metacyclics increased progressively. Following the
development of the parasite in the present study, the procyclic promastigotes were enormously
infrequent (Figure 2.3-a) and infrequently oval amastigotes were also observed in Giemsa
stained smears (Figure 2.3-e).
2.2.3. Division of L. tropica Promastiogtes in Axenic Development
The nucleus, kinetoplast and flagellum provide distinct morphological indicators. A typical L.
tropica promastigote cell have single kinetoplast, nucleus, and flagellum and each of which
duplicates once during the cell cycle (Wheeler et al., 2011). During logarithmic growth, 17% of
the promastigotes were found dividing, so different features of the dividing cells were recorded
(Figure 2.4). Most of the individuals duplicated the flagellum first following division of the
kinetoplast and the nucleus respectively. However, in rare cases, division of the kinetoplast and
nucleus preceded flagellar splitting (Figure 2.4 C, D, and E).
Wheeler et al. (2011) reported interesting events through phase contrast and fluorescence
microscopy in L. mexicana promastigote cell cycle. They presented new flagellar emergence
from the flagellar pocket at 5.2-hour, DNA segregation at 6.3-hour (mitosis) and finally the
kinetoplast partition at 6.6-hour time period respectively. We also observed “rosettes” which are
bigger bunches of cells placed side-to-side with flagella in the direction of the centre of the
cluster. Rosettes are also involved in a rare swarming behaviour, flocking into prolonged
44
clusters. Iovannisci et al. (2010) showed rosettes as an unrecognized stage in the life cycle of
Leishmania and hypothesized that rosettes initiate mating in Leishmania.
Figure 2.4. Division of the L. tropica promastigotes in axenic development. Cells with 2 flagella
single kinetoplast and single nucleus (A and B), Cells with single flagellum, two kinetoplasts and
single nucleus (C), Cells with Single flagellum, two kinetoplasts and two nuclei (D and E) and
Cells with two flagella, two kinetoplasts and two nuclei near to the completion of cytokinesis
(F). Solid arrows show kinetoplasts and the others indicate flagella.
2.2.4. Cultivation of Axenic Amastigotes
After 24 hours of incubation under the above-mentioned conditions, the metacyclic
promastigotes started to change in the overall morphology. In transformation from promastigote
to amastigote stage, major changes have been observed regarding the morphological changes in
the cell shape and length of flagellum (Figure 2.5).
45
Figure 2.5. Promastigote to amastigote transformation. Change in the cell morphology and loss
of flagellum is seen progressively.
2.2.5. Changes in Outline and Flagella during Differentiation of Promastigotes to
Amastigotes
Leishmania experiences differentiation from sandfly promastigote form into intramacrophage
amastigotes, a progression that is vital for its intracellular existence. The changes in shape of the
cell and length of the flagellum during promastigote to amastigote transformation/ differentiation
were documented (Table 2.1). Leishmania are exposed to pH changes through their life-cycle.
Sandfly blood meal contains procyclic promastigotes which reproduce in the pH 7.3-7.4 of the
bloodmeal, while amastigotes are adopted to acidic environment of the macrophage
phagolysosomes (pH 3.2-5.5). Acidification of phagosome is critical defensive mechanism in
phagocytes activating microbial killing.
We observed amastigote stages when Leishmania tropica in the in vitro promastigote culture was
kept starved without fresh medium changes, by chance for the first time, and then after several
46
times when repeated purposely. This was a total accidental happening, which occurred due to
one-week holidays and when, on the safe side, the culture was sub-passaged in 25 mL complete
RPMI-1640 growth medium in 25cm2 non-vented flasks at 26°C, the normal temperature for
promastigotes. The results are presented as percentage of various forms (morphotypes) in 100
cells counted in a Giemsa stained smears. During 10 days period, daily monitoring of the
transformation (differentiation) processes displayed dissimilar morphotypes. There was a steady
loss of the flagella with individual variability. As indicated in table 2.1, interestingly, on day 1,
dissimilar morphotypes were observed and till day 3 a mixture of morphotypes was present. Day
4 and onward only the rounded forms with no flagella and oval forms with no flagella were
observed. However, few rounded forms with scarcely visible flagella were also present in the
culture. Although the transformation process started right on the following day of culture in
starvation (conditioned/used) medium, however, proper amastigote conversion was observed
after day 4 onwards. The fully transformed culture contained the round and oval forms.
However, oval cells predominated the rounded ones throughout the culture of amastigotes of L.
tropica isolated from CL lesions from the type locality of the strain (Figure 2.5).
Cell size as well as length of flagellum experienced tremendous reduction during the
transformation of Leishmania tropica KWH23 promastigotes to amastigotes. There was
significant reduction (10.97 to 4.51µm) in cell length on day one, moderate reduction on day two
(4.51 to 3.93µm) and day three (3.93-3.28µm) in acidified medium respectively. We measured
pH of the medium and interestingly it was from 5.0 to 5.5 and in this acidified medium
(temperature 26°C and pH 5.5) transformation/differentiation was satisfactory. From day four till
day seven the length of the amastigote forms became stable measuring between 3.19 to 3.11µm
respectively (Table 2.2). Fully transformed amastigotes varied from little oval to near rounded
(intermediates) to fully rounded (Table 2.3). The oval amastigotes measured 3.65±0.36 µm in
length and 2.94±0.28 µm in width, while the complete rounded forms were between 3.18±0.31
µm to 3.15±0.30 µm in diameter on day 5 and day 10 in conditioned medium respectively.
47
Table 2.1. Promastigote to amastigote transformation ratio over 10-day duration based on
Leishmania morphology. *****
*****RNF=Rounded no flagellum, RBVF=Rounded barely visible flagellum, RSF=Rounded short flagellum,
RLF=Rounded long flagellum, ONF=Ovoid no flagellum, OBVF=Ovoid barely visible flagellum, OSF=Ovoid short
flagellum, OLF=Ovoid long flagellum, ENF=Elongated no flagellum, EBVF=Elongated barely visible flagellum,
ESF=Elongated short flagellum, ELF=Elongated long flagellum,
Shape
Variation→
Day
↓
RNF RBVF RSF RLF ONF OBVF OSF OLF ENF EBVF ESF ELF
1. 03 05 05 02 04 25 27 09 08 05 04 03
2. 10 09 05 01 23 27 20 02 0 0 02 01
3. 09 08 07 01 31 40 03 01 0 0 0 0
4. 19 13 0 0 68 0 0 0 0 0 0 0
5. 31 06 0 0 63 0 0 0 0 0 0 0
6. 29 09 0 0 62 0 0 0 0 0 0 0
7. 41 0 0 0 59 0 0 0 0 0 0 0
8. 44 0 0 0 56 0 0 0 0 0 0 0
9. 47 0 0 0 53 0 0 0 0 0 0 0
10. 43 0 0 0 57 0 0 0 0 0 0 0
48
Table 2.2. Size distribution of various differentiating and transforming Leishmania tropica
KWH23 amastigotes in acidified medium.
Days in
Culture
Mean Diameter in µm Variance Standard
Deviation
Confidence
Interval (CI) of
the Variance
Standard Deviation of
the Confidence Interval
0 10.97 2.36 1.54 1.4-5.4 1.2-2.1
1 4.51 0.70 0.84 0.4-1.7 1.18-1.45
2 3.93 0.29 0.54 0.17-0.70 0.413-0.840
3 3.28 0.06 0.25 0.036-0.15 0.189-0.387
4 3.11 0.04 0.21 0.025-0.10 0.158-0.316
5 3.19 0.06 0.25 0.035-0.138 0.187-0.371
6 3.11 0.04 0.21 0.025-0.10 0.158-0.316
7 3.14 0.05 0.23 0.030-0.118 0.173-0.343
Table 2.3. Size distribution of the completely round, intermediates and oval forms of
Leishmania tropica amastigotes.
Fully Rounded Amastigotes
Mean
Diameter
in µm
Variance Standard
Deviation
Confidence Interval
(CI) of the Variance
Standard Deviation
of the Confidence
Interval
Day-5 3.18 0.1017 0.32 0.05-0.25 0.224-0.50
Day-10 3.15 0.095 0.31 0.048-0.22 0.219-0.47
Oval Amastigotes
Length 3.65 0.133 0.36 0.071-0.33 0.266-0.57
Width 2.94 0.084 0.29 0.045-0.21 0.212-0.46
49
2.2.6. Viability during Transformation
Viability of cells is a necessary aspect in the drug sensitivity and biological studies. The viability
of Leishmania tropica KWH23 cells during promastigote to amastigote transformation was
checked daily during the study period using trypan blue exclusion and was expressed as
percentage of viable cells (Fig. 2.6). Leishmania tropica differentiation from promastigotes to
amastigotes stage viability was assessed by differential total of lifeless and living parasites using
the trypan blue colourant exclusion technique. Results presented in figure 2.5 corresponds to
three independent experiments performed in triplicate. Results clearly present non-significant
difference in Leishmania tropica amastigotes survival by the 0.4% trypan blue dye exclusion
technique throughout the experimental time (day zero to day 7). We observed maximum
survival rates of 96-98% throughout our experiments when amastigotes were exposed to various
available nutrients in acidified complete growth medium at 37oC.
Figure 2.6. Viability assessment for Leishmania tropica amastigotes during transformation and
differentiation.
0
10
20
30
40
50
60
70
80
90
100
0 1 2 3 4 5 6 7
% V
iab
ilit
y
Days
50
2.2.7. Natural Acquisition of Axenic Amastigotes
During the study period, when the culture was kept starved for one week to ten days at 26⁰C and
in the absence of O2, the promastigotes changed to amastigote stages due to change in pH of
medium (from pH 7.4 to 5.0). This occurrence was purely unintentional for the first time, during
which colour of the RPMI 1640 medium changed from pink to light yellow with pH of 4.8-5.0
(Figure 2.6). The complete culture medium, in the repeat experiments, having 10% HI-FCS
started changing its colour after day 3 with pH falling to 6.8 having only promastigotes. On day
5 the pH dropped to 6.0 but it still was having only promastigotes in late log phase. The pH fell
gradually to 5.5 on day 7 with 82% promastigotes and the rest were partially changed
(intermediates) amastigotes having rounded to oval or stumpy body but long flagellum. On day 9
the pH further dropped to 4.9 to 5.0 and the percentage of amastigotes increased to 80% mostly
having rounded body and diminutive to rudimentary flagellum. On day 10 onwards the pH
dropped to 4.8 and the proportion of amastigotes invigorated to 91% and interestingly viability
of the amastigotes was 97% as assessed by trypan blue exclusion test. The culture had 1.2 x 107
amastigotes per mL. When grown in fresh complete medium for promastigotes at 26oC the
amastigotes successfully transformed back to promastigotes. Natural transformation and
differentiation were slower than the one performed in the defined medium for axenic
amastigotes. However, the results were satisfactory, and the amastigotes yield was enough. Such
transformation yielded enough axenic amastigotes for one-time use in an experiment. The
amastigotes could be successfully propagated through sub-passaging in the axenic amastigotes
defined medium.
C
51
Figure 2.7. Unsurprisingly acquired amastigotes (A), Starved culture medium with pH 4.8-5.0
having naturally changed amastigotes (B). Standard RPMI 1640 growth medium with pink
colour and phenol red as a pH indicator (C).
2.2.8. Requirements for Counting, Fixation and Smearing of Leishmania tropica KWH23
Counting is a necessary process in culturing and propagation as well as for in vitro and in vivo
anti-leishmanial assays for testing various compounds. To count Leishmania parasites,
promastigote form needs inactivation by diluting in 2% formaldehyde in PBS, because they are
fast moving. Amastigotes are, however, stationary and need no such inactivation treatment.
During the present study, 10-25 % ethanol was found acceptable to inactivate them to be easily
studied under microscope in haemocytometer. Ten percent aqueous methanol solution for 10-30
seconds gave excellent results in fixing both the promastigote and amastigote forms of the
parasite. For adhesion of the parasite to the slide surface, egg albumin was used. Fresh album
was less effective in holding the parasite to slide surface but its room storage for one to 7 days
made it much effective in attaching the Leishmania cells to the slide surface. Hen egg albumin
contains 385 amino acids with molecular mass of 42.7 kDa. Rehault-Godbert et al. (2010)
showed that hen egg albumin contains many molecules good for human health and with many
antimicrobial proteins which inhibits growth of Salmonella enterica after its storage at 4, 20, or
37°C for 30 days preceding to inoculation. Amastigotes were found less effective in sticking to
the surface of albumenized slides than the promastigote. A 2 % (V/V) working Giemsa stain
solution for 14-20 minutes at room temperature gave excellent results in staining both
promastigotes and amastigotes. This working Giemsa stain solution functioned reasonably for 2
weeks without any granulation when placed in dark. Fresh stain replacement after two weeks,
however, is recommended.
2.2.9. Revision of Strain Nomenclature
Leishmania tropica KWH23 strain was originally isolated from a 5-year-old boy from Jamrud (an
endemic region for cutaneous leishmaniasis) in Khyber Agency of Pakistan who visited Kuwait
Teaching Hospital, Peshawar, for treatment in 2010. It was isolated by our colleague Dr. Nazma
Habib Khan during her PhD from London School of Hygiene and Tropical Medicine. Since then
it has been labelled with the name where KWH comes from the name of the hospital where the
52
strain originated and 23 refers to the serial number of the patient studied visited on that day.
According to the strain nomenclature for Leishmania isolates, it must contain four parts
separated by slash (/). The first part is a reference to the host from which it is isolated. It is
usually a four-letter abbreviation. The first letter represents the class of the host in the hierarchic
classification and the following are first three letters of the genus of the host. The second part is a
two-letter abbreviation of the country of origin, a list of which is available online. The third part
is the year of isolation. The fourth part is not fixed and is on the choice of the person who has
done the isolation. It might be the original patient ID in the hospital record or of the researcher.
So as per Article 50.1 and Endorsement 50A of the International Code of Zoological
Nomenclature the name of this Leishmania tropica KWH23 strain technically be written as
MHOM/PK/2010/KWH23.
2.3. Discussion
Morphological variability of promastigote stage of different Leishmania species have been
described in the vector host. Several different Leishmania morphotypes occurs during
development in the Phlebotomus sand fly vector. Each of these morphotypes also presents
bichemical and molecular variability with diverse virulence to the host (Molyneux and Killick-
Kendrick, 1987; Schlein, 1993; Ismael et al., 1998; Gossage et al., 2003; Kamhavi, 2006;
Oliveira et al., 2009; Ramalho-Ortigao et al., 2010; Dougall et al., 2011). The description of
such morphotypes in axenic development is apparently difficult partly because of temporal
association of such developmental forms and partly due to great deal of species-specific
variations. For these reasons, limited number of studies have been attempted to assess the
morphological forms in the axenic cultivation of the Leishmania promastigotes. Such studies
have been done on limited number of Leishmania species e.g., L. donovani, L. mexicana and L.
major being the prominent ones (da Silva and Sacks, 1987; Sacks, 1989). However, axenic
growth of the promastigotes reflect several differnet morphotypes realised during the in vivo
dvelopment in the sandfly vector. Different morphotypes found in the sandfly vector are also
reported from the in vitro culture (Sacks et al., 1984; Gossage et al., 2003; Lei et al., 2010a).
Initiating from CL lesion amastigotes, Bates (1994) demonstrated, various morphotypes of L.
mexicana axencally. The initial amastigote to promastigote transformation was achieved in 2
days. Logarithmic growth, with a mixture of dissimilar morphotypes in the multiplicative stage,
53
was noted to occur on days 3 and 4. At this point, very low density of the infective metacyclics
was distinguished. Most of the promastigotes on the late day 3 were longest (15.22 ± 3.49 μM)
and extremely slender than noted on any other day and were carrying long flagella. However,
flagellar and body length was comparable. These were morphologically much similar to the
nectomonads noted in in vivo studies. On day 7th , the promastigotes reached to the stationry
phase and metacyclogenesis was on its peak. In 9 days old axenic culture, however, the density
of metayclics reached to 95%. The metacyclics became shorter, were broader anteriorly and
needle sharp posteriorly and were with long flagella. The flagella were 1-2 times longer than the
cell size (7.59 ± 1.15 μM). These findings are in agood concensus with what we came across in
the present study. Short and slender metacyclic promastigotes with long flagella have been
demonstrated in stationary cultures by other investigators also (Sacks and Perkinn, 1984; Grimm
et al., 1991). The ratio of metacyclics to other forms is in inverse relationship to the number of
passages in our experiments. It usually decreases in long-term axenic cultivation. Fresh cultures
that have gone through few passages get fast metacyclogenesis with higher densities of
metacyclics than the older cultures (Cysne-Finkelstein et al., 1998). Results of the present study
are strongly supported by the in vivo results shown by Dougall et al. (2011), where similar
morphological forms of promastigotes of an unidentified species of Leishmania isolated from
day biting midges of genus Forcipomyia. They reported long slender nectomonads with extended
flagella. The leptomonads they isolated were rather short banana like with relatively short
flagella. Their metacyclics, however, varied from needle long shape with long flagella to
anteriorly broad and posteriorly needle sharp morphotypes having long flagella. Lei et al.
(2010b) also reported procyclic, nectomonad, leptomonad and metacyclic stages in Leishmania
culture. They have shown similar body lenghts for procyclics, leptomonads and metacyclics with
length ranging from 6-11 μm. However, nectomanads were more than 12 μm in length. All of the
stages they recorded have been with similar morphology to what we have found in the present
study.
Lectins obtained from differnet plants including peanut, are used to purify metacyclic
promastigotes from culture. These proteins agglutinates the procyclic and other forms leaving
metacyclics only. Metacyclics and non-metacyclics separated from stationary culture of L.
amazonensis have been reported to be morphologically different. The non-metacyclics have been
long and almost equally broad along their length except near the posterior tip. These have been
54
shown to have extended flagella. These are the characteristics of the nectomonads. The
mectacyclics have been short and stumpy with broader anterior half and much narrower posterior
half. These have been shown to possess very long flagella (Saravia et al., 2005). This study
support our results. Soares et al. (2005) also noted similar results for L. braziliensis. The
procyclic form was found to be much variable in size of the cell and flagellum. They probably
considered all the non-metacyclic forms as procyclic that varied in cell size from rounded to
elongated individuals. However, the metacyclics were defined as smaller and thinner posteriorly,
with long flagella.
Bee et al. (2001) grown promastigotes of L. mexicana under different temperatures and pH and
found metacyclics with different growth and pro-amastigote transformation kinetics. At a
temperature of 26°C and pH of 7.2 no transformation was recorded. In these physiological
conditions, however, in some experiments transitional forms have been seen for short duration.
At temprature of 26°C and pH 5.5, greater degree of transformation have been observed but it
was followed by reversion to promastigotes in 24 hours. When the transformation was checked
at 32° C and pH 7.2, within 12 hours almost all of the cells entered to the intermediate forms that
sustained for 48 hours. Although they did not revert to the promastigote but did not fully
transformed. A temeperature and pH panel of 32°C and 5.5 was the most effective in bringing
the desired transformation over 48-96 hours (Bee et al., 2001). These conditions for
transformation are in close association to our observation. Similarly, the observation of various
trnasitional stages in the above mentioned study share what we noted in this study. However, we
did not see any reversion like they mentioned at the ideal combination they used. A difference
from what they have observed in our study is that we recorded good amastigote transformation at
26°C and 4.8 pH in the natural starvation medium.
Metacyclic promastigotes of L. mexicana grown in Schnider’s medium supplimented with 20%
fetal calf serum at 25°C and 5.5 pH have been successfully transformed to amastigotes by rising
simply the temperature to 32°C in the parent culture. After 24 hours of incubation at 32°C
temperature, more than 80% of the cells changed to rounded amastigotes with complete loss of
flagella. The remaining 20% were the intermediate forms differing in their cell and flagellar
morphology. By the day 2, however, 100% transformation was observed (Bates, 1994). These
findings present a strong correlation of temeterature and pH to the transformation process.
55
Species-specific variability can, however, not be ruled out. Generally, a temeperature and pH
combination of 32-37°C and pH of 4.5-5.5 seems feasible for bringing satisfactory
transformation. Fully trnasformed amastigote may be ovoid to fully rounded in almost all
Leishmania species.
Morphological variability in the in vitro development of L. tropica can be determined
microscopically. The present study will provide guidelines for the subsequent workers in
acquisition of any specific morphotype for their use. This will also help them in acquiring axenic
amastigotes of the species by simply starvation of promastigotes. A comprehensive protocol for
processing the prmastigotes as well as amastigotes for both light and electron microscopy will be
of help to the future projects.
56
Chapter 3.0. In vitro Cytotoxicity Assays of Actinomycins and Isoteviol
Derivatives on Human Peripheral Blood Lymphocytes
The key assays used during in vitro harmfulness estimation are called as cytotoxicity assays,
which concentrate mostly on cell death or to quantity growth weakening. Numerous assays are
established to determine in vitro toxicity such as enumerating cell survival /death by evaluating
integrity of the plasma membrane, overall cell protein content and certain vital functions.
Cytotoxicity testing is intended to assess the inherent capacity of a compound to kill the cells.
Along with dosage, toxicology of a compound depends upon the length of exposure and the
compound’s toxicity mechanism (Ferro & Doyle, 2001).
In sum, life ends in death and death in cells occur either by necrosis or by apoptosis. One of the
life's majestic sarcasms is that it also rests on death. The term necrosis originates from the
Greek core “necros” meaning ‘dead’, and denote accidental demise of cells exposed to extreme
ecological or heritably programmed insults (Walker et al., 1988). Wounded cells experiencing
necrosis show gross morphological and ultrastructural changes that differ sharply with those
displayed by cells enduring apoptosis. Necrotic cell death is escorted by swelling of the cell,
widespread plasma membrane lost integrity and endocytosis, expansion of cell organelles,
clumping and arbitrary degradation of nuclear DNA (Hall et al., 1997). Necrosis terminates with
the removal of cell corpses in the nonexistence of apparent phagocytic and lysosomal
participation (Galluzzi et al. 2018).
Programmed cell death (PCD) is cascade of inherent suicide pathways, vital for tissue
homeostasis, growth, development and pathogenesis. PCD was originally considered identical
with apoptosis; however, in recent times, alternate mechanisms of PCD have been described,
viz. necroptosis, autophagy and apoptosis. PCD can happen in the absence of any exogenous
environmental agitation, henceforth, working as a built-in effector of physiological agendas
for development and cellular turnover. Apoptosis or type I cell death exhibits cytoplasmic
contraction, plasma membrane bulging, chromatin shortening (pyknosis), nuclear
disintegration (karyorrhexis), and ending with the creation of seemingly integral small
vesicles (apoptotic bodies) that are proficiently taken up by adjacent cells by phagocytosis and
their degradation within phagolysosomes. This cascade of activities leads into cell death.
Cytotoxicity can be measured by diverse approaches like vital dyes viz. trypan blue exclusion
57
testing and propidium iodide uptake assay, protease biomarkers activity and MTT assay (Fuchs
and Steller, 2015; Galluzzi et al,. 2018).
Previously performed cytotoxicity assays accompany all classes of drug sensitivity studies. For a
material to be a therapeutic one, it is essential that it must not be damaging to the host cells or at
least should have minimum cytotoxicity at the recommended dose which is bearable and
repairable by the body. Following this principal, all the test compounds and the standard drug
were subjected to prior cytotoxicity evaluation using human peripheral blood lymphocytes, an
easily available celly type for in vitro testing. These cells are the the very first nucleated cells
that come across any material, therapeutic agents for example, that enters the body through
vascular fluid, the blood.
3.1. Materials
3.1.1. Chemicals and Solutions
Ficoll polymer, RPMI-1640 growth medium, Phosphate-Buffered Saline (PBS), Trypan blue
solution (0.4%), XTT (2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-
Carboxanilide), Dimethyl Sulfoxide (DMSO), Heat-inactivated fetal bovine serum (hi-FBS),
Histopaque-1077 (Sigma H8889), Streptomycin-Penicillin solution (All from Sigma-Aldrich,
Europe).
3.1.2. Disposables
Centrifuge tubes (15 and 50 mL), 5-10 mL EDTA tubes, 5-20 mL syringes, 1.5-2.5 mL
Eppendorf tubes, 20-1000 μL pipette tips, Glass slides, Thermo Scientific Nunc™ Cell Culture
Treated Easy-Flasks™ vented (close cap) and filtered cap culture T25 and T75 flasks, Nunc™
96-Well Polystyrene Flat-Bottom Microwell Plates, 5-10 mL pipettes, Aluminium foil
(Thermo-fisher, Germany; Bio Tek GmBH, Germany; Sigma-Aldrich, Europe).
3.1.3. Apparatus
Thermo-Fisher Scientific Direct Heat CO2 Incubator, DAIHAN MaXterile™ Digital Fuzzy-
control Autoclave 80 Litre (Daihan, Korean), Thermo-Fisher Scientific Finn-pipette FpF3
micropipettes (2-200 and 100-1000 μL capacity), Microbiological Laminar flow hood (Technico
Scientific Supply, Lahore), Olympus CKX41 Inverted microscope (Olympus, Singapore),
58
Brightfield compound microscope (Leitz, Germany), Heraeus Centrifuge Megafuge X1R
Refrigerated (Thermo-Fisher, Germany), Bio-Tek Microtiter plate reader with a branded system
(Bio Tek GmBH, Germany), Microbiological Incubator (Gallen-kamp Fistreem International
Limited, UK).
3.2. Methods
3.2.1. Isolation of Human Peripheral Blood Lymphocytes
Human peripheral blood lymphocytes (PBLs) are an easy source for in vitro cytotoxicity
experiments. In the present study, freshly isolated PBLs were used to test the cytotoxic effects of
the compounds. Briefly, fresh 5 mL blood was collected from verbally consented healthy BS and
M.S Zoology students using sterile 5 mL disposable syringe. The blood was immediately added
into 5-10 mL Di Potassium EDTA containing screw capped sterile polystyrene tubes. The blood
was mixed with PBS in 1:1 in a 15mL centrifuge tube. The diluted blood was carefully layered
up on equal amount over a 5 mL Ficoll Histopaque in two other tubes. Layering was done slowly
by tilting the Ficoll/Histopaque containing tube at an angle and pouring blood along the down
facing inner wall of the tube using an automatic pipette filler. This preparation was then
centrifuged at 400xg for 30 minutes at room temperature. The mono-nuclear cells were isolated
through a pipette. The isolated cells were washed three times in PBS at 400xg for 10 minutes
each. The cells were then incubated at 37oC for 2 hours in complete RPMI-1640 medium to
remove the monocytes by their affinity to adhere to the plastic and glass surfaces. The suspended
cells were then collected and pelleted by centrifugation. The pellet was resuspended in 1 mL
RPMI-1640 medium and the cells were counted in haemocytometer. Cells from different
individuals were processed separately. All the steps after the collection of blood were performed
aseptically in a laminar flow hood. As blood is a potential infectious material, therefore all the
disposables used were disinfected overnight in phenyl before proper disposal.
3.2.2. Inspecting Viability of Lymphocytes
The dye exclusion assessment is shaped on the idea that viable cells having intact cell
membranes do not take up colourants like trypan blue, eosin and propidium, however lifeless
cells are permeable and mesmerize the dye. Trypan blue (C34H28N6O14S4) is an azo dye i.e., having
59
the colouring azo function (N=N-), which is an organic molecule containing two neighbouring
nitrogen atoms sandwiched between carbon atoms. To check the viability of the lymphocyte
cells, 10-50 µL of lymphocyte cells suspension was added to 10-50 µL 0.4% trypan blue solution
(in PBS), mixed by pipetting up and down and incubated at room temperature for 2-3 minutes.
The mixture was then placed on a glass slide and the percentage of viable cells was determined
by counting 100 cells. The percentage of viable blood lymphocytes was calculated by dividing
the number of viable cells by quantifying total cells and multiplying by 100. Viable cells were
the ones that did not stain with the trypan blue dye (clear cytoplasm) as viable membranes are
impermeable to the trypan blue while dead lymphocytes cells were with permeable membranes
having blue cytoplasm.
3.2.3. Plating Lymphocytes and Adding Compounds
Lymphocytes were dispensed in flat bottom 96 well polystyrene plate at 2×104 cells/well in
100μL complete RPMI-1640 growth medium. The trial compounds and the standard miltefosine
were applied in 0.02, 0.2, 2, 20, 25, 50, 75 and 100μM in triplicate for each concentration. A
negative control was kept with only 1% DMSO, the solvent of the test compounds and
miltefosine. The concentration of the DMSO, until mentioned otherwise, was always kept as 1%.
The lymphocytes were incubated at 37°C in the CO2 incubator for 48 hours.
3.2.4. Evaluation of Cytotoxicity and Cell Death in vitro
After 24 hours exposure to the test compounds, the lymphocytes cells were incubated with 20μL
of XTT solution for 2 hours at 37°C in the CO2 incubator. After 2 hours, the absorbance was
read in Bio-Tek ELx800 ELISA microplate reader through GEN5 software at 490 and 630 nm
wavelength. The formazan assembly by XTT reduction is soluble in water and therefore was
directly detected in lymphocytes supernatants so was easily calculated by colorimetric assay.
3.1.5. Data Analysis
The 96 well flat-bottomed plate was abstemiously shaken to dispense the orange colour evenly
and absorbance for lymphocyte cells and the blank background control (RPMI-1640 growth
medium only) wells was read at a wavelength between 450-490 and 630nm. The absorbance data
was first manipulated by subtraction of the 630nm value from 490 nm value for each well. Then
values obtained after substracting were then processed in excel and converted to percent
60
inhibition by using the formula (control-treated/control×100). The percent inhibition values were
then processed by GraphPad Prism 6 software to calculate the IC50 values for the test
compounds and the standard drug miltefosine.
3.3. Results
3.3.1. Cytotoxic Impact of Miltefosine on Lymphocytes
Miltefosine (Hexadecylphosphocholine) was used as a standard positive control (as it is the only
drug use for the treatment of all form of Leishmaniasis orally) for evaluating its cytotoxicity
against human peripheral blood lymphocytes. It gave an IC50 value of 272.2 μM (95 % CI=
143.6 to 515.7 μM). In this respect, it may be considered as comparably less cytotoxic to the
lymphocytes as it inhibited less than 50% of the cells even at its highest concentration as
revealed in the figure 3.1 after incubation for 48 hours. Among the test compound, variable
degree of cytotoxicity was observed as compared to the standard miltefosine.
Miltefosine has been shown to bind peritoneal macrophages and Leishmania plasma membrane
membrane-bound proteins in large quantities via a detergent-like action and are causing
structural and organizational alterations showing noticeable increase in dynamics and exposure
to aqueous milieu, so resulting in membrane fluidity (Fernandes et al., 2017).
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Figure 3.1. Concentration dependent cytotoxicity of Miltefosine against human peripheral blood
lymphocytes
3.3.2. Cytotoxicity of 16 (2,4-Dinitrophenylhydrazine) Isosteviol
As evaluated from the half maximal inhibitory concentration IC50, 16 (2,4-dinitrophenyl
hydrazine) isosteviol showed IC50 value of 781.2 μM (95 % CI= 240.8 to 2535 μM). This is
slight toxicity to human peripheral blood lymphocytes (PBLs) after 48 hours exposure. Figure
3.3 shows the concentration dependant inhibitory effects of the compound on PBLs. The
cytotoxicity of 16 (2,4-dinitrophenyl hydrazine) isosteviol with IC50 value of 781.2 μM (95 %
CI= 240.8 to 2535 μM) was considerably less than that of the standard drug miltefosine 272.2
μM (95 % CI= 143.6 to 515.7 μM). Concluding the cytotoxicity estimation against human
peripheral blood lymphocyte cells, it was revealed that, in both cases, these test compounds
along with miltefosine showed minimum tolerable cytotoxicity.
62
Figure 3.2. Cytotoxicity of 16 (2,4-dinitrophenylhydrazine) isosteviol
3.3.3. Cytotoxicity of 17 Hydroxy, 16 (2,4-dinitrophenylhydrazine) Isosteviol
This structurally different Isosteviol derivative containing a hydroxyl group, was found more
cytotoxic than the 16 (2,4-dinitrophenylhydrazine) Isosteviol, its parental skeleton. The IC50
value of this compound was 294.1 μM (95 % CI= 177.4 to 487.5 μM) as compared to that of the
miltefosine 272.2 μM (95 % CI= 143.6 to 515.7 μM) i.e., 9.2% less cytotoxic than miltefosine.
Figure 3.4 displays the concentration dependent percent inhibition of PBLs in a 48 hours
exposure time. The inhibitory effects of the compound increased with concentration.
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Figure 3.3. Cytotoxicity of 17 hydroxy, 16 (2,4-dinitrophenylhydrazine) Isosteviol against
human peripheral blood lymphocytes.
3.3.4. Cytotoxicity of Benzyl ester 16 (2,4-dinitrophenylhydrazine) Isosteviol
The compound showed limited toxicity with an IC50 value of 421.8 μM (95% CI= 211.3 to
842.1 μM) i.e., 35.46% less cytotoxic than that of the miltefosine. Figure 3.5 shows the impact of
this compound on the growth of the cultured PBLs in vitro. The fifty percent inhibitory
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concentration of this compound certifies adequate, if not complete, safety of the compound.
Figure 3.4. Cytotoxicity of Benzyl ester 16 (2,4-dinitrophenylhydrazine) Isosteviol against
human peripheral blood lymphocytes.
3.3.5. Cytotoxicity of Actinomycin D
Actinomycin D (C62H86N12O16) is a recognized antibiotic of the actinomycin group that
demonstrates high antitumor and antibacterial action. This has been extensively used in medical
practice since 1954 as an anticancer medication for handling numerous tumour types and it is
also a valuable contrivance in cell biology and biochemistry. It attaches to DNA and obstructs
RNA production (transcription), so protein synthesis also drops.
As revealed by the IC50 value 195.8 μM (95% CI=135.3 to 283.2 μM), the Actinomycin D
showed greater cytotoxic effects as compared to miltefosine and the Isosteviol derivatives. The
cytotoxicity of the Actinomycin D showed a concentration dependant increase as is revealed in
the figure 3.5. Actinomycin D being more cytotoxic than the standard drug, has inhibitory effects
65
which are still not that much perfect to the host cells. However, the compound can be considered
harmless antileishmanial contender at lower doses.
Figure 3.5. Cytotoxicity of Actinomycin D against human peripheral blood lymphocytes.
3.3.6. Cytotoxicity of Actinomycin Z3
Actinomycin Z3 (C62H83ClN12O18) compound gave an IC50 value of 210.1 μM (95% CI= 145.2
to 304.1 μM) which is comparable to actinomycin D and less than miltefosine (Figure 3.6). As
such the results of this study showed safety of the compound for human PBLs. The fifty percent
inhibitory concentration of cytotoxicity (IC50 value of 210.1 μM) is such that the compound can
be a harmless candidate for its antileishmanial action as well as it may be used for other
antiparasitic activities also. Inhibitory effects of this compound are negligible in the living
system of the host as shown by its effect on PBLs.
66
Figure 3.6. Cytotoxicity of Actinomycin Z3 A against human peripheral blood lymphocytes
3.3.7. Cytotoxicity of Actinomycin Z5
Actinomycin Z5 gave an IC50 value of 234.9 μM (95% CI= 155.5 to 354.9 μM). The compound
is less cytotoxic than miltefosine and Z3. So can be safe for the host cells in vitro. As is revealed
by figure 3.7, the compound gives less than 50% cytotoxicity even at the highest concentration
used at 48 hours incubation.
67
Figure 3.7. Cytotoxicity of Actinomycin Z5 against human peripheral blood lymphocytes.
3.4. Discussion
Nature is an outstanding unit for novel therapeutic challengers, as marvelous chemical diversity
is found in thousands of species of microorganisms, animals, plants and marine organisms. Plant-
derived metabolites and phytochemicals second the microbial metabolites as candidate
therapeutic agents. Although it has been at the advent of 20th century that microbial agents
surpassed the plant extract-based cures and remedies that have a history going back to ancient
Mesopotamian and Egyptian civilizations about 2600 and 2900 BC respectively. There are
records of hundreds of drugs, primarily of plant origin, which have been used by Mesopotamian
and Egyptian societies (Atanaso et al., 2015). Bacterial and fungal secondary metabolites
emerged at the discovery of penicillin and were amongst the major suppliers of the marketed
medications. Most of the available anti-infectious chemotherapeutics are either bacterial or
fungal metabolites. These metabolites, apart from having antibacterial, antifungal, antiviral,
68
anticancer and anti-enzyme potentials, have also been shown to possess a variety of different
bioactivities including their uses as herbicides and source of biodegradable plastics (Duke et al.,
2000; Bérdy, 2005; Mooney, 2009).
Actinomycins are a group of 35-50 secondary metabolite compounds of actinomycetes bacteria
especially Streptomyces spp. Members of the Actinomycin group possess antibiotic, cytostatic,
anti-neoplastic and hormone-like stimulatory effects. Their antimicrobial effects are mediated by
blocking DNA synthesis when they intercalate to the minor grove (Grand et al., 1972; Hollstein,
1974; Cooper and Braverman, 1977; Zotchev, 2012) and prevent DNA mediated RNA synthesis
which is believed to be the possible mechanism behind their anti-neoplastic nature (Quigley et
al., 1980; Nunn et al., 1991).
Actinomycins, regarding their biological activities, are second to none but their cytotoxic effects
are the main hinderance in their path to the list of approved therapeutics (Kamitori and
Takusagawa, 1994). However, being harmful to the DNA of various cell types, they are still in
contention for antimicrobial and anti-cancer research. Restructuring through chemical
modification of the peptide or chromophore components has been found to improve therapeutic
nature of these compounds (Sengupta et al., 1985; Anthony and Helmut, 2011). Actinomycin D
have been found to show higher toxicities to renal, cortical and medullary cells, myocardial cells,
pulmonary histocytes and spleen cells in vitro. When used in combination with human
recombinant TNF-α against various cell types, renal medullary cells showed high sensitivity as
compared to liver cells, cardiac muscle cells and bladder cells (Aoki et al., 1998). Actinomycin
C have been found to cause morphological alterations in lymphocytes at a concentration of 5-
10μg/mL. It has been known to cause obstruction of RNA synthesis in lymphocytes at doses as
low as 1μg/mL (Lundgren and Mӧller, 1969). Actinomycin D has been found to cause inhibition
of bovine oocytes maturation by causing chromosomal modifications at a concentration of 2.5-
5μg/mL after 14-hour incubation. Lower concentration, however, caused little chromosomal
alterations and maturation impairment at the same exposure period (Moura et al., 2008).
Similarly, in mice model at lower dosages, actinomycin D caused reduced suppression of
transcription e.g., 0.25mg/kg body weight, five percent genes were found supressed (Siboni et
al., 2015). The present study reveals more or less comparable findings regarding cytotoxic
69
effects of actinomycins on normal human peripheral blood lymphocytes. These microbial
metabolites deliver both therapeutic and toxic effects at minor concentrations.
The glycoside metabolites of Stevia rebaudiana (also called as sweet leaf is flowering plant in
the family Asteraceae) are one of the major non-caloric sweetening food additives used around
the world. Among these, steviol, stvioside, rebaudioside and Isosteviol are notable metabolites.
Presently these and their natural or artificial derivatives are under trials to assess their
bioactivities beyond sweetening. Regarding their adversities to human health, variable opinions
and evidences exist. They have, however, been characterized in various in vitro and in vivo
studies with no teratogenicity, cytotoxicity, mutagenicity or carcinogenicity (Takasaki et al.,
2009; Momtazi et al., 2016) or behavioural, haematologic, clinical and histopathological effects
(Zhang et al., 2017). Steviosides has been demonstrated to cause no cytotoxicity in peripheral
blood mononuclear cells (Noosud et al., 2017). In the present study for all the three hydrazine
derivatives of Isosteviol the inhibitory effects on lymphocytes were found to be in much higher
micromolar concentrations. The 50% inhibitory concentrations were found to be more than 400
μM giving indication of their nonaggressive nature to human PBLs and tissues. Steviol and
isosteviol derivatives of alkyl amide dimerization have been revealed to possess in vitro anti-
neoplastic potency against different cell lines. One study, however, found these compounds to be
less toxic to normal human embryonic lung cells (Lin et al., 2004). Animal studies have found
crude extracts and polyphenols from stevia to alleviate liver and kidney damage in streptozotocin
induced diabetes (Shivanna et al., 2013). Although hundred percent literature available regarding
health safety of the natural Stevia metabolites and synthetic analogues is not stamping a safe
nature of these compounds, most of the studies have found no or negligible adversities to normal
cells and tissues. Synthetic analogues, however, have been found to be comparatively more
aggressive along with higher therapeutic capabilities against cancer and infectious agents.
3.4. Conclusion
The present study found actinomycins to be more toxic to human peripheral blood lymphocytes
and some with comparable cytotoxicity to standard drug Miltefosine. For all the three candidates
of this group, the fifty percent inhibitory concentrations were less than 1μg/mL in in vitro setup
to human peripheral blood lymphocytes.
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Hydrazine derivatives of Isosteviol were less cytotoxic to human peripheral blood lymphocytes
in vitro. Derivatives of isosteviol are biologically energetic and are one of nature exceptional
compounds with hydrophobic and hydrophilic possessions. Their skeletal structure can easily
be used in the synthesis of a number of other useful derivative compounds and to design
active, selective and polyfunctional Isosteviol analogues for the therapy of leishmaniasis and
other infections.
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Chapter 4. Antileishmanial Compounds Screening in vitro
Cutaneous leishmaniasis has assumed an epidemic status in our province Khyber Pakhtunkhwa
in Pakistan and according to WHO local office and ministry of health about 23,760 patients are
registered in January-February 2019 only. An international medical charity organization
“Medicines Sans Frontieres” (MSF) has started treating cutaneous leishmaniasis patients with
meglumine antimonate but have several side effects (Figure 4.1). There is a dire need to stem the
tide of fast growing cutaneous leishmaniasis in Pakistan. This investigation is an effort to stem
the tide of growing cutaneous leishmaniasis and to balance toxicity with efficacy, as a
predictable dosing plan calls for intermittent application of an antileishmanial compound at or
near the maximum tolerable dose.
Figure 4.1. Cutaneous leishmaniasis patients are treated in Peshawar Hospital with intralesional
injection of meglumine antimonate (https://www.dawn.com/news/1461261).
In vitro screening is an important mean of preliminary evaluation of biological and antiparasitic
or antimicrobial activity of compounds. Such lab dish evaluations can give valuable insights to
the in vivo drug interactions with the living systems (Volpe et al., 2014). Biotechnological
approaches are desirable to improve superior medications against leishmaniasis. Though
paromomycin and miltefosine were registered as therapeutic agents against Leishmania donovani
infection in the previous decade, the leishmanicidal medication arsenal still necessitates
upgrading, mainly in the zone of oral antileishmanials for cutaneous, mucocutaneous and
visceral leishmaniasis. Numerous novel preparations and compounds have demonstrated
72
encouraging efficiency in animal models infected with various Leishmania species. In this
investigation, we tested some existing drugs used for the treatment of other diseases as new
antileishmanials against Leishmania tropica promastigotes and amastigotes in vitro. In the
present study, antileishmanial effects of six compounds and standard drug miltefosine were
analysed against both the promastigotes and axenically transformed amastigotes of Leishmania
tropica in vitro.
4.1. Materials and Methods
4.1.1. Culture of the Leishmania tropica KWH23 Promastigotes
Promastigotes of Leishmania tropica KWH23 were grown aseptically in RPMI 1640 growth
medium supplemented with 10% heat-inactivated foetal bovine serum (HI-FBS) and 100 μg/mL
streptomycin and 100 IU/mL penicillin at a density of 1×105/mL at 26°C under anaerobic
conditions. The Sigma-Aldrich RPMI 1640 was already added with L-glutamine an unstable
necessary amino acid needed at a final concentration of 29.2 mg/mL. For setting up a
Leishmania tropica KWH23 growth curve, a culture was initiated between 1x105 to 1x106
parasites/mL and Leishmania cells were counted daily for 7 days. The growth was monitored for
a week until the culture reached the stationary phase of growth. When mentioning to the phase of
growth in Leishmania tropica KWH23 cultures, stationary stage was obtained at ~2-3 x 107
promastigotes/mL. It was these stationary promastigotes that were subsequently utilized for anti-
promastigote assays and for generation of the axenic amastigotes.
4.1.2. Generation of Axenic Amastigotes
Stationary culture of Leishmania tropica KWH23 was harvested by centrifugation and diluted to
1×106/mL in RPMI 1640 medium fortified with 20% HI-FBS and 1% v/v streptomycin and
penicillin antibiotics. The pH of the RPMI 1640 medium was adjusted to 4.4 using concentrated
phosphoric acid as recommended in Sigma-Aldrich cell culture manual. At the acidic pH the
medium colour turned light yellow. The Leishmania tropica KWH23 promastigotes, during
transformation, were maintained at 37°C in humidified conditions in the absence of CO2. The
transformation process was monitored through Giemesa staining daily. Stationary promastigotes
after 10 days in culture without medium changes (as mentioned previously in section 2.1.4) was
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converted to fully round to oval aflagellated cells (axenic amastigotes) with more than 90%
viability were utilized for sensitivity assays.
4.1.3. Antileishmanial Activity
Both promastigote and amastigote stages of Leishmania tropica KWH23, in separate
experiments, were seeded at 1×106/well in 96-well flat bottom plates in 200 μL of RPMI1640
medium fortified as mentioned above for both the stages of the parasite. The test compounds and
the standard drug miltefosine were added in 8 different concentrations i.e., 0.02, 0.2, 2, 20, 25,
50,75 and 100 μM respectively. All the experiments were performed in triplicate. A negative
control was run with RPMI 1640 growth medium with 1% DMSO, the solvent used in the test
compounds. Both promastigotes and amastigotes were exposed to the compounds for 48 hours
under the respective conditions of temperature and pH for both the stages. After 48 hours
incubation, 20 μL of XTT solution was added per well and plates were covered with alluminium
foil and incubated for 2 hours under the respective conditions of temperature (26-37ᴼC) for both
promastigotes and amastigotes. After 2 hours, the absorbance in terms of viability was read in
Bio-Tek ELx800 ELISA microplate reader through GEN5 software at 490 and 630 nm
wavelength. The formazan assembly by XTT reduction is soluble in water and therefore was
directly detected in promastigotes and amastigotes supernatants so was straightforwardly
calculated by colorimetric examination.
4.1.4. Data Analysis
The absorbance statistics was first manipulated by deduction of the 630nm value from 490 nm
value for each well. The values obtained after subtraction were then processed in Microsoft
Excel 2016 and converted to percent inhibition by using the standard formula (control-
treated/control×100). The percent inhibition values for each compound against promastigote and
amastigote stages of Leishmania tropica KWH23 were then processed by GraphPad Prism 6
software to calculate the IC50 values for the test compounds and the standard drug miltefosine.
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4.2. Results
4.2.1. Antileishmanial Activity of Miltefosine
Miltefosine, the only oral approved therapy for visceral and cutaneous leishmaniasis, was used as
standard positive control in the present study. Miltefosine (C21H46NO4P) is a phospholipid with
elimination half-life of 6 to 8 days and Anatomical Therapeutic Chemical (ATC) classification
number L01XX09 as maintained by WHO. It gave IC50 values of 10.80μM (95% CI=9.114 to
12.81 μM) and 1.245 µM (95% CI=0.7250 to 2.138 μM) against promastigotes and amastigotes
respectively. Figure 4.2 shows the leishmanicidal activity of the drug against both the
promastigotes and amastigotes. As is revealed by the figure below, its killing ability increased
with concentration against both the stages of the parasite. However, greater degree of variability
in the killing of both the forms of the parasite was seen from 0.02-20 μM than from 25-100 μM.
It caused maximum killing of both promastigotes and amastigotes at the highest concentration
over 48-hour exposure. The present investigation reveals miltefosine as potent therapeutic
against Leishmania tropica. The amastigotes, as given by the IC50 values and the figure below,
were found more susceptible to the drug as compared to the promastigotes.
Figure 4.2. Antileishmanial activity of miltefosine against promastigotes (A) and amastigotes (B)
of L. tropica KWH23.
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4.2.2. Antileishmanial Activity of 16 (2,4-dinitrophenylhydrazine) Isosteviol
Figure 4.3 shows the antileishmanial activity of 16 (2,4-dinitrophenylhydrazine) isosteviol. The
IC50 values of the compound against both the promastigotes and amastigotes were 7.098μM
(95% CI=5.328 to 9.455μM) and 4.447μM (95% CI= 2.788 to 7.094μM) respectively.
Leishmanicidal activity of the compound increased with the concentration showing greater
variability when moving from 0.02-20μM to 25-100μM. The compound gave higher activity
against the amastigotes than the promastigotes. As revealed by the fifty percent inhibitory
concentrations (IC50) of the compound 16 (2,4-dinitrophenylhydrazine) isosteviol, it is
comparable with those to miltefosine so it can be a good antileishmanial candidate.
Figure 4.3. Leishmanicidal activity of 16 (2,4-dinitrophenylhydrazine) Isosteviol against
promastigotes (A) and amastigotes (B) of L. tropica KWH23.
4.2.3. Antileishmanial Activity of 17 hydroxy, 16 (2,4-dinitrophenylhydrazine) Isosteviol
As revealed in Figure 4.4, the IC50 values of 5.603μM (95% CI= 4.628 to 6.784μM) and
5.033μM (95% CI= 3.189 to 7.945μM) against promastigotes and amastigotes of L. tropica
KWH23 were detected by the compound 17 hydroxy, 16 (2,4-dinitrophenylhydrazine) Isosteviol.
76
This clearly depicts that 17 hydroxy, 16 (2,4-dinitrophenylhydrazine) Isosteviol possesses
virtuous antileishmanial bustle in vitro as compared to the standard antileishmanial
alkylphospholipid miltefosine. From the present results, it can be concluded that 17 hydroxy, 16
(2,4-dinitrophenylhydrazine) Isosteviol may be proved as an ideal antileishmanial agent
particularly in cutaneous leishmaniasis caused by L. tropica and this may be a central compound
for the development of potent antileishmanial in future. Consequently, development of innovative,
active, and harmless antileishmanial compounds, with fewer side effects, is a main priority of this
health investigation.
Figure 4.4. Antileishmanial activity of 17 hydroxy, 16 (2,4-dinitrophenylhydrazine) Isosteviol
against promastigotes (A) and amastigotes (B) of L. tropica KWH23.
4.2.4. Antileishmanial Activity of Benzyl ester 16 (2,4-dinitrophenylhydrazine) Isosteviol
The antileishmanial activity of the compound benzyl ester 16 (2,4-dinitrophenylhydrazine)
Isosteviol is revealed in figure 4.5. A concentration dependant response of both the
promastigotes and amastigotes of L. tropica KWH23 is clear from the figure 4.5. As revealed in
the figure 4.5, IC50 values of benzyl ester 16 (2,4-dinitrophenylhydrazine) Isosteviol are
10.71μM (95% CI= 8.611 to 13.31μM) and 6.794μM (95% CI= 4.248 to 10.87μM) against the
77
promastigotes and amastigotes of L. tropica KWH23 respectively. Like previously mentioned
compounds used in this investigation benzyl ester 16 (2,4-dinitrophenylhydrazine) Isosteviol is
more active against the amastigote forms as compared to metacyclic promastigotes. The
antipromastigote activity of the compound is almost similar to that of the miltefosine, however,
regarding its antiamastigote activity benzyl ester 16 (2,4-dinitrophenylhydrazine) Isosteviol is
less effective than the miltefosine. The overall results, however, reveal that the compound may
be a potent candidate for the antileishmanial drug development.
Figure 4.5. Antileishmanial Activity of Benzyl ester 16 (2,4-dinitrophenylhydrazine) Isosteviol
against promastigotes (A) and amastigotes (B) of L. tropica KWH23.
4.2.5. Antileishmanial Activity of Actinomycin D
Figure 4.6 represents the leishmanicidal activity of Actinomycin D (M1) against both the
promastigotes and amastigotes of L. tropica KWH23. Actinomycin D has been expansively
exploited in medical exercise since 1954 as an anticancer medicine for management of abundant
cancer types and it is correspondingly a valuable gadget in cell biology and biological chemistry.
It hinders RNA synthesis (transcription), so protein synthesis also drops. As indicated by the IC50
values 8.739 μM (95% CI= 6.675 to 11.44 μM) and 2.135 μM (95% CI= 1.419 to 3.211μM) for
78
promastigotes and amastigotes forms, the compound possesses good antileishmanial activity.
The leishmanicidal effects against both the promastigotes and amastigotes are parallel with that
of the standard drug miltefosine. The higher inhibitory activity against amastigote forms of L.
tropica KWH23 reveals that the compound may probably become a good antileishmanial
candidate after extensive in vitro and in vivo investigations in future.
Figure 4.6. Antileishmanial activity of Actinomycin D (M1) against promastigotes (A) and
amastigotes (B) of L. tropica KWH23.
4.2.6. Antileishmanial Activity of Actinomycin Z3
Figure 4.7 depicts the antileishmanial activity of Actinomycin Z3, also designated as M2. The
compound gave promising antileishmanial activity against metacyclic promastigotes as well as
amastigote stages of the L. tropica KWH23 parasite. Against both the stages of L. tropica,
Actinomycin Z3 gave efficacious results than the miltefosine with IC50 values of 5.5μM (95%
CI= 3.811 to 7.939μM) and 1.760μM (1.136 to 2.728μM) respectively. Activity against
amastigotes was, however, higher as compared to the metacyclic promastigotes. With the current
in vitro results, the compound Actinomycin Z3 is proposed for further antileishmanial activities
and it is expected to come up as a potential leishmanicidal agent.
79
Figure 4.7. Antileishmanial activity of Actinomycin Z3 (M2) against promastigotes (A) and
amastigotes (B) of L. tropica KWH23.
4.2.7. Antileishmanial Activity of Actinomycin Z5
The Actinomycin Z5 (M3) gave the IC50 values of 9.529 μM (95% CI= 7.354 to 12.35μM) and
1.691μM (95% CI= 0.9559 to 2.991μM) against the promastigotes and amastigotes of L. tropica
KWH23 respectively (Figure 4.8). The compound showed less cytotoxicity than miltefosine and
Z3 as tested against human peripheral blood lymphocytes. This Actinomycin Z5 may be
harmless for the host cells as shown by in vitro results as revealed in figure 3.8. The compound
showed greater activity against amastigotes than promastigotes as is clear from the mentioned
fifty percent inhibitory concentrations represented in figure 4.8. The overall IC50 activity of the
compound is comparable to that of the miltefosine. The present in vitro results elucidate that
actinomycin Z5 compound may be an ideal candidate against both the life cycle stages of the
other species of Leishmania genus prevalent in Pakistan like L. donovani, L. infantum and L.
major too.
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Figure 4.8. Antileishmanial activity of Actinomycin Z5 (M3) against the promastigotes (A) and
amastigotes (B) of L. tropica KWH23.
4.3. Discussion
Although search for new therapeutics against leishmaniasis has been there since the discovery of
antimonials and still lingers, but no novel and effective agent has been discovered with limited
side effects. First oral antileishmanial drugs like miltefosine and paromomycin were registered as
clinical compounds against visceral leishmaniasis in the preceding decade, the antileishmanial
medication battery still needs upgrading, predominantly in the area of oral antileishmanial
medications for both cutaneous and visceral form of the disease.
In this hunt for new antileishmanials, phytoproducts and antimicrobial metabolites are on the
uppermost priority among the biological contenders. Traditional ethnobotanic practices propel
inflight to the mission for exploring plant products to skim out new antimicrobials together with
antileishmanials (Iwu et al., 1994).
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4.3.1. Mechanism of Action of Miltefosine
Miltefosine is a main therapeutic development as it is the only oral drug for the treatment of
visceral form of leishmaniasis caused mainly by L. donovani in India and Pakistan. Miltefosine
development displayed that public-private establishment is a practicable model for encouraging
exploration and development in neglected tropical diseases like leishmaniasis (Sunyoto et al.,
2018). Miltefosine obstructs the synthesis of phosphatidylcholine and affects the Leishmania
mitochondrion by cytochrome-c oxidase inactivation. It has been testified that the interruption of
the intra-leishmanial Ca2+ homeostasis characterises a significant entity for the action of
antileishmanial drugs. Pinto-Martinez et al. (2018) have demarcated a plasma membrane
Ca2+ channel in Leishmania mexicana, which is similar to voltage-gated Ca2+channel present in
human cells. This Leishmania Ca2+ channel is activated by miltefosine, particularly in L.
donovani in vitro. Miltefosine strongly affect the acidocalcisomes from L. donovani, making
quick alkalinisation of these organelles. Acidocalcisomes are acidic organelles which resemble
lysosome-related organelles of mammalian cells and contains high concentration of phosphorus
amalgamated with calcium and accompanying cations. Acidocalcisome contribute in calcium
homeostasis, management of intracellular pH and osmoregulation. Acidocalcisomes resemble
lysosome having comparable size distribution, acidic possessions, and together having
phosphorus and calcium. So miltefosine interaction and endocytosis to Leishmania tropica in
present study may have all the above-mentioned mechanism responsible for the demise of
Leishmania tropica promastigotes and amastigotes by quick alkalinisation of acidocalcisomes
and thereby disrupting calcium homeostasis, management of intracellular pH and
osmoregulation.
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Figure 4.9. Leishmania promastigotes (A) and amastigotes (B) showing acidocalcisomes
(https://www.sciencedirect.com/science/article/pii/S0020751907001038#fig1).
4.3.2. Antileishmanial Action of Isosteviol and Actinomycin D
A variety of different diterpenoids and their derivatives have been tested for their biological
activities (Kubo et al., 2004; Zhu et al., 2013; Wang et al., 2018). All the three Isosteviol (an
ent-beyrene diterpenoid) derivative used in the present study showed good activity against both
the flagellated promastigotes and amastigote forms of the Leishmania tropica KWH23. Natural
non-caloric glycosides from Stevia rebaudiana and many other plants which includes stevioside,
steviol and Isosteviol and their derivatives have been variously tested for their anticancer,
antibacterial, antifungal, antiprotozoal and antioxidant potential (Malki et al., 2014). Isosteviol
and its derivatives have been proposed to possess broad spectrum biological activities.
Antituberculotic potential of Isosteviol, steviol and their derivatives have been confirmed by
several in vitro investigations. The minimal inhibitory concentrations (MIC) found in these
studies have been mostly in the range of 0.3-10 μM for different derivatives. Some of these,
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however, showed noticeably lower activity (Kataev et al., 2011; Khybullin et al., 2012;
Garifullin et al., 2016).
Whole extracts of Stevia rebaudiana has been found to effectively inhibit the growth of plague
spirochete Borrelia burgdorferi in vitro. These extracts were also found significantly effective
against biofilms of the microbes which normally contain more resistant cells (Theophilus et al.,
2015). Isosteviol from other plants like Pittosporrum tetraspermum has also been found much
effective against different pathogens including Staphylococcus species, Escherichia coli,
Salmonella typhi, Klebsiella pneumoniae and Pseudomonas aeruginosa. It has shown different
degrees of antifungal activities against Candida albicans, Aspergillus niger and Trichophyton
mentagrophytes (Al-Dhabi et al., 2015). Similarly, ent-kaurane diterpenoids, close relatives of
the Isosteviol, have shown antimicrobial activity against Gram-positive bacteria including
Bacillus subtilis, Streptococcus mutans, Streptococcus aureus, Brevibacterium ammoniagenes,
and Propionibacterium acnes. They, however, not been found effective in inhibiting Gram-
negative bacteria and fungi (Kubo et al., 2004). When tested against hepatits B virus, Isosteviol
inhibited the secretion of both HBsAg and HBeAg giving EC50 values of 4.8 and 5.9μg/mL
respectively. At concentration greater than 5 μg/mL, it reduced the synthesis of viral DNA
(Huang et al., 2014). Another derivative of Isosteviol IN-4 [N-(propylcarbonyl)-4aamino-19-
nor-ent-16-ketobeyeran], effectively inhibited the secretion HBsAg and HBeAg as well as the
DNA production at a concentration as low as 5.67 μg/mL (Huang et al., 2015).
Diterpenoids and terpenes have also been revealed for their antiprotozoal activity against the
major protozoan pathogens including Plasmodium, Trypanosoma, and Leishmania species and
other neglected tropical diseases (de Alencar et al., 2017). These studies have mostly been done
in vitro. Candidates from ent-kaurane diterpenoids, close relatives of Isosteviol, have shown
activity against Plasmodium parasites. Some of the compounds have been of substantial
importance regarding their antiprotozoal effects in micromolar concentrations ranging from 4.5-
10.4μM against chloroquine resistant W2 strain of Plasmodium. It has been noted that the
substitution of carboxylic group enhances the antiplasmodial effects. Methyl kaurenoate and
methyl iso-kaurenoate have been revealed to show higher activity against Plasmodium
falciparum W2 (IC50 8.2 and 8.8 μM respectively) than the kaurenoic acid and grandiflorenic
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acid (IC50 21.1 and 23.3 μM respectively) (Batista et al., 2013). This kind of activity of ent-
kaurane diterpenoids have also been noted against Trypanosoma cruzi (Batista et al., 2007).
Actinomycins have been extensively investigated for their biological potencies including
antimicrobial and antineoplastic activities. Actinomycin D is an approved therapy for several
different types of malignancies. In an in vitro study, actinomycin Z3 showed highest activity
against Streptococcus aureus at a concentration of 50 μg/mL with an inhibition zone of 10 mm
followed by actinomycin Z5 (8 mm), actinomycin Z1 (7 mm) and actinomycin Z6 (0 mm). None
of these compounds, however, showed inhibition of Candida albicans (Dong et al., 2018). As
evaluated by cross-streak assay, actinomycin D caused inhibition of different species of fungi
and Streptomyces mutabilis with inhibition zone from 19-45 mm. Actinomycin D was also found
effective as biocontrol agent against chocolate spot (Botrytis cinerea) and Fusarium wilts and
rots causing Fusarium oxysporum (Toumatia et al., 2015).
Streptomyces metabolites (actinomycin Z5) possesses wide antimicrobial activities including
several important protozoan parasites including Plasmodium, Leishmania, Trypanosoma and
Entamoeba species. When tested against promastigotes of three different species of Leishmania
i.e., L. tropica, L. enrietti, and L. donovani, actinomycin D was found to cause inhibition on
translation of heat shock proteins at 40 μg/mL (Lawrence and Robert-Gero, 1985). The
inhibitory concentrations reported in the present study reflect close similarity to the mentioned
study. Along with protein inhibition, it also found to cause fragmentation of DNA in Leishmania
amazonensis promastigotes (Mendes et al., 2016). Inhibition of translation by preventing
transcription of genes crucial for pre-mRNA maturation of mRNA have been recently revealed
in Plasmodium falciparum. Its major targets were falcipain-2, FP-21-cys peroxiredoxin and
Plasmodium falciparum cleavage and polyadenylation specificity factor subunit 3 (pfCPSF). The
inhibition has been described at 80 and 320 μg/mL for the two chloroquine resistant strains W2
and Dd2 of Plasmodium falciparum (Sonoiki et al., 2017). Actinomycin D based inhibition of
protein and DNA synthesis have been confirmed in other study at picomolar (50 pM)
concentration (Diekmann-Schupert and Franklin, 1990). It also has been proposed to cause
transcription inhibition and mitochondrial dysfunction in P. falciparum (Pérez-Moreno et al.,
2016). Actinomycin D could cause 58% and 53% reduction in Plasmodium vinckei parasitaemia
at 10 μg/kg body weight in mice respectively once and twice a day (Fink and Goldberg, 1969). It
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also caused growth inhibition and morphological changes in Trypanosoma cruzi at 10, 20 and 50
μg/mL for 24-hour treatment in vitro. The parasite however, remained alive for at least 2 weeks
after this treatment (Zaverucha et al., 2003). The parasites treated with actinomycin D at
50μg/mL did not caused immunosuppression of both cellular and humoral immune response in
mice suggesting its potential use as a vaccine candidate (da Cruz et al., 1989). Actinomycin Z
has been found to effectively eradicate Entamoeba histolytica at 100 μg/mL in vitro. In almost
all the mentioned previous investigations, these compounds have been found effective in
inhibiting the various protozoans at low micromolar concentrations showing much similar results
to the present work. Further in vitro as well as in vivo studies are, however, needed to find the
mechanism of action and toxicity of the actinomycins using different cell types and animal
models.
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Chapter 5. Evaluation of the Compounds Genotoxicity against Human
Lymphocytes
5.1. Introduction
Pakistan is rich in traditional remedial flora with an enormous biodiversity which offers a good
occasion to explore the plants to hunt new, effective, less toxic and harmless therapeutic agents.
One of the several challenges faced by the biomedical researchers is the quantification of
physical damage to DNA by many physical (Osipove et al., 2004) and chemical agents. These
agents include food flavours, pharmaceuticals, cosmetics and radiations, to which living system
is either directly or indirectly exposed. In this regard, Comet assay is one of the simple and
convenient technique to assess DNA damage by electrophoresing whole nuclei on microscope
slides from cells, either experimentally exposed to an agent in vitro or isolated from living body
naturally exposed to the agent (Ostling and Johnson, 1984; Reus et al., 2012). The term "comet"
denotes to the design of DNA movement through the electrophoresis gel, that commonly
resemble a comet (bright object with long tail). The importance of Comet Assay can be judged
from the fact that every year International Comet Assay Workshop is held at different countries.
This year 13th International Comet Assay Workshop (ICAW 2019) will be held in Pushchino, a
town in Moscow Oblast in Russia from 24-28 June 2019 (www.mutagenesisambiental.com/13th-
international-comet-assay-workshop/). These workshops are a succession of scientific meetings
dealing with practical and theoretical aspects of the Comet Assay. The original publication
relating such an assay was published 35 years before in 1984 by Swedish investigators O.
Ostling and K. J. Johanson in Biochemical and Biophysical Research Communications. This
assay was further established by Singh et al. (1988) and afterward this assay has been
extensively used in measuring DNA damage. The present study is one such attempt to assess
DNA damage caused by Actinomycin (actinomycetes metabolite) and Isosteviol derivatives
(plant metabolites) in human peripheral blood lymphocytes (PBLs).
5.2. Methodology
Genotoxic effects of the Actinomycin and Isosteviol derivatives on human peripheral blood
lymphocytes were evaluated using alkaline comet assay protocol originally developed by Ostling
and Johnson, 1984, modified by Singh et al. (1988) and further improved by Osipov et al. (2014)
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and Khisroon et al. (2015). In this technique the cells are first entrenched in agarose on a clean
glass slide and are then lysed by using detergent to nucleoids having supercoiled coils of DNA
connected to the nuclear milieu. Then electrophoresis at raised pH results in structures like
comets, as observed under fluorescence microscope; the strength of the comet tail comparative to
the head reflects the quantity of DNA disruptions. A variety of different alternatives including
reverse mutation assay, chromosomal aberration test and micronucleus tests, all with efficiencies
and limitations, are used for genotoxicity evaluation. Comet Assay is, however, easy and
comparable to the rest.
5.2.1. Application of Test Compounds to Lymphocytes for Genotoxicity
To check the genotoxicity of the test compounds, lymphocytes were incubated for 48 hours with
Actinomycin D, Actinomycin Z3, Actinomycin Z5, 16 (2,4-dinitrophenylhydrazine) Isosteviol,
17 hydroxy, 16 (2,4-dinitrophenylhydrazine) Isosteviol, Benzyl ester 16 (2,4-
dinitrophenylhydrazine) Isosteviol and miltefosine (standard positive control). The
concentrations used were100μM, 25μM and concentration caused 50% inhibition (IC50) in
axenic amastigotes. The IC50 concentrations were 2.135, 1.76, 1.691, 4.447, 5.033, 6.794 and
1.245μM respectively for Actinomycin D, Actinomycin Z3, Actinomycin Z5, 16 (2,4-
dinitrophenylhydrazine) Isosteviol, 17 hydroxy, 16 (2,4-dinitrophenylhydrazine) Isosteviol,
Benzyl ester 16 (2,4-dinitrophenylhydrazine) Isosteviol and miltefosine. Only cultured human
peripheral blood lymphocytes and 1% DMSO were used as negative control and were denoted as
“Plain”. Three different experiments were done in triplicate to validate the results.
5.2.2. Slide Preparation and Mounting of Treated Lymphocytes
After required exposure to the test compounds, a 40μL of cell suspension (1x105/mL) was mixed
with 130μL of molten low melting point agarose (LMPA) in a 3 mL sterile Eppendorf tube. This
mixture was then smeared on a normal melting agarose (NMA) pre-coated slide. Pre-coating was
done 15 hours before by simply plunging a degreased slide in molten 0.7% NMA. Two smears
were prepared per slide. Each smear was overlaid by a separate 22x22 mm clean sterile coverslip
avoiding bubbles. The smear was solidified on an ice pack for 5 minutes and the coverslip was
removed by careful side pushing with a finger.
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5.2.3. Lysing of Lymphocyte Cells
Lysis of the human peripheral blood lymphocytes was performed in a working lysing solution
prepared freshly from stock lysing solution (2.5 M NaCl, 100 mM Na2-EDTA, 10 mM Tris,
adjusted to pH 10) by adding 1% Triton X-100 and 10 % DMSO. Lysing was performed for 2-24
hours at 4°C and cellular protein was isolated.
5.2.4. Single Cell (SC) Gel Electrophoresis
Single cell gel electrophoresis was performed in buffer prepared by mixing 300 mM sodium
hydroxide and 1 mM EDTA. The pH of the buffer was then adjusted to 13. To unwind the DNA
and to expose alkali labile regions, slide was kept immersed in buffer for 20 minutes without
switching the electrophoresis unit on. After unwinding, the electrophoresis was performed for 30
minutes at 4°C on 300mA currents at 25 V.
5.2.5. Neutralization and Fixation
Three washes of 5 minutes each were done with neutralization buffer (400 mM Tris, pH 7.5) and
slides were then fixed in 99.5% ethanol for 5 minutes and air dried at room temperature.
5.2.6. Staining
The slides were stained in 10% Giemsa stain for 20 minutes. Giemsa's stain is an associate of the
Romanowski cluster of stains, which was initially intended to integrate cytoplasmic (pink) staining
through nuclear (blue) staining and fixation as a lone stage for smears and thin films of blood and
other tissues. After rinsing under tap water and air drying, the comets were observed under light
microscope at low power objective (100 x magnifications), high power dry objective (400 x
magnifications) and high refractive indexed oil immersion objective (1000 x magnification
which increases the numerical aperture of the objective lens by oiling the specimen and the
objective) to record results (Opisov et al., 2014).
5.2.7. Data Interpretation and Scoring
For recording results, 100 randomly selected lymphocyte cells nuclei were counted from the dual
smear for each slide. The cells were then assorted to 5 different comet classes by intensity of
staining in the tail from zero (0) (no damage) to 4 (maximum/total damage). Comet scoring into
various classes was done visually. Total comet score (TCS) was determined by using formula
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TCS= 0 (n)+1(n)+2(n)+3(n)+4(n), where “n” represents number of cells in each class (Khisroon
et al., 2015).
5.2.8. Statistical Analysis
The Total comet score (TCS) values were analysed in SPSS software using two-way ANOVA
and Tuckey test for comparison of the test compound’s genotoxic potential in relation to 1%
DMSO control, plain control and standard drug i.e., Miltefosine. While at the lowest
concentration, which was the IC50 value of the compounds against axenically grown amastigotes
of Leishmania tropica as described in chapter 4, one-way ANOVA and Tuckey test were used
for comparison of genotoxicity in relation to the mentioned control and standard.
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5.3. Results
5.3.1. Genotoxicity of Miltefosine (Milt)
Miltefosine showed least genotoxicity against peripheral blood lymphocytes (PBLs). As
evaluated by comet assay, it gave total comet scores (TCS) of 10, 8 and 8 respectively at 100, 25
and 1.25μM concentrations. As revealed in figure 5.1 A, B and C, damage to only the category
of class 1 could be seen in the smears treated with all concentrations of the Miltefosine drug. As
compared to the solvent (1% DMSO) and negative (Plain) control, the genotoxicity of
Miltefosine was found to be non-significant at all concentrations used (P>0.05).
Figure 5.1. Genotoxicity of Miltefosine (Milt) at 100μM, 25μM and 1.25μM (concentration
which showed IC50 for Leishmania tropica amastigotes in Chapter 3.0).
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5.3.2. Genotoxicity of 16 (2, 4-dinitrophenylhydrazine) Isosteviol (ID)
Genotoxicity of 16 (2,4-dinitrophenylhydrazine) Isosteviol showed dependence on the
concentration of the compound used as is revealed by the total comet scores (TCS) and classes of
damage (figure 5.2). At 100μM concentration Isosteviol indicated highest damage to human
blood lymphocytes and all the 4 classes of damage were observed. A TCS value of 207.33 was
noted. As compared to the DMSO and plain control where only very little class 1 damage was
seen, the compound caused damage to class 4 level at 100μM concentration (figure 5.2 A). At 25
μM concentration, damage to the level of class 3 was detected in the treated human blood
lymphocytes cells while in DMSO and plain control damage to only class 1 was documented
(figure 5.2 B). A still higher value of 87.333 for TCS was observed.
Figure 5.2. Genotoxicity of 16 (2,4-dinitrophenylhydrazine) Isosteviol (ID) at 100μM, 25μM and
4.447μM (concentration which showed IC50 for Leishmania tropica amastigotes in Chapter 3.0).
Damage in the DMSO and plain control may be attributed to the naturally occurring apoptotic
cells in the inoculum. This little damage was persistently recorded in both types of the control.
When used at 4.447μM concentration Isosteviol (concentration which showed IC50 for
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Leishmania tropica amastigotes in Chapter 3.0), showed least genotoxic damage was observed as
revealed by a TCS value of 10.666 and damage to only class 1 level (figure 5.2-C). The
compound Isosteviol showed non-significant (P>0.05) genotoxic effects as compared to the
standard Miltefosine and 1% DMSO and negative control at all the concentration used.
5.3.3. Genotoxicity of 17 hydroxy, 16 (2, 4-dinitrophenylhydrazine) Isosteviol (ID7)
As revealed in figure 5.3, a concentration dependent variability was noted in the genotoxic
effects of 17 hydroxy, 16 (2,4-dinitrophenylhydrazine) Isosteviol (ID7). A sharp waning was
seen in total comet score (TCS). It showed highest genotoxicity at 100μM concentration
successfully creating class-4 damage. Damage at the level of class 3 and 4 was, however, less
than 20% (figure 5.3 A) and TCS value of 202 was witnessed. At 25μM concentration 17
hydroxy, 16 (2,4-dinitrophenylhydrazine) Isosteviol showed damage to class-3 level but at the
minimum (figure 5.3 B). A TCS value of 85.333 was calculated.
Figure 5.3. Genotoxicity of ID7 at 100μM, 25μM and 5.033μM (concentration which showed
IC50 for Leishmania tropica amastigotes in Chapter 3.0).
At 5.033μM damage to the level of class 2 was created but to a minor extent while around 10%
comets were with class 1 damage (figure 5.3 C) and a TCS value of 15.333 was calculated.
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Therefore, in summary the genotoxic effects of the compound were slightest at the minimum
concentration of the 17 hydroxy, 16 (2,4-dinitrophenylhydrazine) Isosteviol. However, it was
found to possess significant genotoxicity as compared to plain at 100 and 25μM (P<0.05) but
genotoxicity became non-significant when the concentration was reduced to 5.033μM
concentration (P>0.05).
5.3.4. Genotoxicity of Benzyl ester 16 (2,4-dinitrophenylhydrazine) Isosteviol (IDB5)
Genotoxicity of Benzyl ester 16 (2,4-dinitrophenylhydrazine) Isosteviol (IDB5) also showed
altering scores with in consort with the concentration. Greater concentrations of the compound
had more genotoxicity in the human blood lymphocytes (figure 5.4). All the four classes of
genotoxic damage were seen at 100μM concentration while genotoxic damage to class 3 was
detected at 25μM concentration (figure 5.4 A and B). The TCS values of 166.33 and 85.33 at
100μM and 25μM concentration were acknowledged. At 6.794μM concentration Benzyl ester 16
(2,4-dinitrophenylhydrazine) Isosteviol showed a negligible damage at class 2 level with a TCS
value of 15 (figure 5.4-C). As revealed in the figure 5.4, total comet score got steep decline as
the concentration of the compound was reduced. Th compound, however, showed non-
significant level of genotoxicity as compared to the standard Miltefosine, 1% DMSO and
negative controls at all concentrations (P>0.05). Safety of the compound to cause genotoxicity is
tangled to its concentration used and causes negligible and probably restorable harm to human
blood lymphocytes at lower concentration.
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Figure 5.4. Genotoxicity of Benzyl ester 16 (2,4-dinitrophenylhydrazine) Isosteviol at 100μM,
25μM (concentration which showed IC50 for Leishmania tropica amastigotes in Chapter 3.0).
5.3.5. Genotoxicity of Actinomycin D (M1)
Actinomycin D produced greater genotoxic effects in human blood lymphocytes in comparison
to standard drug Miltefosine and the test compound Isosteviol. Genotoxicity was, however,
greatly dependant on the concentration of the compound used. Highest value (327) of total comet
score (TCS) was detected at 100μM along with higher percentage of damage at the levels of
class 3 and 4 as depicted in figure 5.5 A. At 25μM still a higher amount of genotoxicity was
caused on the lymphocyte DNA as is revealed by the TCS of 207 and class 3 and 4 damage
(figure 5.5 B). At 2.135μM concentration, however, the genotoxic effects got much reduced as is
revealed by much lower TCS (16.333) and the absence of damage at class 3 and 4 levels. This
was the concentration that produced 50% inhibition of the axenically grown amastigotes of
Leishmania tropica as shown in chapter 3.0. Actinomycin D caused
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Figure 5.5. Genotoxicity of Actinomycin D (M1) at 100μM, 25μM and 2.135μM concentration
(which showed notable IC50 value for Leishmania tropica amastigotes in Chapter 3.0).
significant level of genotoxicity at 100 and 25μM concentrations on human lymphocytes as
compared to standard Miltefosine, 1% DMSO and negative control as well as Isosteviol
derivatives (P<0.05). At 2.135μM, however, the genotoxicity was non-significant in comparison
to the standard drug and 1% DMSO control as well as Isosteviol derivatives (P>0.05) but was
still significant as compared to negative control. Thus, concentration effective against the
parasite caused minimum and probably repairable genotoxic effects on the human PBLs.
5.3.6. Genotoxicity of Actinomycin Z3 (M2)
Actinomycin Z3 also showed a concentration-based genotoxicity in human peripheral
blood lymphocytes. It was highest at 100μM concentration (figure 5.6 A) followed by 25μM
(figure 5.6 B) and was minimum at 1.76μM (figure 5.6 C). The TCS of 302.33, 213, and 29.66
respectively were recorded which shows steep decrease with the reducing concentration of
compound used. The damage wrecked to class 1 and 2 only at the lowest concentration as is
revealed in figure 5.6-C. At the higher concentrations, class 1 and 2 damage was, however, less
as compared to the class 3 and 4 which occurred at greater rate (figure 5.5-A and B). In the
nutshell, the 50% inhibitory concentration of the compound was found with minimum genotoxic
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associations to the human peripheral blood lymphocytes. The compound was, however, found
causing significant genotoxicity as compared to Miltefosine, 1% DMSO, negative control and
Isosteviol derivative at all concentrations (P<0.05).
Figure 5.6. Genotoxicity of Actinomycin Z3 (M2) at 100μM, 25μM and 1.76μM concentration
(which showed notable IC50 value for Leishmania tropica amastigotes in Chapter 3.0).
5.3.7. Genotoxicity of Actinomycin Z5 (M3)
Actinomycin Z5 showed similar representation of genotoxicity on human blood lymphocytes
like the two other actinomycin family of compounds used. It was however, found more
genotoxic as compared to the Miltefosine. As is revealed in figure 5.7, genotoxicity experienced
a steep decline with the decrease in concentration. At 100μM highest value (337.66) of TCS was
noted and greater proportion of damage was represented at class 3 and 4 levels (figure 5.7-A). At
25μM, still enough genotoxic effects were caused on the lymphocytes with still a higher value of
TCS (214.33) and most of the damage represented at class 2, 3 and 4 levels (figure 5.7-B).
Genotoxic intensity, however, came to the minimum at 1.691μM with much lower TCS (23.666)
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and damage at the level of only class 1 and 2 levels being present. The compound showed
significant genotoxicity as compared to standard Miltefosine, 1% DMSO and negative control at
all the concentrations used in present investigation (P<0.05).
Figure 5.7. Genotoxicity of Actinomycin Z5 (M3) at 100μM, 25μM and 1.691μM concentration
(which showed notable IC50 value for Leishmania tropica amastigotes in Chapter 3.0).
5.4. Discussion
Genotoxic effects of an agent depend on the exposure time and concentration. They show a
direct proportionality to both the time and concentration. As assessed in vitro on human
leukocytes and in mice, exposure to 0.25, 0.5 and 1μg/mL of Miltefosine for a few hours caused
no significant damage but it became significant when exposure time increased to 48 hours
(Branco et al., 2016). In the present study, however, the drug was found less genotoxic to human
peripheral blood lymphocytes at the same exposure time at the lowest concentration. At higher
concentrations, however, genotoxic damage to the level of class 1 was gripped but not with
higher ratio.
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There has been evidence of associated genotoxicity regarding actinomycin which is a DNA
mediated RNA synthesis inhibitor (Daza et al., 2002). The same research group (Daza et al.,
2004), also reported actinomycin-D causing significant DNA damage at 10, 20 and 40μM
concentrations. A similar dose dependant genotoxicity was also recorded in gold fish (Carassius
auratus) skin primary cell culture exposed to actinomycin D (Ishikawa et al., 2005). Results of
our study are in consensus with the findings of these workers at the highest concentrations. At
the lowest concentration, however, different results were obtained as damage up to the level of
class 2 could be seen in this study. Actinomycin D, when used to check genotoxicity on
axenically grown corneal cells, showed dose dependant increase in the genotoxicity causing
highest damage at a concentration of 100μM (Sakaki et al., 2015). Actinomycin D has been
found to cause damage to DNA at 0.05μg/mL with comet tails measurement up to 30μm in
length (Kwaguchi et al., 2010). As revealed in the present study, actinomycin caused longer tails
in the lymphocytes showing consensus with the aforementioned study.
Isosteviol has been found to inhibit the activity of mammalian (including human), polymerase
and topoisomerase II, that play important role in DNA replication (Mizushina et al., 2005).
Stevioside and rebaudioside A, Isosteviol close kin metabolites, have been found to cause no
mutagenesis in certain studies. A few studies, however, like that of Nunes et al. (2007), have
found stevioside to cause genotoxicity at 4 mg/mL as evaluated by comet assay (Brusick, 2008;
Momtazi-Borojeni et al., 2016). At very higher dose i.e., 10 folds the recommended one, it has
been found to cause mutagenicity in bacteria (Suttajit et al., 1993). In other studies, stevioside
have been proved not to be mutagenic but steviol, a more related compound to Isosteviol, was
found to cause dose dependant mutagenic effects in bacteria and Chinese hamster lung fibroblast.
In the latter it also caused chromosomal aberrations (Matusi et al., 1996). A similar dose related
mutagenic response has also been confirmed by Pezzuto et al. (1985) in bacteria through forward
mutation assay. In the present study the Isosteviol derivatives were found to cause DNA
fragmentation in a dose dependant manner. At the highest concentration, all classes of damage
were seen while damage decreased to a minimum and class 01 at the lowest concentration. From
the present results, it can be concluded that chemical modification of Isosteviol and other
compounds from the diterpenoid groups, along with having antimicrobial effectiveness, may also
increase genotoxic and cytotoxic effects on pathogenic protozoa like Leishmania tropica.
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Chapter 6. General Discussion
There is an imperative need to develop novel treatment to combat leishmaniasis, as majority of
the existing medications are highly toxic, present resistance issues and are financially
unaffordable by poor victims. If we identify the intricate life-cycle phases of Leishmania and
mechanism of drug action, then our capability to control leishmaniasis will advance. In the
present investigation variances in pathogenicity, transmittable prospectives and their response to
antileishmanial compounds have been studied. In previous studies, temporal morphological
variability of Leishmania species during life cycle stages was measured with specific metabolic,
physiologic, structural, surface protein and genetic differences (Wheeler et al., 2011).
Predisposed by such differences is variability in pathogenicity, infective potential, vector
specificity and response to antileishmanial agents of these life cycle stages. There have been
several detailed studies to evaluate morphologically variable forms in sand fly vector (Killick-
Kendrick et al., 1996; Gossage et al., 2003; Kamhavi, 2006; Frietas et al., 2012). There are,
however, fewer studies to evaluate phenotypically variable forms in vitro. Such assessment
during axenic cultivation of Leishmania is indispensable prior to experimental infection in model
animals, isolated macrophages and drug sensitivity assays. Recently there have been
improvement of more sensitive tools to select infective metacyclic promastigotes, but they have
associated limitations including requirement of expensive apparatuses, lengthy protocols and
histochemical and molecular preparations (Calcagno et al., 2002). This renders morphological
association of different stages during in vitro cultivation an important task regarding Leishmania
laboratory studies. Sorting of temporally transitional promastigote stages in axenic growth
through microscopy has been performed throughout and is still exercised. Careful follow up
usually ends up with better, if not best, results regarding metacyclic selection for further use.
Light microscopic analysis taken jointly with scanning electron microscopy, can bring precise
correlation of different stages of promastigotes (Chang, 1979). A greater move is seen towards
morphological and morphometric approaches, equalling histochemical and molecular one, for
ready and easy morphotype assessment in axenic cultivation. Axenically grown amastigotes
provide a substitute for intramacrophage stage of the parasite. Different species of Leishmania
require different conditions to transform into amastigotes (Wheeler et al., 2011; Chanmol et al.,
2019). During the present study Leishmania tropica was successfully transformed to amastigotes
100
in culture by just leaving promastigotes in culture without medium changes. It was found that,
while transforming, parasites are not getting completely round but many remain ovoid. Loss of
extracellular flagellum and reduction in cell size were shown to be the main criteria to determine
that transformation has occurred as determined previously by Ismaeel et al. (1998).
Cytocidal effects of an agent in candidature for therapeutics in an infection must be evaluated
before its use in vivo. In vitro studies on both the normal and malignant cells provide a good
model for such assessments and are often crucial in biomedical findings and designing drug
administration and dosage schedules (Valeriote et al., 1973; Akoi et al., 1998). Blood is the
immediate exposed body tissue to any material that enters the body. Blood cells, therefore, are
convenient entities for in vitro chemical and pharmaceutical testing (Andersson et al., 2003;
Faust et al., 2004; Vorobyeva et al., 2007). Findings of the present study may be helpful in future
in vitro as well as in vivo studies on Isosteviol derivatives and actinomycins regarding their use
as antileishmanial agents. Dosage schedules with sub lethal concentrations of these agents may
be designed based on the current finings. In correlation to the current cytotoxic values, future
animal trials may be planned regarding use of Isosteviols and actinomycins against cutaneous
and visceral forms of leishmaniasis.
The re-emergence of leishmaniasis has provoked concerns amongst the researchers to find new
and safer therapeutics for the infection (Mendes et al., 2016). Natural substances have been of
special concern in treating various ailments. Diterpenoids have been used in folkloric medicine
for several different conditions and are now gaining greater attention from researchers
(Nagarajan and Brindha, 2012; Strobykina et al., 2015). Actinomycins are known for their
antimicrobial and anti-neoplastic activities since 1950s. Actinomycin D is the most familiar of
the group (Alenazi et al., 2018). It has shown potential in vitro antiprotozoal activity against
Plasmodium falciparum strain 3D7 (Fink and Goldenberg, 1969). It also has been found to cause
the prevention of heat shock proteins in Leishmania major promastigotes (Lawrence and Robert-
Gero, 1985). However, there has been no comprehensive study of both the diterpenoids and
actinomycins against Leishmania tropica. The present study may therefore be amongst the
pioneering efforts to evaluate antileishmanial effects of diterenoid Isoteviol analogues and
actinomycins. This will provide baseline information regarding the antileishmanial potency of
these candidates. Based on the findings of the current attempt, actinomycins may be of prime
101
significance against cutaneous infection caused by Leishmania tropica. On the other hand, this
investigation may offer a solid base for the strengthening of other antiprotozoal studies and in
principally how other protozoan parasites react to actinomycins and diterenoid Isoteviol
analogues in vitro.
Comet assay is one of the several techniques used to assess DNA damage and mutagenesis
which result in a variety of different conditions including carcinogenesis and teratogenesis (Tice
et al., 2000; Pereira et al., 2012). Comets are generally visualized by using fluorescent dye
technique that need fluorescent microscopy which is expensive device and not affordable in low
budget researches. Giemsa stain has been evaluated by just one earlier study (Osipov et al.,
2014) as a cheaper and effective alternative for comet visualization. The present study supports
the original study positively. We visualized all classes of comets using Giemsa stain. The results
were not less than where comets were visualized with fluorescent techniques. Use of Giemsa
staining technique for comet assay will probably be adopted as an inexpensive readily available
alternative. Careful and expert handling of the stain is expected to bring comparable, if not
superior, results to fluorescent approaches. Use of fresh and double filtered 10% working
Giemsa stain solution has sown greater clarity. Based on the present Comet assay results,
actinomycins, in higher doses, can be used as positive control for genotoxicity studies in future.
102
Conclusion
Leishmania tropica (MHOM/PK/2010/KWH23) grows satisfactorily in RPMI-1640 medium
achieving a density of 1x107/mL in late log phase. Greater density of metacyclic forms is seen
towards the stationary phase which is achieved after 7 days of growth. Metacyclics are
characterized by their specific carrot tuber or cone shaped morphology with broader anterior and
much narrower posterior end. Loss of extracellular flagellum must be used as criterion for
identification of axenic amastigote. Orthophosphoric acid works satisfactorily for medium
acidication for axenic amastigotes as it is not volatilized at 37ᴼC to the free space in culture
flask. Isosteviol derivatives 16 (2,4-dinitrophenylhydrazine) Isosteviol, 17-hydroxy 16 (2,4-
dinitrophenylhydrazine) Isosteviol and Benzyl ester 16 (2,4-dinitrophenylhydrazine) Isosteviol
showed less cytotoxicity and genotoxicity than actinomycin D, Z3 and Z5 in vitro on human
peripheral blood lymphocytes. Genotoxic activity of both the classes of compounds is dependent
on concentration. Actinomycins, however, carry much higher genotoxic characteristics which
may be probable cause of their limited use as therapeutics. Both the classes of compounds, based
on the current in vitro findings, may be potential antileishmanial agents.
103
Recommendations
Considering the present findings, following suggestions are put forward for researchers to
follow;
1. Extensive investigations of actinomycins against Leishmania are needed in vivo in animal
models with broader range of dose regimens and protocols using oral, intramuscular,
intravenous, intraleisional and topical drug delivery routes.
2. Evaluation of actinomycins through intraleisional and topical formulations shall be of
prime concern against cutaneous species of Leishmania. Use of drugs through topical
route may have less probable side effects due to less amount of drug used and by not
bringing drug in direct contact with more delicate internal organs and systems.
3. In vivo evaluation of cytotoxicity, genotoxicity and teratogenicity of actinomycins, again
with broader spectrum of treatment regimen, shall be conducted in association to
Leishmania infection.
4. Establishment of finer optical protocols for morphologic and morphometric analysis of
promastigote morphotypes would be highly appreciable as it provides ready to use
approach. Providing such data for greater number of species and strains is of immediate
need.
5. Derivatives of Isosteviol with a myriad of functional groups may provide antileishmanial
candidates in future studies.
6. In vitro transformation to amastigote is needed to be evaluated further for genotypic and
chromosomal variability and dividing ability.
104
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