journal of ecophysiology and occupational health (j. ecophysiol. occup. hlth.) editor-in-chief: dr....

Volume - 18

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Journal of Ecophysiology and Occupational Health (J. Ecophysiol. Occup. Hlth.)

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Journal of Ecophysiology and Occupational Health (JEOH)

Volume 18 Issue 3 & 4 July-December 2018

CONTENTSPages

Functional Differentiation of Neural Stem Cells into Neuronal Subtypes: A Biological Tool for Developmental Neurotoxicity Studies ................................................................................................................. 59–65Ankita Pandey and Madhulika Singh

Length-Weight Relationship and Condition Factor of Schizothorax plagiostomus found in River Jhelum from Kashmir Valley ........................................................................................................................ 66–72Zubair Ahmad Sheikh and Imtiaz Ahmed

Length Weight Relationship (LWR) and Condition Factor (K) of Brown Trout, Salmo trutta fario .............................. 73–79Muddasir Jan, Neelofar Jan and Imtiaz Ahmed

Habitat Heterogeneity and Spatio-Temporal Distribution of Macrobenthos in in a Tropical Estuarine Mangrove Ecosystem ......................................................... 80–85S. M. Parvez Al Usmani

Monitoring of Nuclear Abnormality Frequencies as Indicators of Environmental Pollution in Peripheral Erythrocytes of Labeo rohita Reared in Lakes of Bangalore ...................................................... 86–102Nazima Noor and Bela Zutshi

Ammonia Intoxication at the Work Place - A Case Report .................................................... 103–105Lorena Maries and Marina Maries

Antioxidant and Anti-Apoptotic Activities of Phytochemically Validated Fruit Extract of Solanum xanthocarpum in Primary Chondrocytes ............................................................... 106–116Neelam Shivnath, Vineeta Rawat, Sahabjada, Asif Jafri, Juhi Rais, Habiba Khan, and Md. Arshad

Association of Cytokine TNF-α in Development of Osteoarthritis: A Comprehensive Study........................... 117–121AmitKumar, Md. Arshad, Ajai Singh, Habiba Khan and Suchit Swaroop

Dr. Umesh Prasad, Lucknow

©2018 The Academy of Environmental Biology, IndiaJournal of Ecophysiology and Occupational Health, Vol 18(3&4), DOI 10.18311/jeoh/2018/17925, 59-65, July-December 2018

ISSN (Print): 0972-4397ISSN (Online): 0974-0805

Functional Differentiation of Neural Stem Cells into Neuronal Subtypes: A Biological Tool for Developmental

Neurotoxicity Studies Ankita Pandey1 and Madhulika Singh2*

1Developmental Toxicology Laboratory, System Toxicology and Health Risk Assessment Group, CSIR-Indian Institute of Toxicology Research (CSIR-IITR), 31, Vishvigyan Bhawan, MG Marg, Lucknow – 226001, Uttar Pradesh, India and Department of Zoology, Maharishi University of Information Technology Sitapur Road (IIM Bypass, Bhitauli Tiraha),

P. O. Maharishi Vidya Mandir, Lucknow – 226013, Uttar Pradesh, India; [email protected] 2Department of Zoology, Maharishi University of Information Technology Sitapur Road (IIM Bypass, Bhitauli Tiraha),

P. O. Maharishi Vidya Mandir, Lucknow – 226013, Uttar Pradesh, India; [email protected]

Keywords: Nerve Growth Factor, Neural Stem Cells, Neuronal Subtypes

AbstractNeural Stem Cells (NSCs), owing to their potential to get differentiated into various mature cell subtypes including neuronal cells have proved to be an indefinite source of ‘raw material’ for their application in developmental neurotoxicity as well as therapeutic intervention in neurodegenerative disorders. However, applications of NSCs for such purposes have been broadly limited by lack of enough methods for their directed differentiation. Herein, we describe a chemically defined protocol for efficient differentiation of rat neural stem cells to neuronal subtypes using an 8-day time period. NSCs, subject to NGF (50 ng/mL) were differentiated into neuronal sub-types supplemented with a cocktail of growth factors and supplements. Differentiating cells revealed a gradual and significant induction in the neuronal markers and a parallel decrease in markers of stemness as confirmed by immunocytochemical and translational analysis. The expression of markers was found to be maximum at day 8 of differentiation. Such selective differentiation of NSCs into neurons could offer an imperative step towards generation of NSC derivatives that could facilitate their utilization for research studies.

*Author for correspondence

1. IntroductionTherapeutic application of NSC differentiation into neurons for tissue transplantation studies has been a significant hall-mark of stem cell research. With capabilities of differentiating into neurons, oligodendrocytes and astrocytes, NSCs, serve as a promising tool for the treatment of many acquired and hereditary diseases of CNS1. More recently, their application has also been extended to elucidate the toxic effects of a vari-ety of environmental contaminants. In addition, the efficient differentiation of NSCs has also raised the possibility that it may provide a novel source of neuronal cells for nervous tissue replacement or repair after injury or neurodegeneration2.

Neurogenesis is a multistep developmental process of pro-ducing functionally active neurons from their precursors3. For a long time, it was believed that no new neurons were gener-ated in the adult Central Nervous System (CNS). The concept of neurogenesis gained momentum with the identification of cell division in the adult brain of birds and rodents4,5. In 1992 NSCs were isolated from the striatum of adult mice and grown in the presence of Epidermal Growth Factor (EGF). Post EGF removal, cells could differentiate and express markers of neural or glial origin, an indication of stem cells in the adult brain6.) These findings gave birth to a very useful in vitro model to understand cell fate determination.

Extensive therapeutic potential exhibited by NSCs has fuelled various attempts to develop and apply protocols to

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Functional Differentiation of Neural Stem Cells into Neuronal Subtypes: A Biological Tool for Developmental Neurotoxicity Studies

direct their differentiated fate towards particular lineage for their utilization in various studies. To recapitulate the differ-entiation process of NSCs in in-vitro has been a Herculian task. Studies revealed, that apart from internal features of stem cells, their external environment, including presence of growth factors, cytokines as well as cell-cell contacts, have played a crucial function in influencing the ultimate fate of these cells. Basic Fibroblast Growth Factor (BFGF) and Epidermal Growth Factor (EGF) have revealed to influence the prolifera-tion and differentiation of NSCs in-vitro7. Neurotrophic factors like Neurotrophin-3 (NT-3) and Brain-derived Neurotrophic Factor (BDNF), in addition, are also crucial role players influ-encing the subtype of NSCs8. Apart from these cell to cell contact signals may also control the differentiation process. For example, co-culturing of ammonic cells and astrocytes led to enhancement of NSC differentiation into neurons9. Electrical stimulation using carbon nanotube ropes might also trigger the differentiation process10. However, these various strategies of NSCs differentiation have few limitations such as chemical toxicity, insufficient cell-specific differentiation and several others11. Therefore, there has been a pressing need for better and more efficient protocols for differentiation of NSCs.

The mammalian brain is enriched with various neurotroph-ins/growth factors essential for the process of neurogenesis. Nerve Growth Factor (NGF) is the main growth factor known to be involved in the cellular functions such as protection from cellular injury and enhancement of cell repairing process12. Since neurogenesis is majorly supported by the neurotrophins in brain, therefore, in the current study, we have attempted to explore the neurogenesis potential of NGF to trigger induction of neuronal differentiation in NSCs.

This study was thus undertaken to develop and standardize a protocol for efficient isolation, characterization, prolifera-tion and subsequent differentiation of NSCs towards neuronal lineage with a high selectivity. The undifferentiated as well as differentiated cell fate was identified by western blot assay analysis and immunofluorescence microscopy. Along with morphological studies, expressional analysis of various cell-specific biomarkers such as nestin (a type-IV intermediate filament: marker for neural progenitor cells), BrdU (prolifera-tion marker), β-III-tubulin (component of the microtubular complex: marker for mature neurons) and ChAT (marker of cholinergic neurons) were employed to determine the different cell lineages.

2. Materials and Method

2.1 Reagents and ConsumablesAll the reagents, chemicals and kits employed in the study were procured from Sigma, unless otherwise stated. Neurobasal Medium, Fetal Bovine Serum, GlutaMAXTM-I (100X), Penicillin- Streptomycin antibiotic solution (100X), Sodium bicarbonate (7.5%), Phosphate Buffered saline were procured from Gibco (Invitrogen, USA) and antibodies from Millipore (USA).

2.2 Isolation of NSCsNSCs were extracted from embryonic day-16 rat foetus. The isolated tissue was then washed and dissected with chilled HBSS (Hanks Balanced Salt Solution), incubated for 30 min in 0.1% trypsin, then in DNase (40 μg/ml) for 10 min, 37°C to get a single cell homogenous suspension. Cells that showed more than 95% viability were seeded at a density of 0.5×106 cells/ml in 75 cm2 flasks in neurobasal medium and were allowed to proliferate as neurospheres in 5% CO2 and 95% air at 37°C.

2.3 Proliferation and Characterization of NSCsExpansion of proliferating NSCs was carried out in neurobasal medium with supplements N-2 (1%), B-27 (2%), EGF (10 ng/ml) and bFGF (10 ng/ml). Small neurospheres were observed post 1 week that matured by day 20. Passaging was carried out after every 10-12 days. To visualize the proliferating activ-ity of NSCs and to identify the undifferentiated progenitor cells, neurospheres were co-immunostained with anti-nestin monoclonal antibody (1:200; a neural stem cell marker) and anti-BrdU antibody (1:500, proliferating cells marker).

2.4 NGF induced neuronal differentiation of NSCs

NSCs were seeded on pre-PLL coated 25cm2 flask (1X106 per flask) and subsequently cultured for 24h. The cells were fur-ther incubated in neurobasal medium (deprived of growth factors and supplements) for 24 h prior to exposure to NGF. For induction of differentiation into cholinergic neurons, cells were incubated in medium containing NGF (50 ng/ml) along with lowered concentrations of EGF and bFGF (1 ng/mL), B-27 (0.1%) and N-2 (1%). Concentration of NGF (50 ng/ml)

Journal of Ecophysiology and Occupational Health 61Vol 18 (3&4) | July-December 2018 | http://www.informaticsjournals.com/index.php/JEOH/index

Ankita Pandey and Madhulika Singh

was selected based on previous studies reporting optimal dif-ferentiation using different cell cultures13. Exposure was given till 8 days and exposure medium was removed and replenished every alternate day. The differentiated cells were characterized for expression of mature neuronal markers using western blot-ting and immunocytochemical analysis.

2.5 Immunocytochemical LocalizationImmunocytochemical studies were carried out employ-ing the protocol briefly described14. Post different exposures, the medium was replaced with 4% paraformaldehyde for 20 min for fixation. Further cells were gently washed with 1X PBS two times and then non-specific sites were blocked with 0.02% Triton X 100 and 0.1% BSA in PBS for 1h at room tem-perature. Post blocking cells were incubated with primary antibodies against specific proteins, viz. Nestin (1:250), BrdU (1:500), ChAT (1:250) and β-III tubulin (1: 150) at 40C over-night. Next, unbound primary antibodies were removed by gently washing cells with 1X PBS. Next, Alexa Fluor second-ary antibodies (1:150) were added and the cells were incubated in dark at room temperature for 1 h. Next, unbound second-ary antibodies were removed by gently washing cells with 1X PBS. Next, the cells were observed under Nikon Eclipse 80i using particular filters and captured which were analyzed for their fluorescent intensity by Leica Qwin 500 Image Analysis Software (Leica, Germany).

2.6 Translational StudyWestern blotting was carried out employing the protocol briefly described15. Of the total protein, 30–40 µg was elec-trophoresed in a 10%-12% SDS-PAGE and transferred onto

PVDF membrane using transfer buffer (25mM Tris [pH 8.3], 190mM glycine, and 20% methanol) for 2h with a current of 250 mA. Nonspecific sites were blocked with 5% BSA in TBST (20mM Tris-HCl [pH 7.4], 137mM NaCl, and 0.1% Tween 20) for 2 h at 37°C. Post blocking the membrane was incubated with primary antibodies (Nestin (1:1000), ChAT (1:1000) and β-III tubulin (1: 1000) and β-actin (1:2000)) against specific proteins at 40C overnight. After washing with 1X TBST thrice for 10 min the membrane was next incubated with secondary antibodies (conjugated with HRP, Chemicon, 1:2000) at room temperature for 2 h. Post washing with 1X TBST, expression levels for different proteins were developed using chemilu-minescent Substrate (Thermo Fisher Scientific) and Bio-Rad Versa Doc Imaging System 4000 (Bio-Rad, Philadelphia, PA).

2.7 Statistical AnalysisResults have been expressed as mean ± Standard Error of Mean (SEM) from the values that were obtained from at least three independent experiments. Statistical analysis was computed using one-way analysis of variance (ANOVA) and Dunnett’s Multiple Comparison test employing Graph Pad prism (Version 5.0) software.

3. Results

3.1 Isolation, Characterization and Proliferation of NSCs

Rat brain NSCs were successfully isolated from embryonic day-16 rat foetus (Figure 1a). The proliferating cells grown in neurobasal medium could be seen as small neurospheres by

Figure 1. Figure 1: (a) Rat brain NSCs isolated from embryonic day-16 rat foetus (b) Proliferating cells observed as small neurospheres by day 7 (c) The neurospheres gained maturity by day 20.



day 7 (Figure 1b) that gained maturity and grew significantly both in size and number by day 20 (Figure 1c). Cells in the neurospheres expressed both progenitor cell marker-nestin (green) as well as proliferative cell marker-BrdU (red) indica-tive of their stemness nature (Figure 2 a-c).

3.2 Morphological Analysis of Neuronal Differentiation Induced by NGF

We next analysed NSCs for their potential to undergo neural differentiation under the influence of 50ng/ml of NGF. On day 2, we observed few morphological alterations such as cells becoming longer as compared to Day 0. By day 4 and 6, cells revealed neurite like extensions that became more prominent by day 8 (Figure 3a-d). Quantitative analysis revealed that

NGF had the potential to induce significant neurite outgrowth in a time dependent manner. (Figure 4A (a-e) and Figure 4B).

3.3 Expressional Analysis of Neuronal Differentiation Induced by NGF

To further confirm the neurotrophic effect of NGF, protein expression of β-III-tubulin and ChAT was determined over an 8-day period via immunocytochemical localization (Figure 4A and 4C). Western blot analysis (Figure 5A and 5B) revealed time dependent significant increase in expression of β-III-tubulin and ChAT as compared to the day 0. Simultaneously there was a decrease in expression of the stemness marker nes-tin, indicative of differentiation of neural stem cells towards neuronal lineage.

Figure 2. NSCs showing immunoreactivity for both (a) nestin (green) and (b) BrdU (red) (c) counterstained.

Figure 3. (a-d) Representative microphotographs revealing alterations in morphology on neuronal induction by NGF (50ng/ml).



Figure 4. A. (a-e) and B Morphological and quantitative analysis of relative neurite length. (A (f-t)) Differentiated NSCs showed neuronal morphology and stained positive for neural marker β-III tubulin (green) and cholinergic specific marker ChAT (red) counter stained with DAPI. (C) Relative fluorescence intensities expressed in fold change. * = p< 0.05, * * = p< 0.01, * * * = p< 0.001.

4. DiscussionNSCs have been one of the potential candidates for restoration of central nervous system injury, such as neurodegenerative diseases, stroke and traumatic brain injury16. More recently, their application for developmental neurotoxicity studies has received much attention in identification of stage specific markers activated in response to a range of toxicants that have shed light on the underlying toxicity mechanisms17.

The main objective of culturing NSCs in vitro has been to regulate the proliferation and ultimate differentiation into different cell types. Unfortunately, such efforts have been met with only limited success. A plethora of studies have revealed that exposure to NT-3, BDNF and Shh, sequentially, signifi-cantly enhanced the neuronal differentiation from NSCs both in vitro or in vivo after transplantation8. Moreover cytokines, chemicals, electrical stimulation have all shown to affect the differentiation process. These results were suggestive of the fact that the external environment has a significant involvement in triggering NSCs to differentiate into neurons18.

NGF is a major biologically active molecule that is actively involved neurogenesis. As a neurotransmitter, NGF has been

Figure 5. Expressional analysis of markers of stemness (Nestin) and neuronal markers (β-III tubulin and ChAT) under NGF exposure over an 8-day time period. * = p< 0.05, * * = p< 0.01, * * * = p< 0.001.



convincingly demonstrated to control proliferation and dif-ferentiation of NSCs19 and has been earlier used to induce many cells such as PC12, SH-SY5Y into mature neurons under in vitro conditions20,21. More recently, exogenous NGF supplementation on Human Derived Mesenchymal Stem Cells (hMSCs) was also studied13. Among the different concentra-tions of NGF that were tested, NGF at 50 ng/mL concentration could most significantly trigger differentiation of hMSCs into neuronal lineage.

In an attempt to find new approaches, we have standardized a protocol to isolate, characterize and differentiate neural stem cells to neuronal lineages that have been confirmed through a panel of characterization techniques. Specific markers were selected to characterize particular cell lineages. Nestin, an intermediate filament protein commonly used to label undif-ferentiated NSCs was found to be down-regulated over the 8-day time period. β-Tubulin III, a cytoskeletal protein whose expression elevates during axonal outgrowth, is expressed by mature neurons (Lee et al., 1990) and ChAT, a choliner-gic marker, both simultaneously significantly elevated. Stage specific increase in expression of neuronal markers indicated the effective differentiation over an 8-day time period. These experiments support our conclusion that was NGF is an impor-tant factor in determining stem cell differentiation. These stage specific alterations could be used to study the effects of toxi-cants by monitoring changes in expressional levels.

In this study, it was thus identified that exogenous supple-mentation of NGF could direct the differentiation of NSCs towards the neuronal subtypes. As indicated by immuno-cytochemical staining and Western blotting examination, it was observed that NGF had the potential to promote the growth of processes and distinct differentiation of NSCs into neurons from embryonic rat brain cultured neural stem cells. Furthermore, the mechanism and signaling pathway of neuronal differentiation of NSCs in NGF supplementation conditions are still to be further investigated.

5. AcknowledgementThe authors thank Dr AB Pant, Principal Scientist, Developmental Toxicology Division, CSIR-Indian Institute of Toxicology Research, Lucknow for taking keen interest and providing the state of the art facilities to conduct this research work.

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©2018 The Academy of Environmental Biology, IndiaJournal of Ecophysiology and Occupational Health, Vol 18(3&4), DOI : 10.18311/jeoh/2018/v18i3&4/19991, 66-72, July-December 2018


Length-Weight Relationship and Condition Factor of Schizothorax plagiostomus found in River Jhelum

from Kashmir ValleyZubair Ahmad Sheikh and Imtiaz Ahmed*

Fish Nutrition Research Laboratory, Department of Zoology, University of Kashmir, Hazratbal, Srinagar – 190006, Jammu and Kashmir, India; [email protected]

Keywords: Condition Factor, Jhelum, Length Weight, Schizothorax plagiostomus

AbstractLength weight relationship is important in describing several biological aspects of fish species found under cultured and natural condition. Length weight relationship provides information about whether somatic growth was isometric or allometric. While condition factor provide information about the well being fish. In the present study the individuals of Schizothorax plagiostomus were collected from river Jhelum from different locations for the determination of length weight relationship and condition factor during the period of 2014–2016. Length weight relationship was computed using the equation W = aLb, which was further transformed into LogW = Loga + bLog L. Results showed that the value of b in length weight relationship of male S. plagiostomus ranged from 2.316–2.965, while in female fishes the value of b ranged from 2.01–3.66. The results clearly show allometric type of growth in male and isometric type of growth in females. The regression coefficient between males and female did not shows any significant difference, (p > 0.05) whereas significant difference (P < 0.01) could be noticed between males and between females (p < 0.01). However, condition factor (K) of S. plagiostomus fluctuates between 0.82–1.58 in male and between 0.870–1.31in female, indicating the robustness of the fish inhabiting in river Jhelum.

1. IntroductionLength-weight Relationship (LWR) studies of fishes is con-sidered as an important tool for understanding of fish health status. Length-weight Relationship of fishes is also important in fisheries and fish biology as its allow the estimation of the average weight of the fish of a given length group by establish-ing a mathematical relation between them6. Length–weight Relationships have broadly been used for the conversion of growth-in-length equations to growth-in-weight for use in stock assessment models in order to estimate the stock assessment, relate the life histories of certain species and other changing aspects of fish population2,32,48,31,51 Nile et al., 2013;. Like any other morphometric characters, the LWR can be used as a character for the differentiation of taxonomic units and the relationship changes with the various develop-mental events. In addition, the Length-weight Relationship indicates the degrees of stabilization of taxonomic characters in fish species and very useful in the management and exploi-tation of fish populations39. Growth of fish, usually indicated

through increase in length and weight which is considered as the most appropriate characteristic to determine the pop-ulation analysis at a particular time21. Now a days study of Length-weight Relationships (LWRs) of threatened fish spe-cies are the most important biological parameters to provide information about the growth and condition of fish species as well as entire fish community and are highly significant for management and conservation of natural populations43,33. Fulton’s condition factor (k) is extensively used in fisheries and fish biology studies. This factor refers to the well-being of a certain species and its degree of fatness which depends on the weight of the fish38,13. Condition factor reflects the variations by interaction among feeding conditions, para-sitic infection and physiological factors and recent physical and biological circumstances24,5,49,28. The study of condition assumes that heavier organisms of a given length are in better physical state, therefore, condition indices are used in fishery sciences as indicators of the length-weight-relationship of a population. Condition factor also helps to reflect the feeding conditions of the species.


Journal of Ecophysiology and Occupational HealthVol 18 (3&4) | July-December 2018 | http://www.informaticsjournals.com/index.php/JEOH/index

Length-Weight Relationship and Condition Factor of Schizothorax plagiostomus found in River Jhelum from Kashmir Valley

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2. Material and MethodOur study estimates LWRs of indigenous cold water species Schizothorax plagiostomus belonging to family Cyprinidae and order Cypriniformes. The samples of S. plagiostomus were collected from 2014–2016 from locations of river Jhelum of Kashmir valley with the help of fisherman. After collection, the samples were brought into wet laboratory Department of Zoology, University of Kashmir and the required measurement of length and weight were taken by using digital caliper and digital weighing balance, respectively. The total length of fish was measured to its nearest 0.01 cm and total body weight was measured to its near 0.01 g. The total length of fish was taken from the tip of snout to the extended tip of the caudal fin and the relationship was analyzed by measuring length and weight of fish specimen collected from study area. The statistical rela-tionship between these parameters of fishes was analyzed with the help of algometric equation by Forese (2006).

W= aLb

Where W = Total weight (g).L = length of fish (cm).a = Initial growth coefficient. B = Slope or the growth coefficient. The value of constants ‘a’ and ‘b’ was estimated by linear

regression after logarithimic transformation of weight and length data by using formula:

LogW = Loga + bLog L

2.1 Determination of Condition FactorCondition factor is used for comparing the condition, fatness or well-being of fish, based on the assumption that heavier fish of a given length are in better condition. The coefficient of con-dition ‘K’ was calculated using15 equation:

K = W/L3 × 100Where, W = weight (g), L = length (cm) and 100 is a factor

to bring the value of K near unity.

3. Results The Length-weight Relationship of S. plagiostomus representing male, female and pooled (both sexes) are presented in Figure 1, Figure 2 and Figure 3, respectively. The equations thus derived in respect of Length-weight Relationship are as follows:

Females: logW = 1.764 + 2.895 logLMales: logW = 1.690 + 2.736 logLCombined: LogW = 1.656 + 2.7145 logLIn the present study increase in ‘b’ value in male fish showed

deviation from cube law throughout the annual period as nega-tive allometric growth was observed throughout the annual period i:e b < 3. The growth coefficient was minimum in March (2.315) and maximum was observed in July (2.965). The

coefficient of determination ‘r2’ fluctuates from 0.90 (January) to 0.990 in (June) as shown in Table 1. In case of females the value of ‘b’ also indicated deviation from cube law throughout annual period except, May, July and August were value of b > 3. However, this was to be attributed to the fact that during these months presence of high food availability and favorable envi-ronmental temperature, as compared to rest of months. This has led to almost ideal growth pattern of the fish. During annual period the female S. plagiostomus, shows coefficient of deter-mination oscillates between (July) 0.901 to 0.975 in October (Table 2). The month wise Fulton’s condition factor, of male, S. pla-giostomus ranged from 0.82 to1.583 and it was observed highest in the month of June followed in the month of July and December, respectively while lowest value was recorded in the month of September followed by January. Whereas in case of female, S. plagiostomus the condition factor values are in the range of 0.870–1.31 with highest condition factor was recorded in the month of June followed by July, October and lowest value was recorded in the month of April followed March and January, respectively (Table 3). The variation of condition factor in both male and female Schizothorax plagiostomus is depicted in Figure 4.

Figure 1. Depicts Length-Weight Relationship of Schizothorax plagiostomus (Male).

Figure 2. Depicts Length-Weight Relationship of Schizothorax plagiostomus (Female).


Zubair Ahmad Sheikh and Imtiaz Ahmed

68

Table 1. Month wise Length-Weight Relationships of S. plagiostomus (Males) in river Jhelum from Kashmir valley

Months NTotal Length

(cm)Total Weight (gm)

Regression Parameters W= aLb r2

Min. Max. Min. Max. a b

January 12 30 37 295 323 1.789 2.564 0.905

February 12 26 31 210 310 1.876 2.435 0.935

March 12 28 36 290 360 1.764 2.315 0.956

April 12 24 32 230 310 1.564 2.316 0.946

May 12 30 38 300 375 1.734 2.356 0.976

June 12 23 29 240 280 1.489 2.563 0.990

July 12 27 39 290 370 2.670 2.965 0.908

August 12 29 34 280 340 1.785 2.435 0.911

September 12 31 36 285 325 1.297 2.927 0.959

October 12 29 37 295 310 1.786 2.433 0.921

November 12 31 35 295 335 1.234 2.315 0.901

December 12 30 35 288 310 1.456 2.198 0.965

Mean ± SD 28.16 ± 2.65 34.91 ± 2.96 274.83 ± 30.21 329 ± 28.30 1.70 ± 0.36 2.48 ± 0.23 0.93 ± 0.03

Table 2. Month wise of length-Weight relation of S. plagiostomus (Female) in river Jhelum from Kashmir valley

Months NTotal Length cm Total Weight (gm)


Min. Max. Min. Max a b

January 12 32 37 326 360 1.573 2.897 0.936

February 12 28 35 272 415 1.786 2.887 0.949

Figure 3. Depicts Length-Weight Relationship of S. plagiostomus Combined.

Figure 4. Depicts month wise condition factor of both male and female S. plagiostomus



69

Months NTotal Length cm Total Weight (gm)


Min. Max. Min. Max a b

March 12 31 38 310 345 1.674 2.967 0.917

April 12 32 36 320 365 1.789 2.980 0.938

May 12 31 37 312 390 1.897 3.665 0.917

June 12 26 30 240 295 1.906 2.762 0.926

July 12 28 35 245 340 2.098 3.016 0.975

August 12 29 38 285 370 2.790 3.013 0.921

September 12 28 34 225 290 1.798 2.091 0.911

October 12 29 36 320 335 2.765 2.011 0.901

November 12 28 34 295 345 1.980 2.754 0.913

December 12 27 35 210 310 1.786 2.781 0.933

Mean ± SD 29.08 ± 1.97 3541 ± 2.19 280 ± 40.79 346.66 ± 3694 0.39 ± 1.98 2.81 ± 043 0.92 ± 0.01

Table 3. Month wise condition factor of male and female S. plagiostomus in river Jhelum from Kashmir valley

Months Male (K) Female (K)

January 0.935 ± 0.263 0.890 ± 0.156

February 1.067 ± 0.084 1.021 ± 0.160

March 0.984 ± 0.307 0.882 ± 0.229

April 1.034 ± 0.484 0.870 ± 0.143

May 0.937 ± 0.248 1.086 ± 0.360

June 1.583 ± 0.381 1.318 ± 0.151

July 1.257 ± 0.173 1.277 ± 0.269

August 1.026 ± 0.186 1.149 ± 0.106

September 0.829 ± 0.123 1.139 ± 0.261

October 1.054 ± 0.087 1.221 ± 0.018

November 1.088 ± 0.234 1.164 ± 0.176

December 1.218 ± 0.208 1.216 ± 0.271



70

4. DiscussionThe Length Weight Relationship of fish has significant importance in studying the growth, gonadal development, general well being of fish population and for comparing life history of fishes24,38,40. Fisheries management and research often requires the use of bio-metric relationships in order to transform data collecting from the field into appropriate indices37 . Studies on the Length Weight Relation of fishes constitutes an important tool in fishery biology and helps to understand whether variations from the expected weight for the known groups are the indicators of fatness, well-being and gonadal development in relation to the environment 4,24,50.I n the present study, the ‘b’ value was found higher in females as compared to males. The highest ‘b’ value in females of S. plagios-tomus implies that the females gain weight at a faster rate in relation to its length. The b value of males indicates negative allometric, which indicates that, the increase in length is not in accordance with increase in weight. Similar results were observed by Dar et al. (2012) in Schizopyge esocinus. Le Cren (1951) had reported that females are heavier than males of the same length probably because of difference in fatness and gonadal development. The slope value of regression line less than ‘3’ has been reported in Tortor (Malhotra, 1982), Labeo dero (Malhotra and Chauhan, 1984), Labeo dyocheilus (Malhotra, 1985), Cyprinus communis and Ctenopharyngodon idella (Dhanze and Dhanze. 1997) and Rasbora daniconius (Sunil, 2000). According to Ruz Campos, 2010, the b value ranged from a minimum of 2.863 for reef fin spot (Paraclinus integripinnis) to 3.404 for the fluffy sculpin (Oligocottus snyderi). All the earlier reports are in compliance with the present study in which the b value was very close to isometric value of 3 and this indicates that S. plagiostomus in the present study showed an isometric growth. Also it is well known that the functional regression b value represents the body form and is directly related to the weight affected by ecological factors habitat, area, seasonal effects, degree stomach fullness, gonad maturity, sex, health, pres-ervation techniques and differences in the observed length ranges of the captured specimens29,17,18 (Ricker, 1973. Several workers reported positive allometric growth in freshwater fish; notable among them are20,34,23,9,19,45,30,42,35 in Cirrhina mrigala, Catla catla, Labeo bata, Labeo rohita, Pristolepis fasciata, Pangasius pangsius, Pseudorasbora parva and Macrognathus aculeatus respectively. The greater value of ‘b’ mainly depends on shape and fatness of individuals of fish. Isometric growth was also reported by

(Haniffa et al., 2006; Serajuddin et al., 2013; Kashyap et al., 2015) in Channa punctatus collected from lentic and lotic water bodies. According to to Copp et al. (2013), the isometric body growth of Barbatula barbatula changes to allometric type when it reaches a certain standard length in order to become an adult. This indi-cates that body growth type sometimes changes accordingly to their physiology needs. Allen, (1938) have reported that the cube law is applicable only for those species which maintain the form and specific gravity throughout their life, but the shape and

the form of fish may change with time, so the Length-weight relationship of most of fish species may deviate the cube law. The value of ‘b’ reported by Bhat et al. (2010) for S. labiatus differs from the present study, which is possibly due to several factors such as habitat, number of specimens examined and length ranges and length types used. Qadri and Mir (1980) reported the value of ‘b’ as 2.4487 for S. plagiostomus from the peripheral water bodies of Dal Lake, where as7 have reported the ‘b’ value of S. plagiostomus (2.928) for the same fish from Jammu water bodies. Our results are in conformity with the earlier reports of Bhagat and Sunder (1983); Qadri and Mir (1980); Bhat (2010). In general it has been observed that the ‘b’ values reported of snow trout species from this part of the world are within the range of 2.5 to 3.5 which are considered as normal ‘b’ value as reported by14. The b values observed in this study were significantly below 3 which mean that the S. plagiostomus in river Jhelum exhibited negative allometric growth pattern except few months in case of females. Finally, the Length-weight relationships and condition factor presented here will provide useful information for fisheries management and fish population dynamic studies. Therefore, the results of the present study can serve as baseline data for these species and for compari-sons with future studies.

5. AcknowledgementsThe author is grateful to the Head, Department of Zoology, University of Kashmir, Hazratbal, Srinagar, India for providing the laboratory facilities and gratefully acknowledge the finincal support from the Department of Biotechnology (DBT), Govt of India, New Delhi on Fish Nutrition and Diet development pro-gram. Thanks are also due to Mr. Mufti Buhran, University Chief Executive Engineer for helping the construction of new Feed Technology Laboratory (Wet-Lab.) in the Department of Zoology.

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Length Weight Relationship (LWR) and Condition Factor (K) of Brown Trout, Salmo trutta fario

Muddasir Jan, Neelofar Jan and Imtiaz Ahmed*

Fish Nutrition Research Laboratory, Department of Zoology, University of Kashmir, Hazratbal, Srinagar – 190006, Jammu and Kashmir, India;

[email protected]

Keywords: Condition Factor, Length-Weight Relationship, Salmo trutta fario

AbstractLength-weight relationships and condition factor of Salmo trutta fario (Brown trout) at Kokernag trout fish farm, Anantnag, Jammu and Kashmir was estimated for a period of one year. During the present study the fish samples was within the range of 30cm to 45.8cm in length and 250g to 750g in weight were originally used to provide information on the condition of fish and to determine whether somatic growth was isometric or allometric. The relationship was analysed using the formula W = a Lb which was further transformed into Log W = a + b log L. The equation obtained for females was log W= 1.61 + 3.33 logL and for males was log W = 1.81 + 3.22 logL. Females show ʻbʼ value slight more than males. Studies on condition factor revealed that the fluctuations in K values can be attributed to the spawning cycle. The condition factor ‘K’ was above 1 indicating robustness or well being of the experimental fish.


1. IntroductionSalmo trutta fario (brown trout) is one of the most important fish species which is a native of European waters and now it has become extensively distributed throughout many of the fresh waters of the world including Jammu and Kashmir. It was introduced in Jammu and Kashmir due to its high aquacul-ture potential, economic value, good taste and high nutritional value. Brown trout (Salmo trutta fario) and Rainbow trout (Oncorhynchus mykiss) constitute the trout fishery in the streams, Lakes and reservoirs in the Indian uplands23. Trout is highly nutritious and it contains omega-3 poly unsaturated fatty acid that is needed for the development of brain and ret-ina in infants2. This fish prefers wild type of environment and accepts less amount of artificial feed which is a big challenge for its culture practice. Kokernag trout hatchery is doing lot of efforts for artificial propagation of this fish.

Length-weight relationship of fishes is an important aspect in fisheries and fish biology because it is used in estimation of the average weight of the fish of a given length group by estab-

lishing a mathematical relation between them21,17,18. The length weight data has two main purposes; it helps to express the rela-tionships between length and weight, so that one of them can be converted into another. It helps to measure the variation of fish condition from the observed weight in relation to the length of the individual fish11. The length weight relationship can be extended for the estimation of fish condition assuming that heavier fish of a given length is in better condition1.

The data on length-weight relationship of some fish spe-cies from Kashmir valley has also been reported by different workers19,4,5,12,16. Fulton’s condition factor (K) is widely used in fisheries and fish biology studies. This factor is calculated from the relationship between the weight of a fish and its length, with the intention of describing the condition of that individual fish10. The condition factor is used for comparing the condi-tion, fatness or wellbeing of fish, based on the assumption that heavier fish of a given length are in better condition17. As per existing literature, not so much work has been done on length-weight relationship and condition factor of a highly demanded fish Salmo trutta fario. Therefore, the study provides baseline



information on this fish species, which may serve as a tool for management and culture practices.

2. Materials and Methods

2.1 Study Sites Kokernag is located within geographical coordinates of 33.584721°N and 75.308601°E and is famous for its trout stream and trout hatchery where trout is reared. The study site was selected at Department of Fisheries Kokernag trout fish farm, Anantnag, Jammu and Kashmir. The species of trout fish which are bred as well as propagated are Rainbow trout (Oncorhyncus mykiss) and Brown trout Salmo trutta fario.

2.2 Collection and Identification of SpecimensThe method for identification of fish was used as described ear-lier by Day 1878 and Kullander et al., 1999.

2.3 Collection of Fish for Measurement of Length-Weight Relationship (LWR)

The samples were randomly collected from the raceways. Total length (TL) was measured to the nearest 0.01 cm and the length of the fish was taken from the tip of snout (mouth closed) to the tip of the caudal fin and the weight was taken on digital electronic balance (Shimadzu UX320G) with 0.01g accuracy. The statistical relationship between these parameters of fishes was established using the parabolic equation as described by9:

W = aLb

Where, W = weight of fish (g)L = total length of fish (cm)a = constantb = an exponential expressing relation between length and

weight

The relationship (W=aLb) when converted into the loga-rithmic form gives a straight line relationship graphically

Log W = Log a + b Log L

Where b represents the slope of the line, Log a is a constant.

2.4 Condition Factor (K)The coefficient of condition K was calculated by using Fulton10, equation:

Condition factor (K) =(W/L3) x 100

Where, W = weight in grams, L = length in cm and 100 is a factor to bring the value of K near unity9.

3. Results

3.1 Length-Weight RelationshipThe monthly data on length-weight relationship of female and male fish is given in Table 1 and Table 2, respectively. During the present study length weight relationship showed some variation throughout the year. The mean value of (b) in both sexes showed positive allometric growth i.e. b>3. In case of females the growth coefficient (b) was minimum in May (3.12) and maximum in November (3.79). The coeffi-cient of determination (r2) ranged from 0.72 in April to 0.96 in November in females. Whereas in case of males the growth coefficient (b) was minimum in May (3.00) and maximum in November (3.76). The coefficient of determination (r2) ranged from 0.39 in October to 0.98 in January. The coefficients a, r2 and b differs due to variations in the length classes. The values were obtained through SPSS statistical software by using linear regression. Length-weight relationship of females and males of Salmo trutta fario can be expressed by the equations: log W = 1.61 + 3.33logL and log W = 1.81+ 3.22logL respectively as shown in Table 1 and 2..

3.2 Condition FactorThe condition factor was calculated month-wise, it ranged from 0.99 ± 0.10 to 1.87 ± 0.08 in females. The highest condi-tion factor (K) in case of females was reported in November i.e. 1.87 ± 0.08, while lowest condition factor was reported in January 0.99 ± 0.10 (Table 3). In case of males, it ranged from 0.98 ± 0.126 to 1.77 ± 0.40 with highest in the month of November 1.177 ± 0.40, whereas lowest was recorded in the month of January 0.98 ± 0.12.


Muddasir Jan, Neelofar Jan and Imtiaz Ahmed

Mon

ths

Tota

l Len

gth

(cm

)To

tal W

eigh

t (g)

Regr

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n Pa

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s

W

= aL

b

r2

Min

Max

Min

Max

ab

Janu

ary

22.5

42.3

104

760.

31.

613.

210.

95

Febr

uary

21.9

4015

465

00.

793.

200.

94

Mar

ch22

.534

.512

048

01.

673.

160.

81

Apr

il24

.734

.415

7.2

440

1.47

3.22

0.72

May

24.7

41.1

157

646.

21.

963.

120.

94

June

24.7

43.8

166.

484

0.4

1.67

3.24

0.95

July

23.2

38.2

108

420

1.83

3.41

0.90

Aug

ust

2638

.114

041

01.

583.

610.

89

Sept

embe

r23

43.5

166.

484

0.4

1.31

3.33

0.95

Oct

ober

23.5

39.5

120

535.

21.

863.

430.

89

Nov

embe

r24

.243

124

840.

42.

113.

790.

96

Dec

embe

r26

42.8

42.8

182

1.79

3.24

0.91

Mea

n±SD

23.9

0±1.

3540

.1±3

.28

129.

98±3

5.41

587.

07±2

10.8

61.

61±0

.31

3.33

±0.2

00.

906±

0.07

Tabl

e 1.

M

onth

ly le

ngth

-wei

ght r

elat

ions

hips

of S

alm

o tr

utta

fari

o fe

mal

e (B

row

n tr

out)



Mon

ths

Tota

l Len

gth

(cm

)To

tal W

eigh

t (g)

Reg

ress

ion

Para

met

ers

W=

aLb

r2

Min

Max

Min

Max

ab

Janu

ary

2344

.216

091

5.7

1.63

3.21

0.98

Febr

uary

2844

316

860

1.11

3.23

0.87

Mar

ch28

4416

6.4

840.

41.

223.

120.

92

Apr

il24

.743

.816

6.4

840.

41.

533.

211.

53

May

24.5

4316

480

02.

113.

000.

96

June

29.3

42.9

252

700

1.80

3.21

0.89

July

2638

.114

041

01.

583.

160.

89

Aug

ust

2344

.210

491

5.7

2.85

3.13

0.97

Sept

embe

r29

.845

200

840

1.77

3.20

0.88

Oct

ober

2944

230

830

1.68

3.01

0.39

Nov

embe

r33

4230

567

21.

393.

760.

79

Dec

embe

r33

4730

510

302.

703.

480.

90

Mea

n±SD

27.6

0±3.

4543

.51±

2.09

209.

066±

71.4

804.

51±1

55.8

1.81

±0.5

153.

22±0

.10.

917±

0.24

Tabl

e 2.

M

onth

ly le

ngth

-wei

ght r

elat

ions

hips

of S

alm

o tr

utta

fari

o m

ale

(Bro

wn

trou

t)



Figure 1. Showing regression line for (LWR) of female fish Salmo trutta fario.

Figure 2. Showing regression line for (LWR) of male fish Salmo trutta fario.

Months Females K±SD Males K±SD

January 0.99±0.10 0.98±0.12

February 1.02±0.06 1.16±0.12

March 1.05±0.14 1.14±0.40

April 1.07±0.14 1.08±0.12

May 1.08±0.09 1.04±0.11

Table 3. Month wise condition factor of female and male of salmo trutta fario (Brown trout)



4. DiscussionStudies on the length weight relation of fishes constitutes an important tool in fishery biology and it helps to determine whether somatic growth was isometric or allometric15,13. It is useful in fish stock and population assessment12. The param-eter length-weight relationships (LWR) is affected by a series of factors viz. season, habitat, gonad maturity, sex, diet, stom-ach fullness, and health22,3,7. The length-weight relationship (LWR) is obtained monthly throughout a complete annual cycle and hence was followed same way in present study for appropriate results. The growth coefficient (b) estimated in the present study was within the range of 3.12-3.79 in case of females, where as in case of males it was found to be in range of 3.00-3.76. The b value was found slight higher in females as compared to males. The higher b value in female implies that the females gain weight at a faster rate in relation to its length15. Similar results were also reported by Rawat et al. (2014) on salmo trutta fario from river Asiganga and found b value 3.04 in females and 3.09 in case of males. Whereas Bagenal and Tesch (1978) also reported that the ‘bʼ value fluctuates between 2 to 4. Similar results were observed by Dar et al (2012) in Schizopyg esocinus. According to Le Cren (1951) ecological conditions of the habitat, temperature, food supply, spawning, sex, age or variation in the physiology of the animals are responsible for growth rate variations in the same species in different months of a year. The b values observed in the present study were above 3 which mean that salmo trutta fario in Kokernag trout fish farm exhibit positive allometric growth.

June 1.09±0.06 1.03±0.13

July 1.10±0.08 1.09±0.41

August 1.21±0.13 1.11±0.31

September 1.41±0.12 1.32±0.23

October 1.62±0.10 1.51±0.21

November 1.87±0.08 1.77±0.40

December 1.72±0.11 1.62±0.12

Condition indices have been widely used as indicators of relative health and robustness (Brown and Murphy, 1991). The condition factor is also used for comparing the condition, fatness, or well being of fish, based on the assumption that heavier fishes of given length are in better condition1,17. It is strongly influenced by both biotic and abiotic environmental conditions and can be used as an index to assess the status of the aquatic ecosystem. Condition factor can also be affected by factors like sex, season, age and maturity stages of fish8.

In the present study the condition factor of Salmo trutta fario showed variation in different months. In case of females it ranged from 0.99 ± 0.10 to 1.87 ± 0.08, with its peak value in November and minimum in the month of January. Similarly in case of males it ranged from 0.98 ± 0.126 to 1.77 ± 0.40, and the highest value of K was recorded in the month of November and the lowest value was again recorded in the month of January. Finally, the length weight relationship and condition factor presented here will prove useful information for fisheries man-agement, research and fish population dynamic studies.

5. AcknowledgementsThe authors are grateful to the Head, Department of Zoology, University of Kashmir, Hazratbal, Srinagar, India for provid-ing necessary laboratory facilities and project officer, trout fish farm Kokernag for his kind approval and help to carry out this work and also gratefully acknowledge the Department of Science and Technology (DST), Govt of India, New Delhi for providing the financial support for the establishment of Fish



Nutrition Research and Feed Technology Laboratory (Wet-Lab.) in the Department of Zoology.

6. References1. Abowei JFN. The condition factor, length-weight relationship and

abundance of Elops senegalensis (Regan, 1909) from Nkoro river, Niger Delta, Nigeria. Advance Journal of Food Science and Technology. 2010; 2:16-21.

2. Ackman RG. Nutritional composition of fats in sea food. Progress in Food and Nutrition Science. 1989; 13:161-241. PMid:2699043

3. Bagenal TB and Tesch FW. Conditions and growth patterns in fresh water habitats. Blackwell Scientific Publications, Oxford, UK. 1978; p. 75- 89.

4. Bhagath MJ and Sunder S. A preliminary notes on length-weight relationship and condition factor Schizothorax plagiostomus (Heckel, 1883) from Jammu region. Journal of Inland Fisheries Society of India. 1983; 15:73-4.

5. Bhat FA, Yousuf AR, Balkhi MH, Mahdi MD and Shah FA. Length-weight relationship and morphometric characteristics of Schizothorax spp. in the river Lidder of Kashmir. Indian Journal of Fisheries. 2010; 57:73-6.

6. Brown ML and Murphy BR. Standard weight (WS) development for striped bass, white bass and hybrid bass. North American Journal of Fisheries Management. 1991; 11: 451–467.

7. Dav SA, Najar AM, Balkhi MH, Rather MA and Sharma R. Length weight relationship and relative condition factor of Schizophyge escocinus (Heckel 1838) from Thelum river Kashmir. International Journal of Aquatic Science. 2012; 3: 29–36.

8. Day F. The fishes of India, being a natural history of the fishes known to inhabit the seas and fresh waters of India, Burma and Cylone reproduced in 1958. London: Willaim Dowen and sons. 1878; p. 778.

9. Demirel N and Dalkara EM. Weight-length relationships of 28 fish species in the Sea of Marmara. Turkish Journal of Zoology. 2012; 36:785-91.

10. Edah BA, Akande AO, Ayo-Olalusi C and Olusola A. Computed the wet weight-dry weight relationship of Oreochromis niloticus, Tilapia. International Journal of Food and Safety. 2010; 12:109-16.

11. Froese R. Cube law, condition factor and weight-length relationship: history, meta-analysis and recommendations. Journal of Applied Ichthyology. 2006; 22:241-53. https://doi.org/10.1111/j.1439-0426.2006.00805.x

12. Fulton TW. The rate of growth of fishes. Twenty-second Annual Report Part III. Fisheries Board of Scotland, Edinburgh. 1904; 3:141-241.

13. Gumanao GS, Saceda-Cardoza MM, Mueller B and Bos AR. Length-weight and length-length relationships of 139 Indo-

Pacific fish species (Teleostei) from the Davao Gulf, Philippines. Journal of Applied Ichthyology. 2016; 32:377-85. https://doi.org/10.1111/jai.12993

14. Khan MA and Sabah. Length-weight and length-length relationships for five fish species from Kashmir valley. Journal of Applied Ichthyology. 2013; 29:283-4. https://doi.org/10.1111/j.1439-0426.2012.02061.x

15. Koutrakis ET and Tsikliras AC. Short communication on length-weight relationships of fishes from three northern Aegean estuarine systems (Greece). Journal of Applied Ichthyology. 2003; 19:258-60. https://doi.org/10.1046/j.1439-0426.2003.00456.x

16. Kullander SO, Fang F, Delling B and Ahlander E. The fishes of the Kashmir valley, River Jhelum, Kashmir valley. Impacts on the aquatic environment. Nyman L ed. Swedmar, Goteborgs, Lanstryckeri AB, Swedmar. 1999; p. 99-162.

17. Le Cren ED. The length-weight relationship and seasonal cycle in gonad weight and condition in the pearch (Perca fluviatilis). Journal of Animal Ecology. 1951; 20:201-19. https://doi.org/10.2307/1540

18. Mir FA, Mir JI, Patiyal RS and Kumar P. Length-weight relationships of four snow trout species from the Kashmir Valley in India. Journal of Applied Ichthyology. 2014; 30:1103-4. https://doi.org/10.1111/jai.12482

19. Mir JI, Shabir R and Mir FA. Length-weight relationship and condition factor of Schizopyge curvifrons (Heckel, 1838) from river Jhelum, Kashmir, India. World Journal of Fish and Marine Sciences. 2012; 4:325-9.

20. Ndiaye W, Diouf K, Samba O, Ndiaye P and Panfili J. The length-weight relationship and condition factor of white grouper (Epinephelusaeneus, Geoffroy saint Hilaire, 1817) at the south-west coast of Senegal, west Africa. International Journal of Advanced Research. 2015; 3:145-53.

21. Qadri MY and Mir S. Length-weight relationship of Orienus plagiostomus (McCl). Geobios. 1980; 7:158-9.

22. Rawat MS, Bantwan B, Singh D and Gusain OP. Length-weight relationship and condition factor of Brown trout (Salmo trutta fario) from river Asiganga, Uttarakhand (India). Journal of Environmental Conservation. 2014; 15:41-6.

23. Sarkar UK, Deepak PK and Negi RS. Length-weight relationship of clown knife fish Chitala chitala (Hamilton, 1822) from the Ganga basin. Journal of Applied Ichthyology. 2008; 25:232-3. https://doi.org/10.1111/j.1439-0426.2008.01206.x

24. Tesch FW. Age and growth. Methods for Assessment of Fish Production in Fresh waters. Ricker WE ed. Blackwell Scientific Publication, Oxford. 1971; p. 98-130.

25. Vass KK. Breeding biology of trout: Cold water aquaculture and fisheries, Narendra publishing house, Delhi. 2000; p. 155-68.

ISSN (Print) : 0972-4397 02018 f ie Academy of Environmental Biology, India ISSN (Online) : 0974-0805 Journal of Ecophysiology and Occupational Health, Vol18(3&4), DOI : 10.1831 l/jeoh/2018/vl8i3&4/21374, 80-85, July-December 201 8

Habitat Heterogeneity and Spatio-Temporal Distribution of Macrobenthos in in a Tropical

Estuarine Mangrove Ecosystem 5. M. Parvez Al Usmani*

D. M(s College and Research Centre, Assagao, Mapusa - 403507, Goa, India; [email protected]

This paper deals with spatio-temporal distribution of macrobenthos and their relationship with physico-chemical environment and sediment properties in estuarine mangrove ecosystem of Chorao, Goa. The complex mangrove habitats provide nutrient and play vital role in the establishment of healthy mangrove fauna. The mangrove of Chorao is inhabited by 14 species of mangrove and associated flora in different substrata. The macrofaunal density in the four study stations varied significantly. Total numerical count was 7960 at C1,3300 at C2,6705 at C3 and 7075 at C4. The wet weight biomass was 27.18 at C1,17.62 at C2,15.85 at C3 and 24.30 at C4. There was strong seasonality in the occurrence of fauna in the study area. The average macrofaunaltaxa was dominated by polychaeta (55%), crustacea (26%) and mollusca (14%). The other groups like nematode, echiurida and nemertenea was insignificant. The epibenthic feeding forms of polychaetes and scavengers/browsers of crustacean were more common in the sandy substratum, while deposit feeders were common in muddy substratum at Chorao mangrove. The result of the present study suggests that mangrove ecosystem with different substrata and physico-chemical parameters supports varied fauna and had different effect on the benthic community.

Keywords: Abundance, Distribution, Environmental Parameters, Macrobenthos, Mangroves

1. Introduction The mangrove ecosystem is ideally situated at the inter phase between terrestrial and marine environment. They are predominant habitats in the tropics and subtropics and have been reported to be of immense ecological and socioeconomic significance and hence categorized under ecologically sensitive zon*. As a detritus-based ecosystem, leaf litter from the mangroves provides the basis for adjacent aquatic and terrestrial food web&. It also serves as breeding, feeding and nursery grounds for most of the commercially important finfish and shellfishes, on which thousands of coastal people depend for their livelihood (Manson, et al. 2005). India has approximately 2.7% of the world's mangroves, covering an estimated area of 4,827 sq km. Almost 80% of the mangrove forests are located

along the east coast and the remaining 20% are located on the west coastu.

Benthic macro fauna are important components of coastal food webs. Many fish species in estuarine ecosystems are strongly dependent taxa available that reside on the sediment surface, such as amphipods, mysids and surface deposit feeders=). Studies on benthic diversity, population dynamics and species diversity of mangrove ecosystem and their relationship to the environmental conditions are important for understanding the mangrove ecosystem and their sustainable useB. The climate change and the anthropogenic processes are bringing sequential changes in the coastal marine environment. The understanding of such changes is essential for resource managements. Water quality and benthos characteristics have been investigated in coastal ecosystems around the


Habitat Heteroaeneity and S~atio-Tem~oral Distribution of Macrobenthos in in aTro~ical Estuarine Manarove Ecosystem

world2'j. The health of benthic communities is related to water quality conditions in fringing communities, such as mangroves. Therefore a study on structure, composi-

Sonnertia alba zone - Grows with S. caseolaris spreads in the north western side of the sanctuary (station C4) in a sandy silt sediment.

tion and seasonal distribution of macrobenthic fauna in the intertidal areas of the a tropical mangrove ecosystem at Chorao Island Goa was carried out with reference to environmental characteristics. The result is reported in this communication.

2 Materials and Method

2.1 Study Area The Mandovi estuary lies within latitude 15O09 ' and 15O33 ' North and longitude 73045' and 74014' east in Goa. It is seasonally influenced by semi diurnal tides that flow west- wards and empty into the Arabian Sea at Aguada Bay on central west coast of India, carrying the waste from the adjacent agriculture lands and mining industries, in addition to domestic municipal effluents. The present study Figure 1. Map of Chorao island showing location of study

area of Chorao island (150 32 ' 50.00"N, 730 52 '45.8"E) falls area.

within Mandovi estuary. The Chorao is the largest among the islands of Goa and had great history behind. The west- 3 Sample Collection and Analysis ern part of the island (15030'53"N, 73O51'27"E) is covered with thick mangrove forest and is declared a bird sanctuary Sediment in replicate were collected monthly (March called Salim Ali Bird sanctuary. The total area of the sanc- 2014-February 2015) using a metal quadrant of 25 cm x

tuary is 178 hectares. There are 14 species of mangrove with 25 cm size up to a depth of 15 cm. The macrofauna was

low vegetation having zonation of Rhizophoramucronata, recovered after sieving through a 0.5 mm mesh sieve in Avicenniaofficinalis, Bruguiraapiculata Sonnertia alba the field and the sample was preserved in 5% buffered for-

clearly identifiable within the sanctuary. It provides refuge malin and brought to the laboratory. Animals were sorted to wide variety of avifauna6. The details of stations are pre- and identified to taxa level. Interstitial water from each sent& in Figure 1. The present study was undertaken in station was collected for the dissolved oxygen, salinity four stations, occupied by dominant mangrove forest. The temperature and pH. The environmental parameters were four sites in the fringe of mangrove varied in microclimatic analyzed by following the method given in3. Sediment - - nitche. The distributions of stations include one each in the texture was determined by the method OF. The organic following area: carbon of the sediment was analyzed by wet oxidation

methods. Fauna were identified to the lowest 1 taxonomic Avicennia oficinalis zone - Includes small level with the help of literature; The density of benthos patch of A. marina, A. alba growing in the was expressed as ~o. lm' . edge of the eastern side (station C1) dominated by the silty sand. Rhizophora mucronata zone - Includes

4. Results patches of R. apiculata on the western side (station C2) dominated by muddy sediment.. 4.1 Phvsico-Chemical Characteristics Bruguiera apiculata zone - Forms dense stand

d

Table 1 shows the environmental parameters in the study with B. cylindrica in the central part (station C3)

area; Total rainfall during June 2014-October 2014 was in a sandy muddy sediment.

2810.9 mm and relatively high rain fall was observed in

1 Vol18 (3&4) 1 July-December 2018 1 http:l/www.informaticsjo~~nals.comlindex.php/JEOH/index Journal of Ecophysiology and Occupational Health

S. M. Parvez Al Usmani

Table 1. Seasonal variation in average value of environmental parameters at the study stations

July-August 2014 which is expected. The temperature of interstitial water ranged from 29.5"C-30.2"C in pre- monsoon 22.8 to 26.0 "C in monsoon and 28.2 to 29.0 "C in post-monsoon season. The highest temperature was in April-May and lowest in the monsoon season. Salinity ranged from 29.65-31.55 psu in pre-monsoon 19.66 to 23.57 in monsoon and 27.15 to 29.14 psu in post- monsoon season. Low salinity in monsoon is the result of precipitation and freshwater runoff; Dissolved oxygen was in the range of 3.02 to 3.98 ml in pre-monsoon, 4.36 to 5.18 ml in monsoon and 4.14 to 5.22 ml in post-monsoon season. Stations 1 and 2 exhibited the highest DO values throughout the study period. Low DO concentrations recorded during the summer season which in part is attributable to higher water temperature. pH ranged from were 6.8-7.7 in pre-monsoon, 7.2 to 8.0 in monsoon and 7.4 to 8.2 in post-monsoon season. The lowest pH was at Station C2 during the monsoon season and the highest was 8.2 at Station 1 during the post monsoon season.

4.2 Mangrove Sediment Characteristics The percentage sediment percentage is given in Table 2. Mangrove substratum was mainly composed of sand with an admixture of silt and clay in different proportion (percentage). The average sand fraction ranged between 12.5-60.4 % in pre-monsoon, 18.8-69.2 % in monsoon and 16.2-70.5 % in post monsoon. The fraction of silt was in the range of 28.5-45.5 % in pre-monsoon, 21.4-40.3 % in monsoon and 25.3-47.3 % in post monsoon respec-

tively. Similarly, the fine particles of clay mud was in the range of 11.1-58.9 % in pre-monsoon 8.3-46.7 % in monsoon and 4.2-55.3 % in the post-monsoon. The sediment organic carbon was in the range of 19.4 to 34.6 % at C1, 19.8 to 38.6 % at C2, 19.8 to 37.4 % at C3 and 21.9 to 38.2 % at C4 respectively. Changes in sediment composition were mainly due to transport of sediments by tides and currents. The sediment type was silty sand, muddy, silty clay and sandy silt. The average organic carbon in sediment was similar at all stations with seasonal variations. The values were in the range of 19.4 to 34.6 at C1, 19.8 to 38.4 at C2, 19.8 to 37.4 at C3 and 21.9 to 38.2 myg at C4, respectively (Table 3).

4.3 Macrofaunal Abundance The density and abundance of macrofauna is presented in Table 4. The average density (no./m2) ranged from 280- 1350 at C1, 125-450 at C2, 180-875 at C3 and 225-1025 at C4 respectively. There was a strong seasonal variation in the abundance of macrobenthic fauna. The lowest benthic fauna was observed in the wet monsoon season while the highest was observed in the pre-monsoon season. The summer season is the time of breeding of most of the intertidal fauna in the mangrove environment that is why the highest density was recorded in this period. It is also responsible in the significant variation of density of macrofauna. Station-wise, the maximum density was recorded in the silty sand at C1 and minimum at C2 in muddy sediment. Total wet weight biomass also varied significantly.

Vol 18 (3&4) 1 July-December 2018 1 http:llwww.informaticsjournals.comlindex.phplJEOHIindex Journal of Ecophysiology and Occupational Health 1 82 -

Habitat Heteroaeneity and S~at io-Tem~ora l Distribution o f Macrobenthos in in aTro~ ica l Estuarine Manarove Ecosystem

The larger heavier organisms were responsible for signifi- and isopoda. Decapos were common at most stations cant variation in the standing crop of benthos. and the post larvae of shrimp and crabs were found at

all the stations. The mollusca was mainly represented by Table 2. Average value (%) of sediment characteristic gastropoda and bivalvia. The other group is represented of the mangrove of Chorao by Echiurid worms, nemertean worms, nematode and

Table 3. Total sediment organic carbon (mglg) at the study station

monsoon

Clay

4.4 Faunal Composition

January '1 5

February '1 5

Figure 2 illustrates that there was a preponderance of

4.2

marine forms in the macroinvertebrate assemblage of

21.5

23.5

the present study. The dominant groups representing the

20.0

fauna were polychaeta crustacean and mollusca (Figure 2). The polychaetes contribution was in the range of 42 to 56 % and crustaceans contributed from 28 to 42 %. The mollusca contribution was 11 to 15 %. The other group

22.6

27.2

oligochaeta.

55.3

Table 4. Seasonal abundance in macrofaunal density (no./m2) at the study sites of the Chorao mangrove

18.2

29.2

26.3

8 Polychaet Crustacia 8 Mollusca 8 Others

25.9

27.5

contributed from 5 to 11 %. The polichaetes had the more Fig. 2: Percentage composition of major taxa at the study stations.

representation of deposit feeding and predatory forms while the crustacean had the dominance of amphipoda Figure 2. Percentage composition of major taxa at the study

stations.

83 1 Vol18 (3&4) 1 July-December 2018 1 http:l/www.informaticsjo~~nals.comlindex.php/JEOH/index Journal of Ecophysiology and Occupational Health

S. M. Parvez Al Usmani

5. Discussion

5.1 Water Quality Parameters Mangrove sediments are highly anaerobic rich in sulphide and organic matterE. Variations observed in water quality parameters at the study stations can be attributed to hydrographical patterns in climate and biological activityu. Generally, water temperature is influenced by the intensity of solar radiation, evaporation, freshwater influx and cooling and mixing up with ebb and flow from adjoining waters of Mandovi estuaryu. In the present study, summer peaks and monsoonal troughs in water temperatures were similar to those reported by others". Salinity acts as a limiting factor in the distribution of liv- ing organisms and its variation caused by dilution and evaporation influences faunal distribution in the intertidal zone". In the present study, salinity at all the four stations was high in summer and low in the monsoon season indicating that variation in salinity at study sites was affected by freshwater runoff entering the creek systems of estuarine mangrove at Chorao. The high organic carbon associated with finer sediment is a common feature observed by others also=.

Higher pH observed in summer season could be attributed to the removal of CO, by the photosynthetic organisms and the lower pH during monsoon season could be due to the dilution of saline water by freshwater inflow'-. The relatively low DO values observed in the summer are attributable to the prevalence of high saline waters in the mangrove channels from Mandovi estuary as well as fluctuations in temperature and salinity. By contrast sediments with a mixture of organic matter, sand and clay, but low sulphide, seem to support higher abun- dances of benthic marcofauna. Distribution and ecology of benthic communities in relation to station and season of the present study showed influence of the environmental parameters. This is in support of earlier studiesm.

The mangrove fauna basically derive from adjacent estuarine and marine environment and its stratification depends mainly on tidal inundation by saline water'. Limited quantitative information has been published on estuarine mangrove habitats of Goa including macroinvertebrate fauna and their relationship to environmental factors. This study provides a baseline for the distribution, abundance and diversity of benthic macroinvertebrate fauna of Chorao mangroves in Goa. The occurrence of different taxa in the present study is similar to that

observed by others on the east and west coast of Indiam. However the macrobenthicdensity observed in this study (140-1113 no./m2) were higher than that reported by2 in Mandovi-Zuari estuarine system. The densities were comparable to those observed in the other mangrove swamps of Indiax. The high densities recorded in the pre- and post-monsoon season could be due to high temperatures and turbidity coupled with stable environmental conditions2. Post-monsoon season (Nov-Feb) peaks in density and higher abundance in pre-monsoon were also reported for the west coast of Indiaz. These studies have reported the dominance of polychaetesover mollusks in mangrove fauna due to silty clay and muddy substratum. Soft mangrove substrates favor tube dwellers over diggers and burrowing animals, such as bivalves. The present study corroborates it. The high diversity of polychaete indicates favorable ecological conditions that exist in mangrove ecosystems (Ajmal and Murugesan; 2005).

6. Conclusions This study provides insights into the effects of a range of environmental parameters on macro benthic communities of Chorao mangroves in Goa. Altogether 76 species of benthic macroinvertebrate fauna, belonging to five major groups, were identified at the four sampling stations. Station 1 was dominated by sandy sediment, high salinity, high DO and relatively low sulphide levels. The region displayed the high abundance and standing crop biomass. The temporal distribution of benthic macro invertebrate fauna exhibited the highest density during pre-monsoon season. The decrease of benthos during the monsoon may be attributable to low temperatures and salinities. Considering the importance of flora and fauna of mangrove and constant threat regular monitoring of this ecosystem is required to assess its health.

7. Acknowledgement The author is thankful to the Principal Dr. D. B. Arolkar for encouragement.

8. References 1. Alongi DM, Christoffersen P. Benthic infauna and organ-

ism-sediment relations in a shallow, tropical coastal area: Influence of out welled mangrove detritus and physical disturbance. Mar Eco Prog Ser. 1992; 81:229-45. https://doi.org/l0.3354/meps081229

Vol18 (3&4) 1 July-December 2018 1 http:llwww.informaticsjournals.comlindex.phplJEOHIindex Journal of Ecophysiology and Occupational Health 1 84 -

Habitat Heteroaeneity and S~atio-Tem~oral Distribution of Macrobenthos in in aTro~ical Estuarine Manarove Ecosystem

2. Ansari, ZA, Ingole BS, Banerjee G, Parulekar AH. Spatial and temporal changes in benthic macrofauna from Mandovi and Zuari estuaries, west coast of India. Indian J Mar Sci. 1986; 15:223-9.

3. APHA. Standard methods for the examination of water and wastewater 20th Ed. Washington, DC: APHA, 1998.

4. Bandekar PD, NaikUG, Hara SB. Diversity status of benthic macro polychaetes species in estuarine region of Karwar, West Coast of India. International Journal of Fisheries and Aquatic Studies. 2017; 5(1):216-9.

5. Bhat UG, Studies on Benthos of Kali estuary, Karwar. [Ph.D. Thesis]. Marine Biol, Karnatak University. 1984.

6. Borges SD, Shanbhag AB. Additions to the avifauna of Goa, India. Journal of the Bombay Natural History Society. 2007; 104(1):98-101.

7. Chong VC, Sasekumar A, Leh MUC, Cruz RD. The fish and prawn communities of a Malaysian coastal mangrove system with comparisons to adjacent mud flats and inshore waters. Estuarine, Coastal and Shelf Science. 1990; 31:703-22. https://doi.org/lo. 1016/0272-7714(90)90021 -I

8. El Wakeel SK, Riley JP. The determination of organic carbon in marine muds. J Cons Perm Int Explo Mer. 1956; 22: 180-3. https://doi.org/lo. 1093/icesjms/22.2.180

9. Folk RL. Petrology of sedimentary rock. Hemphills Ausdty in Texas. 1968. 170.

10. Jagtap TG, Chavan V, Untawale AG. Mangrove ecosystems of India: A need for protection. Ambio. 1993; 22(4):252-4.

11. Jagtap TG. Ecological studies in relation to the mangrove environment along the Goa Coast, India. [Ph. D. Thesis]. Shivaji University, Kolhapur. 1985.

12. Jagtap TG. Seasonal distribution of organic matter in mangrove environment of Goa. Indian Journal of Marine Sciences. 1987; 16:103-6.

13. Kathiresan K. A review of studies on Pichavaram mangrove, southeast India. Hydrobiol. 2000; 430:185-205. https://doi.org/lo. 1023lA: 1004085417093

14. Kathiresan K, Bingham BL. Biology of mangroves and mangrove ecosystems. Advances in Marine Biology. 2001; 403-25 1. https://doi.org/lo. 1016/S0065-288 1 (01)40003-4

15. Khan AS, Murugesan P. Polychaete diversity in Indian estuaries. Indian J Mar Sci. 2005; 34(1): 114-9.

16. Kumar RS. Distribution of organic carbon in the sediments of Cochin mangroves, south west coast of India. Indian J Mar Sci. 1996; 2556-61.

17. Kumar RS. A review of biodiversity studies of soil dwelling organisms in Indian mangroves. Zoos Print. 2000; 15(13):221-7. https://doi.org/lo. 11609/JoTT.ZPJ. 15.3.221-7

18. KUMAR RS. Macrobenthos in the mangrove ecosystem of Cochin backwaters, Kerala (Southwest coast of India). Indian J Mar Sci. 1996; 2456-6 1.

19. Kumar RS. Intertidal zonation and seasonality of benthos in a tropical mangrove. Int J Ecol Environ Sci.2001; 2:199- 208.

20. Kumar RS. Vertical distribution and abundance of sediment dwelling macro invertebrates in an estuarine mangrove biotope, southwest coast of India. Indian J Mar Sci.1997; 26:26-30.

21. Kurnar PS, Khan AB. The distribution and diversity of benthlc macroinvertebrate fauna in Pondicherry mangroves, India. AquaticBiosystem. 201 3;9: 1- 15. https://doi.org/lo. 1 18612046- 9063-9- 15 PMid:23937801 PMCid:PMC375 1066

22. Onuf CP, Teal JM, Valiela L. Interactions of nutrients, plant growth and herbivory in a mangrove ecosystem. Ecology. 1997; 58:514-26. https://doi.org/l0.2307/1939001

23. Parulekar A. Benthic fauna of mangrove environment. Conservation of Mangrove Forest Genetic Resources: A training manual. Sanjay VD, Balaji V, eds. CRSARD; Chennai, 128. 1994.

24. Patra KC, Bhunia AB, Mitra A. Ecology of macrobenthos in a tidal creek and adjoining mangroves in West Bengal, India. Environ Ecol. 1990; 11 8539-47.

25. Saravanan KR, Ilangovan K, Khan AB. Floristic and macro faunal diversity of Pondicherry mangroves, South India. TroEco. 2008; 49(1):91-4.

26. Samidurai K, Saravanakumar A, Kathiresan K. Spatial and temporal distribution of macrobenthos in different mangrove ecosystems of Tamil Nadu Coast, India. EnviroMoni Asses. 2012; 184(7):4079-96. https://doi.org/l0.l007/ ~10661-011-2245-x PMid:21833734

27. Thilagavathi B, Varadharajan D, Babu A, Manoharan J, Vijayalakshmi S, Balasubramanian T. Distribution and diversity of macrobenthos in different mangrove ecosystems of Tamil Nadu Coast, India. J Aquaculture Research Development. 20 13; 4:6- 12.

85 1 Vol 18 (3&4) 1 July-December 2018 1 http:Ilwww.informaticsjournals.comlindex.phplJEOHlindex Journal of Ecophysiology and Occupational Health



Monitoring of Nuclear Abnormality Frequencies as Indicators of Environmental Pollution in Peripheral

Erythrocytes of Labeo rohita Reared in Lakes of Bangalore

Nazima Noor and Bela Zutshi*1Department of Zoology, Bangalore University, Bangalore – 560056, Karnataka, India; [email protected]

Keywords: Blood, Erythrocytes, Genotoxicity, Labeo rohita, Pollutants

AbstractThe present study was aimed to evaluate genotoxicity in peripheral erythrocytes of Labeo rohita reared in two lakes viz., Vengaiah lake - sewage polluted (Lake A) and Yellamallappa Chetty lake - industrially polluted (Lake B) of Bangalore. To assess the micronuclei and nuclear abnormalities in such erythrocytes, blood samples were collected from heart of the freshwater fish, L. rohita anesthetized by MS222. The results were compared with the fish reared in the Hebbal fish farm (Control). The data revealed significantly high frequencies of erythrocytic abnormalities including nuclear as well as cytoplasmic in the fish blood sampled from lake B when compared to those of lake A. Such abnormalities in erythrocytes of fish varied seasonally also with summer exhibiting maximum deformities which can be attributed to the presence of genotoxic pollutants in the selected water bodies. The values were statistically significant at P<0.0001.


1. IntroductionSurface waters, such as lakes, rivers, and seas contain com-plex mixtures of pollutants including genotoxic compounds due to the anthropogenic action, which cause adverse effects on public health and aquatic ecosystems26. Aquatic environ-ment serves as convenient repositories for man’s biological and technological wastes and current awareness of the potential hazards of pollutants in the aquatic environment has stimu-lated much interest in the use of fish as indicators due to their position in the trophic chain, their sensitivity to low concen-trations of genotoxic substances and their ability to metabolize xenobiotics and accumulate pollutants40. Hematological study is important in toxicological research because a hematologi-cal alteration is a good method for rapid evaluation of the chronic toxicities of a compound. Blood parameters are useful for the measurement of physiological disturbances in stressed

fish and thus provide information about the level of damage in the fish28. A thin epithelial membrane separates fish blood from the water and any unfavorable change in the water body is reflected in the blood22. The study of blood characteristics may corroborate important subsidies of diagnoses and prog-noses of morbid conditions in fish populations and therefore, contribute to better comprehending comparative physiology, phylogenetic relations, feeding conditions and other ecological parameters28.

Among the currently available procedures, micronuclei and nuclear abnormalities assays are the most widely applied methods due to its proven suitability for fish species7,21. Nuclear abnormalities, such as micronuclei and other nuclear malfor-mations are considered good indicators of cytotoxicity and genotoxicity, respectively21. For the determination of genotoxic effect in fish, the micronucleus test as well as the study of the abnormal shape of nuclei is a suitable measure with which the

Journal of Ecophysiology and Occupational Health87 Vol 18 (3&4) | July-December 2018 | http://www.informaticsjournals.com/index.php/JEOH/index

Monitoring of Nuclear Abnormality Frequencies as Indicators of Environmental Pollution in Peripheral Erythrocytes of Labeo rohita Reared in Lakes of Bangalore

presence or absence of genotoxins can be detected in water. The detection of MN and NAs in fish helps us to assess the sta-tus of water quality as well as the health of a particular species and any potential risk it might have after consumption37.

Schroder (1966) studied the formation of micronuclei in mammalian bone marrow cells for the first time subsequently this assay was developed by Schmid (1975) in mammalian systems. The MN are also known as Howell-jolly bodies in mammals. Like mammalian species, MNT has also been adopted to study genotoxicity in fishes. The formation of mor-phological nuclear alterations (NAs), was first described in fish erythrocytes by Carrasco et al. (1990). The unique information offered by MNT as a bioindicator for chromosomal aberra-tions is not available from other methods such as the integrated effect of a variety of environmental stresses on the health of an organism and the population, community, and ecosystem; and the effectiveness of remediation efforts in decontaminat-ing waterways42. NAs including blebbed, lobed and notched nuclei and binucleated cells, have been used by several authors as possible indicators of genotoxicity9.

The present investigation was conducted to analyse the blood samples for erythrocytic abnormalities in edible fish Labeo rohita reared in fresh water lakes of Bangalore as a con-sequence of variation in physico chemical parameters of water including detected trace metals.

2. Materials and Methods

2.1 Study AreaBangalore also called as Bengaluru is the capital of Karnataka state in South India (Figure 1). It is located at 12.97°N 77.56°E and covers an area of 741 km2. The two lakes, Vengaiah lake (Lake A - Area 65 acres; depth 8-10 feet) and Yellamallappa Chetty lake (Lake B - Area 110 ha; depth 10-12 feet) situated near Krishnarajpuram - Hoskote taluk, Bangalore District, Karnataka were selected for the study (Figure 2). Lake A received domestic sewage from an adjacent storm-water drain and lake B those of effluents from pharma-industry and other sources. Hebbal fish farm, which is maintained by the fisheries department was taken as reference site (Control).

2.2 Analysis of Water SamplesWater along with the test fish were sampled from Control site, Lake A and Lake B during early morning hours (6:30-7:30

a.m.). The physico-chemical parameters like temperature, pH, BOD, COD, DO, TDS, conductivity, acidity, alkalinity, phos-phates, sulphates, nitrates and trace metals such as mercury, lead, aluminium, cadmium, etc of the water samples collected from control, lake A and B were determined by following stan-dard methods by APHA et al., 2005 and atomic absorption spectrophotometry (USEPA, 1983). The values obtained were compared with the Bureau of Indian Standards BIS:10500-1991 (Revised 2012) for lakes. Water quality index was calculated by following Brown et al., 1972; Chatterjee and Razi uddin, 2000.

2.3 Analysis of Fish Blood SamplesLabeo rohita was selected as animal model for the present study. Blood was drawn out by Cardiac puncture with a hepa-rinized syringe of test fish previously anesthetized in MS222 by stabbing body wall exactly in midline from the posterior mar-gin of opercular cover and directed dorso-caudally at an angle of 450,24. A thin blood smear was spread on a clean dry slide (2x4x1mm), fixed in methanol and was left to air-dry. It was then stained with Leishman’s stain32. The sample’s smear was inspected using Zeiss Axidskop Plus microscope connected with camera and computer, equipped with image viewing anal-ysis system. 1,000 erythrocytes of blood smear sampled from fish of lake A, lake B and control site were viewed and classi-fied. For the scoring of micronuclei, the criterion was adopted from Fenech et al. (2003). MN if present should have similar staining as the main nucleus. They should be separated from or marginally overlap with main nucleus as long as there is clear identification of the nuclear boundary. Cellular and nuclear anomalies observed were registered and photographed, and their frequency was calculated.

3. Statistical AnalysisStatistical analysis was carried by using MS Excel and statis-tical software - Graphpad prism 6.05 to evaluate the physico chemical parameters with respect to three water bodies and erythrocytes of fish. Mean of the water and frequency of eryth-rocytic abnormalities [size (n = 6)] and standard deviation (mean ± SD) were calculated to quantify their variability which was followed by one way ANOVA to compare significant mean differences of the above mentioned groups. This was followed by Tukey’s post-hoc test to compare pair of groups mean of water parameter in water and with erythrocytic abnormalities in fish blood of control group, lake A and lake B. p value at a

Journal of Ecophysiology and Occupational HealthVol 18 (3&4) | July-December 2018 | http://www.informaticsjournals.com/index.php/JEOH/index 88

Nazima Noor and Bela Zutshi

Figure 1. Map of India showing location of Karnataka state and the capital city, Bangalore.



significant level of p<0.05 or less indicated significant relation-ship within variables. Pearson’s correlation coefficient between physico chemical parameters of water sampled from control site and lakes (A & B) and frequency of erythrocytic abnormal-ities in blood samples of fish collected from these water bodies was also studied.

4. Results and DiscussionPhysico-chemical parameters are the basic parameters to determine whether a lake is polluted or non polluted. Physico-chemical parameters of water sampled from control site were compared with lake A and lake B and inturn with the standard BIS: 10500-1991 (Revised 2012) taking into consideration the three different seasons viz., winter, summer and rainy season. The data was statistically analyzed and is represented in table I & II showing the significant mean differences at p<0.001 and 0.01. The data revealed high level of pollution in lake B (dur-

ing all seasons) when compared to lake A, control site and BIS standard. The high levels of temperature, total suspended solids, chemical oxygen demand, biological oxygen demand, conductivity, turbidity, alkalinity and of trace metal content such as, aluminium, cadmium, copper, iron, lead and mer-cury was recorded in water samples of lake B. These results might be due to the discharge of industrial effluents from the pharma industry present on the banks of lake B and the agri-cultural runoff, idol immersion during festival season and discharge of domestic sewage and solid waste through various sources into the water body. The water analysis of lake B indi-cated significantly high level of trace metals along with other water parameters which were recorded above the BIS limits. This rendered the water quality to be very poor as per water quality index (WQI) (Table III) suggested by Chatterji and Raziuddin (2002) (Table IV) and unsuitable for drinking pur-pose. Heavy metal contamination may have devastating effects on the ecological balance of the recipient environment and on

Yellamallappa Chetty lake (Lake B)

Vengaiah lake (Lake A)

Figure 2. Representation of sampling location: Vengaiah lake (Lake A) and Yellamallappa Chetty lake (Lake B).



Para

met

ers

Stan

dard

sBI

S: 1

0500

-19

91(R

evis

ed 2

012)

Con

trol

site

Win

ter s

easo

nSu

mm

er se

ason

Rai

ny se

ason

Lake

ALa

ke B

Lake

ALa

ke B

Lake

ALa

ke B

Tem

pera

ture

22

- 28

26 ±

0.6

321

± 0

.89

(-19

.23)

22 ±

1.2

6(-

15.3

8)26

± 0

.63

(0.0

0)28

± 1

.26

(7.6

9)22

± 1

.26

(-15

.38)

24 ±

2(-

7.69

)

Col

our

5 - 2

53.

1 ±

0.63

4.8

± 0.

63(5

4.05

)7.

3 ±

0.84

(136

.22)

4.3

± 0.

63(3

9.46

)6.

1 ±

0.49

(98.

38)

5.1

± 0.

45(6

3.78

)8.

6 ±

0.39

b

(178

.38)

Odo

urU

OB

UO

BU

OB

Fish

yU

OB

Fish

yU

OB

Fish

y

pH V

alue

6.5-

8.5

7.87

± 0

.08

7.09

± 0

.33

(-9.

89)

7.22

± 0

.69

(-8.

26)

7.65

± 0

.16

(-2.

75)

6.85

± 0

.74

(-12

.92)

7.65

± 0

.30

(-2.

75)

7.34

± 0

.54

(-6.

67)

Turb

idity

5

- 20

7.8

± 0.

1524

± 2

(207

.69)

33 ±

2.6

2(3

22.0

1)21

± 0

.89

(169

.23)

34.2

± 2

.14

(338

.03)

31 ±

1.2

6(2

97.4

4)42

.7 ±

2.7

3b

(447

.01)

Con

duct

ivity

300

644

± 64

.70

654

± 12

.61

(1.6

3)98

8 ±

1.64

b

(53.

42)

837

± 42

.80

(29.

98)

1207

± 3

5.15

b

(87.

57)

704

± 5.

47(9

.43)

1087

± 2

.07b

(68.

80)

TDS

500

- 200

042

0 ±

7.69

523

± 32

.86

(24.

62)

793

± 2.

42b

(89.

04)

750

± 1.

41(7

8.71

)98

5 ±

2.93

b

(134

.75)

570

± 3.

56(3

5.90

)72

4 ±

3.25

b

(72.

48)

Tabl

e 1.

Phy

sico

-che

mic

al p

aram

eter

s of w

ater

sam

pled

from

Heb

bal fi

shfa

rm (C

ontr

ol si

te),

Veng

aiah

lake

(Lak

e A

) and

Yel

lam

alla

ppa

Che

tty

lake

(Lak

e B)

dur

ing

win

ter,

sum

mer

and

rain

y se

ason



TSS

100

92 ±

0.8

216

2 ±

0.55

(76.

18)

287

± 1.

64b

(212

.55)

150

± 0.

82(6

4.00

)26

0 ±

4.73

b

(183

.64)

180

± 2.

10(9

6.36

)37

6 ±

2.88

b

(310

.55)

Aci

dity

20

32 ±

0.5

231

± 0

.82

(-1.

05)

76 ±

2.3

4b

(138

.95)

48 ±

1.2

1(5

0.53

)95

.7 ±

1.8

6b

(202

.11)

33.2

± 1

.17

(4.7

4)70

.4 ±

1.0

2b

(122

.37)

Tota

l alk

alin

ity

200

- 600

202

±1.3

822

1 ±

0.82

(9.8

4)47

0 ±

6.79

b

(133

.17)

290

± 1.

60(4

4.00

)54

4 ±

11.0

7b

(174

.86)

215

± 0.

84(6

.45)

464

± 2.

32b

(130

.36)

Nitr

ates

45

- 10

02.

13 ±

0.2

83.

17 ±

0.1

7(4

8.94

)5.

2 ±

0.30

(145

.89)

2.25

± 0

.20

(5.8

7)3.

87 ±

0.1

0(8

1.68

)2.

58 ±

0.1

6(2

1.38

)4.

8 ±

0.10

(123

.18)

Sulp

hate

s 20

0 - 4

0062

± 0

.52

128

± 0.

55(1

06.7

6)25

3 ±

1.75

b

(310

.81)

103

± 1.

21(6

6.49

)21

0 ±

0.52

b

(241

.08)

154

± 0.

55(1

48.9

2)27

6 ±

0.55

b

(346

.76)

Tota

l ph

osph

orus

-

0.35

± 0

.01

2.08

± 0

.01

(500

.00)

3.98

± 0

.01

(104

6.63

)1.

02 ±

0.0

1(1

92.7

9)2.

42 ±

0.0

1(5

98.5

6)1.

75 ±

0.0

1(4

03.8

5)3.

52 ±

0.0

2(9

15.8

7)

DO

4

- 63.

7 ±

0.05

3.8

± 0.

08(3

.20)

1.7

± 0.

10(-

54.3

4)3.

7 ±

0.05

(0.0

0)1.

2 ±

0.08

(-68

.04)

4.3

± 0.

09(1

7.81

)2.

8 ±

0.08

(-24

.20)

BOD

2

- 66

± 0.

7621

± 1

.17

(262

.32)

97 ±

2.0

7b

(157

8.26

)24

± 1

.21

(323

.19)

113

± 1.

33b

(186

8.12

)18

± 0

.98

(210

.14)

92 ±

1.2

1b

(150

5.80

)

CO

D

200

76 ±

1.7

215

3 ±

1.51

(102

.20)

377

± 1.

37b

(396

.70)

126

± 2.

25(6

6.59

)37

4.7

± 2.

88b

(394

.07)

164

± 0.

89(1

16.2

6)48

4 ±

1.67

b

(538

.24)

Valu

es a

re e

xpre

ssed

in m

g/l e

xcep

t -Te

mpe

ratu

re -

(o C),

Col

our -

(Pt-

Co

scal

e), O

dour

, pH

, Tur

bidi

ty -

(NTU

) and

Con

duct

ivity

- (m

mho

/cm

). Va

lues

are

exp

ress

ed a

s mea

n ±

SD w

here

, n =

6. V

alue

s in

pare

nthe

sis re

pres

ent p

erce

nt ch

ange

(%).

UO

B -

U

nobj

ectio

nabl

e. Th

e su

pers

crip

ts a

, b, c

and

d in

dica

te st

atist

ical

mea

n di

ffere

nces

at p

< 0

.000

1, 0

.001

, 0.0

1 an

d 0.

05 re

spec

tivel

y.

Tabl

e 1 C

ontin

ued



Para

met

ers

Stan

dard

sBI

S: 1

0500

-199

1(R

evis

ed 2

012)

Con

trol

site

Win

ter s

easo

nSu

mm

er se

ason

Rai

ny se

ason

Lake

ALa

ke B

Lake

ALa

ke B

Lake

ALa

ke B

Ars

enic

0.

050

00.

008

± 0.

001

00.

003

± 0.

001

0.00

10.

0027

Cop

per

0.05

– 1

.50.

013

0.03

(133

.77)

0.39

± 0

.01

(291

2.99

)0.

03(1

33.7

7)0.

32 ±

0.0

1(2

419.

48)

0.02

8(1

18.1

8)0.

25 ±

0.0

1(1

874.

03)

Zinc

5

- 15

0.54

± 0

.02

2.88

(438

.32)

3.34

± 0

.03

(523

.99)

1.88

± 0

.01

(251

.09)

2.68

± 0

.01

(401

.56)

1.67

± 0

.01

(212

.46)

3.12

± 0

.01

(482

.87)

Alu

min

ium

0.

03 –

0.2

00.

043

± 0.

001

3.9

± 0.

055

0.06

7 ±

0.00

23.

7 ±

0.08

9 b0.

067

± 0.

001

3.7

± 0.

089b

Cad

miu

m

0.01

0.00

10.

04 ±

0.0

1(5

150.

00)

0.18

4(2

7550

.00)

0.04

± 0

.01

(515

0.00

)0.

124

(184

50.0

0)0.

04 ±

0.0

1(5

150.

00)

0.10

3(1

5375

.00)

Iron

0.

3 - 1

0.04

± 0

.008

0.14

± 0

.024

(269

.57)

3.92

± 0

.056

(101

13.0

4)0.

13 ±

0.0

22(2

43.4

8)3.

68 ±

0.0

04(9

504.

35)

0.11

± 0

.008

(191

.30)

3.02

± 0

.006

(777

8.26

)

Lead

0.

050.

004

± 0.

001

0.04

± 0

.012

(947

.62)

0.51

(144

71.4

3)0.

04 ±

0.0

12(9

47.6

2)0.

37 ±

0.0

20(1

0376

.19)

0.04

± 0

.008

(995

.24)

0.23

(647

1.43

)

Mer

cury

0.

001

00

0.03

± 0

.01

00.

028

00.

023

Valu

es a

re e

xpre

ssed

in m

g/l e

xcep

t -Te

mpe

ratu

re -

(o C),

Col

our -

(Pt-

Co

scal

e), O

dour

, pH

, Tur

bidi

ty -

(NTU

) and

Con

duct

ivity

- (m

mho

/cm

). Va

lues

are

ex

pres

sed

as m

ean

± SD

whe

re, n

= 6

. Val

ues i

n pa

rent

hesis

repr

esen

t per

cent

chan

ge (%

). U

OB

-

Uno

bjec

tiona

ble.

The

supe

rscr

ipts

a, b

, c a

nd d

indi

cate

st

atist

ical

mea

n di

ffere

nces

at p

< 0

.000

1, 0

.001

, 0.0

1 an

d 0.

05 re

spec

tivel

y.

Tabl

e 2.

Tra

ce m

etal

s in

wat

er sa

mpl

ed fr

om H

ebba

l fish

farm

(Con

trol

site

), Ve

ngai

ah la

ke (L

ake

A) a

nd Y

ella

mal

lapp

a C

hetty

lake

(Lak

e B)

dur

ing

win

ter,

sum

mer

and

rain

y se

ason

.



Water bodySeasons

Winter Summer Rainy

Control 1.07

Lake A 36.45 36.52 36.53

Lake B 2802.19 2570.12 2110.99

Table 3. Water quality index (WQI) of control site, lake A and lake B during three seasons

Water Quality Index level Water quality status

0 – 25 Excellent water quality

26 – 50 Good water quality

51 – 75 Poor water quality

76 – 100 Very poor water quality

>100 Unsuitable for drinking

Table 4. Water Quality Index (WQI) and status of water quality (Chatterji and Raziuddin 2002)

the diversity of aquatic organisms11 and are detrimental to the aquatic inhabitants, including fishes27.

Micro nuclei test is recommended to be conducted as part of the monitoring protocols in aquatic toxicological assessment programs26, 39. The in vivo micronuclei frequency assay has been widely used as a technique for genotoxicity monitoring of polluted aquatic media and in the screening for the presence of toxic compounds suspected to be genotoxic29, 45. Morphological alterations in the nuclear envelope in erythrocytes of fish as described by Carrasco et al. (1990) are considered indicators of genotoxic damage and constitutes a complementary analysis to the scoring of micronuclei.

Change in the general structure of erythrocytes such as deformation and swelling along with few nuclear abnormalities

was observed in the blood samples of fish from lake A (Figure 1a - 1b). Large number of nuclear abnormalities which can be classified as blebbed, notched, lobed6, eight shaped14 and pear shaped nucleus along with ruptured nuclear membrane and oozed out nuclear mass (Figure 1a - 1c) were noted from fish of lake B and are in similar lines with Moharram et al. (2011). They recorded the high percentage of deformed erythrocytes, pear and tear shaped erythrocytes, swelling erythrocytes with fading cytoplasm and indicated a decrease in haemoglobin con-tent of Siganus rivulatus due to polluted water from Egyptian Eastern Mediterranean coast. Cytoplasmic abnormalities were also observed and classified into granulated, ruptured and vacuolated cytoplasm and irregularities in cell membrane of erythrocytes during the present piece of work. Deformed



Figure 3a. Erythrocytic abnormalities : a- Normal RBC, b- Deformed RBC, c- blebbed nucleus, d- Notched nucleus and e- Lobed nucleus of Labeo rohita sampled from Hebbal fishfarm (control site), Vengaiah lake (lake A) and Yellamallappa Chetty lake (lake B).



Figure 3b. b. Erythrocytic abnormalities : f- Eight shaped nucleus, g- Pear shaped nucleus, h- Ruptured nuclear membrane and i-oozing of nuclear mass of Labeo rohita sampled from Hebbal fishfarm (control site), Vengaiah lake (lakeA) and Yellamallappa Chetty lake (lake B).



Figure 3c. Erythrocytic abnormalities : j- Disintegrated nucleus and nuclear material, k- Granulated cytoplasm, l- Ruptured cytoplasm and m-Vacuolated cytoplasm of Labeo rohita sampled from Hebbal fishfarm (control site), Vengaiah lake (lake A)and Yellamallappa Chetty lake (lake B).



RBCs were also detected in tench on short term exposure to cadmium by Witeska et al. (2006) and due to environmental pollution by Pacheco and Santos (2002). NAs recorded in the present investigation were in conformity with the reports by Furnus et al. (2014) on native fish from Parana river, Argentina. Juliana de Souza et al. (2012) observed two other NA in cat-fish Cathorops spixii (Ariidae) sampled from different sites on the south-eastern Brazilian coast. They named these NAs as “others” (OT); “heart” or “clover-leaf ” shaped which were not classified by Carrasco et al. (1990). Such Nuclear abnormalities (NAs) are a consequence of exposure to environmental and chemical contaminants of genotoxic action7.

In the present investigation, erythrocytes of fish sampled from control site were in good condition as the water param-eters were within BIS limits. Erythrocytic abnormalities were frequent in samples from lake B (winter -51.87 ± 1.9, summer - 58.34 ± 2.3 and rainy 43.08 ± 2.2) when compared to control site (0.495 ± 0.11) and lake A (winter - 11.61 ± 1.65, sum-mer - 8.62 ± 1.24 and rainy 6.19 ± 0.80) (Table V). Maximum abnormalities were observed in blood of fish during summer season in lake B where as such abnormalities were observed during winter followed by summer and rainy season in lake A. Such variations in frequency of erythrocytic abnormalities within the three water bodies are clearly attributed to the type and level of pollution during all the seasons throughout the year.

Heavy metals induced such changes in fishes which are not reversed and caused cytotoxic damage resulting in death of fishes44. Erythrocyte swelling, poikilocytosis, vacuolation, amitosis, deformation, and deterioration of cell membranes in Barbus conchonius exposed to chromium were reported by Gill and Pant (1985). Nuclear aberrations such as chromatin condensation, nuclear puffs, and chromatin leakage in the same fish species subjected to cadmium intoxication was again reported by Gill and Pant (1987). Karuppasamy et al. (2005) also reported similar observations as increased fragility, rup-ture of erythrocyte membrane, and hemolysis and increase in the frequency of nuclear and cytoplasmic abnormalities in Channa punctatus when exposed to sublethal concentration of cadmium. Nuclear abnormalities (NA) in erythrocytes are con-sidered a useful parameter for assessing the genotoxic effects of environmental pollutants in fish, and have been applied successfully in various species such as Anguilla Anguilla, Dicentrarchus labrax, Oncorhynchus mykiss and Centropomus parellelus when exposed to polycyclic aromatic hydrocarbons

and resin acids30, β-naphthoflavone17, metals3 and from aquatic polluted environment21 respectively.

In fish, both micronuclei and erythrocytic nuclear abnor-malities appear spontaneously and their frequency may be seasonally dependent36. The present study also showed simi-lar results. Variation was observed in the frequency of nuclear abnormalities from summer to winter and rainy since, excess evaporation during summer might cause the pollutants to concentrate in water followed by winter which had a post rain effect (Figure 1a - 1c and Table V). During rainy season influx of rain water and outpouring of lakes resulted in lowering of concentration of pollutants. These results were in agreement with Ergene et al. (2007) who reported increase in frequencies of nuclear anomalies such as irregular nucleus shape, vacuola-tion, binuclei and micronuclei indicating increase in genotoxic effects in fish exposed to water pollution; fluctuation of such abnormalities was reported by Strunjak-Perovic et al. (2009).

Nuclear abnormalities to be considered as precursors of micronuclei was suggested by Walia et al. (2013). Guner and Muranh (2011) reported differences in the erythrocyte micronucleus frequencies to be related to cell kinetics and replacement. They explained that individual and combination exposure of fishes to heavy metals led to an accumulation in the body but this accumulation did not assure an increase in MN frequency in peripheral blood erythrocytes. Since Cu and Cd induced only NAs when used alone and in combination but did not induce MN in Gambusia affinis. Similar results were observed in the present study in peripheral blood erythrocytes of rohu fish with a high frequency of nuclear and cytoplasmic abnormalities but absence of micronuclei in lake B which was polluted due to the presence of metals like aluminium, cad-mium, copper, iron, lead and mercury.

Further, Von Sonntag (1987) and Steenken (1989) hypoth-esized that these abnormalities arise due to damage caused to the genetic material by free radical produced under oxidative stress due to the toxicants. Ventura et al. (2008) and Fernandes et al. (2007) reported aneuploidy, an abnormality caused by aneugenic actions of toxicants resulting in formation of bi-nucleated cells and notched nuclei due to tubulin failure and mitotic fuses as was also seen frequent in the rohu fish in the present work. Elaborate sequence of cellular degradation under the impact of toxicants caused hypoxic conditions which resulted in depression of ATP may lead to abnormal shape of erythrocytes was reported by Ateeq et al. (2002). Various eryth-rocytic abnormalities, Heinz bodies, poikilocytosis, clumped



Veng

aiah

lake

(Lak

e A

) and

Yel

lam

alla

ppa

Che

tty la

ke (L

ake

B) d

urin

g w

inte

r, su

mm

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Eryt

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ab

norm

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es (N

ucle

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and

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te

Win

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Rain

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Lake

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Lake

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Lake

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Bleb

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0.28

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0.05

2.33

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8.2

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07 a

2.33

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9.85

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.18 a

1.83

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7.53

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Not

ched

nuc

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0.21

± 0

.02

2.29

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.07 a

8.87

± 0

.05 a

3.5

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06 a

9.2

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14 a

2.05

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8.58

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Lobe

d nu

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01.

03 ±

0.0

5 a4.

8 ±

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a1.

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5 a5.

305

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21 a

0.8

± 0.

09 a

3.5

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06 a

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t sha

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06 ±

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48 ±

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55 ±

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3.38

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Rupt

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57 ±

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35 ±

0.1

2 a0

8.33

± 0

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05.

87 ±

0.0

5 a

Cyt

opla

smic

gra

nule

s and

ru

ptur

e0

05.

45 ±

0.0

5 a0

5.38

± 0

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02.

57 ±

0.0

5 a

Cyt

opla

smic

vac

uole

s 0

03.

52 ±

0.0

7 a0

3.47

± 0

.05 a

02.

53 ±

0.0

5 a

Tota

l abn

orm

aliti

es0.

495

± 0.

1111

.61

± 1.

65 a

51.8

7 ±

1.9 a

8.62

± 1

.24 a

58.3

4 ±

2.3 a

6.19

± 0

.80 a

43.0

8 ±

2.2 a

Valu

es a

re e

xpre

ssed

in %

and

as m

ean

± SD

whe

re, n

= 6

. The

supe

rscr

ipts

a, b

, c a

nd d

indi

cate

stat

istic

al m

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diffe

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es at

p <

0.0

001,

0.0

01, 0

.01

and

0.05

re

spec

tivel

y.

Tabl

e 5.

Fre

quen

cy (%

) of e

ryth

rocy

tic a

bnor

mal

ities

of

Labe

o ro

hita

sam

pled

from

Heb

bal fi

sh fa

rm (C

ontr

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te),



chromatin, ragged cell membranes, altered staining properties, and hemolysis in erythrocytes of Oncorhynchus kisutch from water contaminated with chlorinated sewage was reported by Buckley (1976). Zeni et al. (2002) reported erythrocyte echi-nocytosis in Ictalurus melas sub-lethally exposed to anionic detergent. It is well known that blood sampling, laboratory techniques, seasonal variations, size, genetic properties, sex, population density, lack of food supply, environmental stress and transportation could affect hematological data22.

5. ConclusionTo conclude our study on water parameters and Micronuclei test in fish sampled from lake A, lake B and control site during winter, summer and rainy season, it can be stated that variation in levels of physico-chemical parameters due to environmental contaminants including trace metals interfered with fish physi-ology, disrupted normal processes, induced stress, toxic effects which inturn caused cyto- and geno-toxicity. Thus keeping in view health of these water bodies remedial measures should be undertaken to combat water contamination and its manage-ment.

6. References1. APHA, AWWA, and WPCF. Standard methods for examination

of water and wastewater (21st Eds: Andrew D Eaton, Clesceri LS, Rice EW, Greenberg AE). The Edition American Public Health Association, Washington, D.C. 2005.

2. Ateeq B, Farah MA, Ali MN and Ahmed W. Induction of micronuclei and erythrocyte alterations in the catfish, Clarias batrachus by 2,4-dichlorophenoxyacetic acid and butachlor. Mutation Research. 2002; 518:135-44. https://doi.org/10.1016/S1383-5718(02)00075-X

3. Ayllon F and Garcia-Vazquez E. Micronuclei and other nuclear lesions as genotoxicity indicators in rainbow trout Oncorhynchus mykiss. Ecotoxicology and Environmental Safety. 2001; 49:221-5. https://doi.org/10.1006/eesa.2001.2065. PMid:11440474

4. Brown RM, Mc cleiland NJ, Deiniger RA and O’ Connor MF. A water quality index - crossing the physical barrier (Jenkis SH ed). Proceedings of International Conference on Water Pollution Research. 1972; 6:787-97.

5. Buckley JA. Heinz body hemolytic anemia in Coho salmon (Oncorhynchus kisutch) exposed to chlorinated wastewater. Journal of the Fisheries Research Board of Canada. 1976; 34:224.

6. Carrasco KR, Tilbury KL and Myers MS. Assessment of the Piscine Micronucleus Test as an in situ biological indicator of chemical contaminant effects. Canadian Journal of Fisheries and Aquatic Sciences. 1990; 47:2123-36. https://doi.org/10.1139/f90-237

7. Cavas T and Ergene-Gozukara S. Evaluation of the genotoxic potential of lambda-cyhalothrin using nuclear and nucleolar biomarkers on fish cells. Mutation Research. 2003; 534(1-2):93-9. https://doi.org/10.1016/S1383-5718(02)00246-2

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22. Kori-Siakpere O and Ubogu Ewoma O. Sublethal hemato-logical effects of zinc on the freshwater fish, Heteroclarias sp. (Osteichthyes: Clariidae). African Journal of Biotechnology. 2008; 7(12):2068-73. https://doi.org/10.5897/AJB07.706

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26. Ohe T, Watanabe T and Wakabasyashi K. Mutagens in surface waters: a review. Mutation Research. 2004; 567:109-49. https://doi.org/10.1016/j.mrrev.2004.08.003. PMid:15572284

27. Olaifa FG, Olaifa AK and Onwude TE. Lethal and sublethal effects of copper to the African cat fish (Clarias gariepnus). African Journal of Biomedical Research. 2004; 27:9-15.

28. Osman A, Ali E, Hashem M, Mostafa M and Mekkawy I. Genotoxicity of two pathogenic strains of zoosporic fungi (Achlya klebsiana and Aphanomyces laevis) on erythrocytes of Nile tilapia Oreochromis niloticus niloticus. Ecotoxicology and Environmental Safety. 2010; 73:24-31. https://doi.org/10.1016/j.ecoenv.2009.08.021. PMid:19811832

29. Ossana NA, Casta-e PM, Poletta GL, Mudry MD and Salibian A. Toxicity of waterborne copper in premetamorphic tadpoles of Lithobates catesbeianus (Shaw, 1802). Bulletin of Environmental Contamination and Toxicology. 2010; 84:712-5. https://doi.org/10.1007/s00128-010-0014-0. PMid:20440472

30. Pacheco M and Santos MA. Induction of EROD activity and genotoxic effects by polycyclic aromatic hydrocarbons and resin acids on the juvenile eel (Anguilla anguilla L). Ecotoxicology and Environmental Safety. 1997; 38:252-9. https://doi.org/10.1006/eesa.1997.1585. PMid:9469877

31. Pacheco M and Santos MA. Biotransformation, genotoxic and histopathological effects of environmental contaminants in European ell (Anguilla anguilla L.). Ecotoxicology and Environmental Safety. 2002; 53:331-47. https://doi.org/10.1016/S0147-6513(02)00017-9

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34. Schroder TM. Cytogenetische and cytologische befunde bei enzy-mopenischen panmyelo pathien and pancytopanien (Familiare panmyelopathien fanconi glutathionreduktasemangel anamie megalobelastare Vitamin B). Humangenzetik. 1966; 2:287-316.

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37. Talapatra SN and Banerjee SK. Detection of micronucleus and abnormal nucleus in erythrocytes from the gill and kidney of Labeo bata cultivated in sewage-fed fish farms. Food and Chemical Toxicology. 2007; 45:210-15. https://doi.org/10.1016/j.fct.2006.07.022. PMid:17034922

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39. Udroiu I. The micronucleus test in piscine erythrocytes. Aquatic Toxicology. 2006; 79:201-4. https://doi.org/10.1016/j.aqua-tox.2006.06.013. PMid:16846653

40. Van der oost R, Beyer J and Vermeulen NPE. Fish bioacumula-tion and biomarkers in enviromental risk assessment: a review. Environmental Toxicology and Pharmacology. 2003; 13:57-149. https://doi.org/10.1016/S1382-6689(02)00126-6

41. Ventura BC, Angelis DF and Marin-Molares MA. Mutagenic and genotoxic effects of the atrazine herbicide in Oreochromis nilot-ics (Perciformes, Cichlidae) detected by the micronuclei test and the comet assay. Pesticide Biochemistry and Physiology. 2008; 90:42-51. https://doi.org/10.1016/j.pestbp.2007.07.009

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Ammonia Intoxication at the Work Place - A Case ReportLorena Maries1 and Marina Maries2

1Occupational Medicine Department, Sanador Medical Center, Bucharest, Romania; [email protected] 2Occupational Diseases Compartment, Sibiu Clinical County Hospital, Romania

Keywords: Ammonia, Gas, Intoxication, Irritant Vapours, Work Accident

AbstractObjectives: We report a case of intoxication with irritant vapors (ammonia). Methods: The patient was admitted to the Occupational Diseases Compartment of the Sibiu Clinical County Hospital, Romania, monthly- in January, February, March, April in 2015, for respiratory symptomatology, which was initially included in the manifestations of the intoxication with irritant vapors, later for the chronic post-intoxication residual respiratory effects. Results: The patient, though young (36 years old), is now retired on medical ground, because of the pulmonary complications which occurred despite the intensive treatment. Discussion: The intoxication occurred because of a work accident at a preserved meat product factory in the city. We emphasize the importance of preventing such situations.

1. IntroductionAmmonia is a colorless gas, stable at room temperature, lighter than air, with a pungent smell. At −33°C or at a pressure of at least 10 bars, it liquefies to a colorless fluid. It is highly soluble in water, forming ammonium hydroxide (NH4OH). It is a strong alkaline that displays high corrosive properties. Because of its chemical properties, it is toxic to human in all forms of exposure. It causes alkaline chemical burns to the skin, to the eyes and particularly to the respiratory system, proportional to the duration and type of exposure, to the strength and pH of the gas or fluid1.

Ammonia is used mainly as fertilizer in agriculture and as refrigerant in industrial cooling equipment. It is also used in the production of nitrous fertilizers, of nitric acid, the synthe-sis of urea, the manufacture of dyes and paints, in refineries2.

Generally, the clinical manifestations of acute exposure are immediate1. The respiratory tract is the most exposed one, because of the inhalation of ammonia2. Acute exposure to high ammonia concentrations may lead to tracheobronchitis with the severe obstruction of the air flow, followed by its chronic obstruction with bronchiectasis3. Exposure to a considerable concentration of ammonia gas may be fatal in a matter of min-utes; asphyxiation may appear after exposure in confined and poorly ventilated spaces1.

2. Case PresentationThe patient H.I.C., 36 years old,a refrigeration technician, was the employee of a preserved meat product factory in the city, being in charge with the maintenance of the fac-

tory’s cooling equipment. According to the patient’s own statement, in December 2014, he went, accompanied by a rescuer, to check the company’s ammonia gas installation, which suffered a failure. During the checks, he dropped a hose, which led to the release of very highly concentrated ammonia gas. Thus, although he did wear a mask and despite the fact that the exposure only took several minutes, the patient blacked out, being removed by the rescuer and transported to the Emergency Admission Unit. He was then transferred in the care of the internist who consulted him, in the Medicala I section of the Sibiu County Hospital, because the Occupational Health physician was on annual leave. At this initial admission in hospital, the major symptoms were treated (oropharyngeal ulcerations, dysphonia, mixed ven-tilatory dysfunction with obstruction on the large airways and severe on the small airways, conjunctival hyperemia), the event was marked and declared occupational disease and work accident.

The patient returned on the 5th of January, this time directly in the Occupational Diseases Compartment, com-plaining about marked dysphonia, mixed dyspnea, sweats, and asthenic-vegetative syndrome. From a clinical viewpoint, at the admission, we noted as pathological-sibilant rhonchi on both pulmonary surfaces, accentuated dysphonia.

The chest X-ray did not show any evolving lesions, con-densation processes. An ENT consultation, noted mobile vocal folds, however with an accentuated bilateral congestion; the recommendation was to administer hydrocortisone hemisuc-cinate i.v., Ampicillin, Fluconazole, Vitamins A and E, Omeran. The spirogram showed mixed ventilatory defects, mainly severe


Ammonia Intoxication at the Work Place - A Case Report

obstructive. All the blood tests that were performed (complete blood count, urinalysis, biochemistry) showed normal results; also, the pharyngeal exudate did not detect bacteria or fungi. In this phase, the case was interpreted as - status after occupa-tional intoxication with irritant vapors (ammonia); irritation syndrome of the upper respiratory tract - acute secondary lar-yngitis; acute secondary tracheal bronchitis; mixed ventilatory dysfunction, mainly severe obstructive. The patient followed antibiotic therapy in association (Ampicillin, Gentamicin), massive corticoidparenteraltherapy, and also treatment with antimycotics, proton pump inhibitors, vitamins, NSAIDs. The patient was discharged in an improved state of health with recommendation of reassessment in 90 days.

The patient returned to the Occupational Diseases Compartment on 19.01.2015, accusing marked dyspnea, dys-phonia, asthenia, fatigability, sweat. Clinically, at the admission, we noted sibilant rhonchi mostly on the right side, dysphonia, productive cough, sweaty teguments. The chest X-ray showed an accentuated bilateral peribronchovascularinterstitium, increased hila, general hyper-transparency.

We recommended an Ophthalmology consultation, which showed spastic angiopathy, and a Cardiology consultation-which showed stage II high blood pressure, for which the recommendation was Nebilet 5mg per day. It is known that the systemic effects after acute exposure to high concentrations of ammonia include high blood pressure, bradycardia, cardio-respiratory arrest, cyanosis1. He also underwent a Pulmonology consultation, following which he was diagnosed with “chronic bronchitis with accentuated functional deficit”, he received indication of lung CT and treatment with Aeriusand Clenil jet. At the stress test-going up and down 2 floors 2 times - he did not present any pulmonary auscultation or BP changes. The blood count showed lymphocytopenia with monocytosis and high ESR, and the biochemistry showed high fibrinogen values. In this phase, the case was interpreted as “Status after occupational intoxication with irritant vapors (ammonia); acute secondary laryngitis; spastic secondary bronchitis, right basal intersti-tial lung disease, stage I/II HBP under treatment”. The patient received therapy with antibiotics (Amoxicillin, Cefuroxime), massive corticoid therapy parenteral, bronchodilators, proton pump inhibitors, vitamin therapy. He was discharged with rec-ommendation of admission in Pulmonology for the lung CT and specialized evaluation. We consider that this symptom-atology corresponded to the second stage of the intoxication with irritant vapors.

The patient returned to the Occupational Diseases Compartment on 26.02.2015, complaining about marked dyspnea at small effort, productive cough with aeration. Clinically, at the admission-lungs-sibilant rhonchi, oscillating blood pressure values. The chest X-ray showed a pulmonary interstitium within normal ranges. The patient was admit-ted in the meantime at the Pulmonology ward where the CT

scan was performed, which did not show any modifications. The blood count indicated neutrophilia with lymphocyto-sis and monocytosis. In this phase, the case was interpreted as “Status after occupational intoxication with irritant vapors (ammonia); acute respiratory failure; acute secondary obstruc-tive lung diseasewith spastic component, HBP stage II under treatment”. The patient continued the therapy with antibiotics (Gentamicin, Cifran), parenteral corticoids, proton pump inhibitors, bronchodilators. The patient was discharged from hospital on 20.03.2015 in an improved state of health, with a sick leave of 31 days.

The patient returned again to the Occupational Diseases Compartment, in April, complaining about the same symp-tomatology. Clinically, at the admission - sibilant rhonchi, wheezing, high blood pressure (BP = 150/90 mmHg), without any other modifications in the clinical examination. The blood tests continued to indicate a high fibrinogen value, otherwise the blood count was normal. In this phase, the case was inter-preted as “status after occupational intoxication with irritant vapors (ammonia); chronic secondary respiratory failure, secondary asthmatic bronchitis, HBP stage II under treatment”. The patient followed therapy with antibiotics(Doxycycline), bronchodilators, antihypertensives.

3. DiscussionsThis is a rather severe form of intoxication with irritant vapors, which went through all the 3 stages of the diseases (irrita-tion syndrome of the upper airways, occlusive bronchiolitis, acute toxic pulmonary edema), despite the insistent treatment. The disorder occurred after an exposure of several minutes (we could not determine the time precisely from the patient’s description), but at a very high concentration of ammonia.

Massive inhalation of ammonia gas may be fatal or it may lead to severe pulmonary impairment, which could require assisted respiratory support (mechanical ventilation)4,5, or even indicate the necessity of a lung transplant2.

In the case that we have described, the impairment was not so severe to require such measures. Nevertheless, com-plications did occur, despite the fact that the victim had been removed quickly from the contaminated environment, admit-ted to the hospital and treated intensively with corticosteroids in oral and parenteral administration. In fact, it is known the lesions caused by ammonia gas inhalation are followed by the installation of a chronic pulmonary disease1,6.

The case was signaled and declared acute intoxication, occupational disease and work accident. The employer asked the patient to come back to work from the last sick leave, the competent authorities announced that the employee resumed his activity and the case was closed. Later, because the employee could not fulfill his tasks, he had to resign and then to retire.At present, the patient is retired on medical ground; because of


Lorena Maries and Marina Maries

the fact that he had returned to work, he cannot benefit from a pension of disability. We do note that this is a young person (36 years old), who is left with complications that require long-term treatment and special conditions should he ever wish to work again; apart from its medical characteristics, the case also has a legislative and social peculiarity.

We emphasize the importance of preventing such situ-ations. The workers should be familiar with the work environment, and comply with occupational safety measures; we also stress the importance of the presence of well-trained and equipped rescue teams, able to act quickly if an accident were to occur.

4. References1. Makarovsky I, Markel G, Dushnitsky T, Eisenkraft A. Ammonia-

When Something smells wrong- toxic chemical compounds. IMAJ. 2008 Jul; 10.

2. Lalic H, Djindjic-Pavicic M, Kukuljan M. Ammonia intoxica-tion on workplace- Case report and a review of literature- Coll. Antropol. 2009; 33(3):945–9. PMid:19860130

3. Sundblad B-M, Larsson B-M, Acevedo F, Ernstgard L, Johanson G, Larsson K, et al. Acute respiratory effects of exposure to ammonia on healthy subjects. Scand J Work Environ Health. 2004; 30(4):313–21. https://doi.org/10.5271/sjweh.800

4. O’Kane GJ. Inhalation of ammonia vapour. A report on the manage-ment of eight patients during the acute stages. Anaesthesia. 1983; 38:1208–13. https://doi.org/10.1111/j.1365-2044.1983.tb12527.x PMid:6660462

5. Bhalla A, Mahi S, Sharma N, Singh S. Glycopyrrolate in toxic exposure to ammonia gas. J Emerg Trauma Shock. 2011; 4:140–1. https://doi.org/10.4103/0974-2700.76830 PMid:21633586 PMCid:PMC3097567

6. Leduc D, Gris P, Lheureux P, Gevenois PA, De Vuyst P, Yernault JC. Acute and long term respiratory damage following inhalation of ammonia. Thorax. 1992; 47:755–7. https://doi. org/10.1136/thx.47.9.755 PMid:1440475 PMCid:PMC474816



Antioxidant and Anti-Apoptotic Activities of Phytochemically Validated Fruit Extract of Solanum

xanthocarpum in Primary Chondrocytes Neelam Shivnath1*, Vineeta Rawat1, Sahabjada1,2, Asif Jafri1, Juhi Rais1 Habiba Khan1, and Md. Arshad1*

1Molecular Endocrinology Lab, Department of Zoology, University of Lucknow, Lucknow – 226007, Uttar Pradesh, India; [email protected]; [email protected]

2Department of Biochemistry, Era’s Lucknow Medical College and Hospital, Lucknow – 226003, Uttar Pradesh, India

Keywords: Apoptosis, Chondrocytes, Non-Steroidal Anti-Inflammatory Drugs (NSAIDs), Osteoarthritis, Phytochemicals

AbstractThe chondrocyte death may contribute in progression of osteoarthritis (OA). Solanum xanthocarpum (Family: Solanaceae) fruits were known for antioxidant activity. This study demonstrates that the phytochemically validated Solanum xanthocarpum fruits (SXF) extract has inhibitory activities on nitric oxide (NO) induced cell death and ROS formation in primary cultured chondrocytes. Chondrocyte death was induced by 1.5 mM of Sodium Nitroprusside (SNP). The Cell viability was measured by MTT assay and nuclear changes were observed by DAPI and Hoechst-PI. Antioxidant activity of SXF was demonstrated in H2O2 induced ROS generation in chondrocytes. Indomethacin (IM) (25μM), a NSAID was taken as positive control. Phytochemical analysis revealed the presence of flavonoids, anthraquinone glycosides, steroids, alkaloids, terpenoids and tannins. SXF significantly reduces the cell death induced by SNP in a dose dependent manner. The fluorescent photomicrograph of DAPI, Hoechst-PI and ROS also revealed the decreased rate of apoptosis in a dose-dependent manner. This study suggests that SXF shows anti-apoptotic and antioxidant activity in chondrocytes.


1. IntroductionOsteoarthritis is associated with the breakdown and ultimate loss of articular cartilage of joints1 and is commonly occurs among the elderly population in the world2. Several etiologi-cal risk factors like age, gender, trauma, overuse, genetics and obesity are associated with pathophysiologic processes that contribute disease progression3. In the pathological condition the cells of articular joints are subjected to complex envi-ronmental control. In addition to various cytokines, growth factors, and mechanical stimuli, reactive oxygen specie (ROS) contributes in pathological condition. Therefore, a functional change in chondrocytes of articular cartilage is related to the progression of OA4. Overproduction of oxidants (reactive oxy-gen species and reactive nitrogen species) in the human body is responsible for the pathogenesis of some diseases. Nitric Oxide (NO) and superoxide anion (O2

−) are the main ROS

produced by chondrocytes5. ROS like superoxide anion (O2−),

Hydrogen Peroxide(H2O2), and hydroxyl radicals (OH_) are

the byproduct of aerobic metabolism6 and are associated with principal oxidative stress molecules. The enzyme complex NADPH catalyzes the reduction of molecular oxygen to super-oxide anion radicals4. The production of NO is stimulated by various cytokines including interleukin (IL)-β, tumor necrosis factor (TNF)-α, interferon (IFN)-γ and lipopolysaccharides (LPS), and inhibited by Transforming growth factors (TGF)-β, IL-4, IL-10 and IL-137–9. It is believed that NO is an important mediator of dedifferentiation and apoptosis of chondrocytes in arthritic cartilage10.

Non-steroidal anti-inflammatory drugs are commonly used drugs in the entire world for the treatment of osteoar-thritis. Long-term use of these NSAIDs leads to significant side effects on liver, stomach, gastrointestinal tract and heart11. Therefore it becomes essential to explore alternative medi-


Neelam Shivnath, Vineeta Rawat, Sahabjada, Asif Jafri, Juhi Rais, Habiba Khan and M. Arshad

cine derived from herbal plants with a potential drug that is effective in terms of both efficacy and safety. Medicinal plants provide a significant source of chemical compounds that have a great importance on the health of individual and commu-nity. There is wide diversity of chemical compounds that have been isolated from plant especially secondary metabolites that were shown to have anti-cancer, anlegesic, anti-inflammatory, anti-bacterial and including some other activities12,13. These phytochemicals include flavonoids, phenols and phenolic gly-cosides, saponins and cyanogenic glycosides, tannins, nitrogen compounds (alkaloids, amines, betalains), terpenoids etc12.

Antioxidants have importance regarding reducing oxidative stress that could otherwise affect and damage biological mol-ecules14. Bioactive components such as flavonoids are natural antioxidant due to its indigenous origin and have strong effi-cacy to scavenge free radicals15.

Solanum xanthocarpum Schrad. Wendl. is commonly known as yellow-berried nightshade (Syn: Solanum surat-tens Burm. F.; Solanum virginium Linn) that belongs to family solanaceae. It is prickly diffuse bright green perennial herb, somewhat woody at the base. The stem is zig-zag with numer-ous branches. The berries are globular with green and white stripes when young but yellow when mature and surrounded by the enlarged calyx16. In Hindi, it is called Kantkari. Its other names are Choti Katheri, Kateli, Bhatkatiya and Bhachkatiya. It has been reported to occur in Ceylon and Malacca through South-East Asia, Malaya, Australia and Polynessia17. It is a wild plant mainly grown in Uttar Pradesh, Uttaranchal, Bihar, Punjab, West Bengal, Assam and other North-Eastern states18. It is a commonly used Ayurvedic medicine for treatment of asthma and bronchitis. Fruit juice of the plant is useful in treatment of sore throats and rheumatism, Decoction of the plant is used in gonorrhea, paste of leaves is applied to relieve pains, seeds act as expectorant in a cough and asthma, roots are expectorant and diuretic and useful in the treatment of catarrhal fever, coughs, asthma and chest pain19.

This study is designed to evaluate the antioxidant and anti-apoptotic efficacy of phytochemically validated SXF extract in primary chondrocytes isolated from rat articular cartilage.

2. Material and Methods

2.1 MaterialsDulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12, 1:1 mixture), 2’,7’-dichlorofluorescein diacetate

(DCFH-DA) and Propidium Iodide (PI) were from Sigma-Aldrich Inc. St. Louis, USA. Fetal Bovine Serum (FBS), sodium pyruvate, Non-Essential Amino Acids (NEAA), sodium bicarbonate, L-glutamine, antibiotic solution (penicillin/streptomycin), MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-nyltetrazolium bromide) dye, were all purchased from Himedia Laboratories Pvt. Ltd. Mumbai, India. Dimethyl Sulfoxide (DMSO), was from Merck Specialities Pvt. Ltd. Mumbai, India. All other reagents were of analytical grades.

2.2 Collection, Identification and Preparation of Plant Extract

The fruits of Solanum xanthocarpum were collected from roadsides in Gomti-Nagar and Kursi Road, Lucknow, India in month from September to February. The plant is identified by Prof. S. Lavania, Deparment of Botany, University of Lucknow, Lucknow. A reference of specimen (Voucher No. LWU-2016-4) has been deposited in the herbarium of Department of Botany, University of Lucknow, Lucknow.

The fresh plant material was collected, washed twice with double distilled water, then shade-dried and turned into pow-dered. The 95% ethanolic extract of plant was prepared with the help of Soxhlet apparatus (Borosil Glass Works Limited, India).

2.3 Phytochemical Screening Before evaluating antioxidant and anti-apoptotic activity, the ethanolic extract of SXF was tested for the presence of phy-toconstituents by standard biochemical tests for alkaloids, steroids, tannins, saponins and glycosides. The qualitative results are expressed as (+) for the presence and (-) for the absence of phytochemicals.

2.3.1 Test for Alkaloids About 15 mg of SXF extract was taken in a test-tube and stirred with 1% HCl (6 mL) on a water bath for 5 min and filtered. These filtrates were divided into three equal parts.

• Dragendorff ’s Test: To the first portion of the filtrate, 1 mL of Dragendorff ’s reagent (Potassium bismuth iodide solution) was added. Formation of an orange-red precipitate shows the presence of alkaloids.

• Mayer’s Test: To the second portion of the filtrate, 1 mL Mayer’s reagent (Potassium mercuric iodide solution) was added. A cream-colored precipitate indicates the presence of alkaloids.

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Antioxidant and Anti-Apoptotic Activities of Phytochemically Validated Fruit Extract of Solanum xanthocarpum in Primary Chondrocytes

• Wagner’s Test: About 2 g Potassium iodide and 1.27 g iodine were dissolved in 10 mL distilled water and diluted to 100 mL with distilled water. To the third portion of the filtrate, a few drops of prepared solution were added. The appearance of a brown colored pre-cipitate indicates the presence of alkaloids20,21.

2.3.2 Tests for Steroids and Terpenoids• Salkowski Test: About 100 mg of SXF extract was

taken in a test-tube. Dissolve the extract in 2 mL of chloroform (2 mL) by shaking followed by the addition of 2 mL concentrated H2SO4 along the side of the test tube. The appearance of reddish-brown coloration of the interface indicates the presence of terpenoid22.

• Liebermann-Burchard Test: About 100 mg of extract was shaken with chloroform in a test tube. A few drops of acetic anhydride was added to the test tube and boiled in a water bath, which is rapidly cooled in iced water. A 2 mL concentrated H2SO4 was added along the sides of the test tube. Formation of a brown ring at the junc-tion of two layers and turning the upper layer to green indicates the presence of steroids while the formation of deep red color shows the presence of triterpenoids21.

2.3.3 Test for Tannins About 0.5 g of SXF extract was separately stirred with 10 mL distilled water and filtered. A few drops of 5% ferric chloride were added to test-tube. Black or blue-green coloration or pre-cipitate indicates the presence of tannins23.

2.3.4 Test for SaponinsAbout 5 g of SXF extract was separately shaken with 10 mL distilled water in a test tube. The formation of frothing, which remains persist on warming the test-tubes in a water bath for 5 min, indicates the presence of saponins23.

2.3.5 Tests for Glycosides • Anthraquinone glycoside (Borntrager’s Test): To the

1 mL of SXF extract solution, 1 mL of 5% H2SO4 was added. The mixture was boiled in a water bath for 5 min and then filtered. The filtrate was then shaken with an equal volume of chloroform and kept to stand for 5 min. A 1 mL of dilute ammonia was shaken with the

lower layer of chloroform. There is formation of rose pink to red-color of the ammoniacal layerthat indicate-santhraquinone glycosides21.

• Cardiac glycoside (Keller-Killiani Test): About 0.5 g extract was shaken with 5 mL distilled water. A 2 mL glacial acetic acid containing a few drops of ferric chlo-ride was added, followed by 1 mL of H2SO4 along the side of the test tube. The formation of a brown ring at the interface gives a positive result for cardiac glycoside and a violet ring may appear below the brown ring22.

2.3.6 Tests for Flavonoids • Shinoda Test: About 1 g of SXF extract was taken in

test-tube and mixed with pieces of magnesium ribbon and concentrated HCl for few minutes. The appearance of pink color showed the presence of flavonoid.

• Alkaline Reagent Test: About 1 gm of SXF extract was taken in test-tube andmixed with 2 mL of 2.0% NaOH. The intense yellow color was produced that became col-orless when 2 drops of diluted acid was added to this mixture showed the presence of flavonoids.

2.3.7 The Culture of Primary Chondrocyte CellsThe primary chondrocytes were isolated from knees of 2-3 days old rat pups. Isolated cartilage was transferred to phosphate buffer saline (PBS) with 500 U/mL penicillin and 500 µg/mL streptomycin. Then the cartilages were cut into small pieces, and subjected to digestion with 0.25% trypsin/EDTA and kept at 37°C, 5% CO2 incubator for 30 min. The supernatant was centrifuged and resulting pellet was digested twice with 0.2% type II collagenase for 1 h each and kept in a CO2 incubator and centrifuged at 1200 rpm for 6 min to obtain a final cell pel-let. Cells were re-suspended in DMEM/F-12 complete culture medium containing 10% FBS, 100 U/mL penicillin and 100 µg/mL streptomycin and placed in 50 mL culture flask24.

When the cells reached up to 80-90% confluency, the cell morphology was observed under phase contrast microscope (Nikon ECLIPSE Ti- S, Japan).

2.4 MTT Assay for NO Induced Cell DeathChondrocytes were suspended in Chondrocyte Growth Medium at a density of 1×104 cells/mL and cultured in 96-well plates at 370C in 5% CO2 for 1 day. After medium change with DMEM/F-12 supplemented with 100 U/mL penicillin, 100

108



μg/mL streptomycin, and 20 μg/mL gentamicin, chondro-cytes were pretreated with 25 μM indomathacin (IM) SXF at concentrations (50, 100, 250 μg/mL) for 1 day. Cell death was induced by treatment of cells with 1.5 mM of SNP for further 24 h. The cell viability was evaluated with a soluble tetrazolium salt MTT [3-(4,5-dimethylthiazole-2-yl)- 2,5-diphenyltetrazo-rium bromide]25.

2.5 Nuclear Apoptosis Assay4,6-Diamidino-2-Phenylindole-2-HCl (DAPI) binds dsDNA that provides a blue fluorescence when viewed under the ultra-violet light. Apoptotic cells are visualized as a small, condensed nucleus. The cells were seeded and treated for 24 h in 96-well plate in medium containing 10% FBS and 1% penicillin/strep-tomycin solution. Then, the different dose of SXF (50, 100, 250 µg/mL) and 25 μM IM was added to each well with complete media. After the treatment period, cells were exposed to SNP and further incubated for 24 h. The cells were washed with PBS and fixed in 4% PFA for 10 min. Subsequently, the cells were permeabilized with permeabilization buffer (3% PFA and 0.5% Triton X-100) and stained with DAPI. After the stain-ing images were taken with the fluorescent microscope (Nikon Eclipse Ti-S, Japan)26.

2.6 Hoechst-Propidium Iodide (PI) Double Staining

This dye is used to detect normal, apoptotic and dead cells in same culture well. Hoechst is used to stain chromatin of apoptotic cells with fluorescence than normal cells. The PI on the other hand is used to stain chromatin of dead cells. The staining procedure was according manufacturer’s protocol (GenScript). The cells were treated with different concentra-tions of SXF (50, 100, 250 µg/mL) and 25 μM of IM. Further

the cells were exposed to SNP for 24 h in CO2 incubator. A 1 μL of Hoechst 33342/100 μL PBS was loaded in each well and incubated in CO2 incubator for 10 min. After aspiration, a 100 μL of 1X buffer A mixed with PI was loaded. Plate was then incubated at room temperature in dark for 5 min. Cells were immediately visualized under inverted fluorescence micro-scope (Nikon, ECLIPSE Ti-Series).

2.7 DCFH-DA Staining for Reactive Oxygen Species (ROS)

ROS generation was assessed by 2’,7’-dichlorofluorescein diacetate (DCFH-DA) dye. Chondrocyte cells were seeded in black bottom culture plate for 24 h and incubate the plate at 37 °C, 5% CO2 maintained in the CO2 incubator. Cells were then exposed to 20 μL (10 μM stock solution) of H2O2 for 24 h. Cells were treated with 25 μM of IM and SXF at concentrations 50, 100 and 250 μg/mL for 24 h in triplicate. The cells were further incubated with DCFH-DA dye (stock 10 mM) for 30 min. The reaction mixture was kept on the shaker for 10 min at room temperature in dark. Fluorescence intensity was measured with a multi well plate reader (Synergy H1 Hybrid Multi-mode microplate reader, BioTEK) at an excitation wavelength of 528 nm. Photomicrographs of another set of cells seeded in 96 wells plate were taken by fluorescence microscope (Nikon Eclipse Ti-S, Japan) to analyze intracellular fluorescence inten-sity of ROS production26.

3. Results

3.1 Phytochemical Screening The outcome through phytochemical screening shows that the whole SXF ethanolic extract contains flavonoids, anthraqui-

Phytochemical Test Investigation (Present/Absent)

1

Test for Alkaloids

(a) Dragendorff ’s Test(b) Mayer’s Test(c) Wagner’s Test

+++

Table 1. Phytochemical validation of locally collected Solanum xanthocarpum fruit extract through standard biochemical tests

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none glycosides, steroids, alkaloids, terpenoids and tannins. The result has been demonstrated in Table 1.

3.2 Inhibition of NO induced cell deathThe production of NO is an important component that involves in the pathogenesis of OA. We address, whether the

given extract reduces the cell death due to induction of NO. The exposure of chondrocytes to the prepared extract before exposure to SNP reduces the cell death significantly (p<0.05%) in dose dependent manner. The increase in viability of cells to approximately must be (50%), 61%, 75% were observed at IM, 50, 100 and 250 μg/mL SXF as shown in Figure 1.

2.

Tests for Steroids/terpenoids

(a) Salkowski test(b) Liebermann-Burchard Test +

+

3. Test for Tannins +

4. Test for Saponins +

5.

Test for Glycosides

(a) Anthraquinone Glycoside (Borntrager’s Test)

(b) Cardiac Glycoside (Keller-Killiani Test)

+

+

6.

Test for Flavonoids

(a) Shinoda test(b) Alkaline reagent test

++

Present = (+) and Absence = (-)

Table 1 Continued

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3.3 Nuclear Apoptosis AssayIt was observed from photomicrograph (Figure 2), the cells exposed to only SNP (NO-control) shows deep blue fluores-cence with condensed nuclei as compared to normal control

cells with no fluorescence. The reduction of fluorescence is visualized in IM treated cells and cells treated with 50 μg/mL of SXF compared to NO-control and it further reduces significantly at concentration 100 and 250 μg/mL of SXF.

Figure 1. (i) Phase contrast microscopic pictures of chondrocyte at different concentration of SXF extract. (A) Control, (B) NO-Control, (C) IM, (D), (E), (F) at different oncentration of extract (50, 100, 250 μg/mL) (Magnification: 20X; Scale bar: 0.1 mm). (ii) Graph represents the effect of SXF on decreased apoptosis and increased % cell viability in dose dependent manner at different concentrations. Values are obtained from three independent experiments and expressed as mean SEM. *P< 0.001 compared to control and #P< 0.05 compared to NO induced control (NO-Control). (Magnification = 20X and scale bar = 0.1 mm).

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Furthermore, about approximately 31% condensed cells were observed in SNP-control cells compared to control. The numbers of apoptotic and condensed cell reduced to 21.50%, 16.47%, 8.18% and 2.67% in treated groups IM 25 μM, 50, 100 and 250 μg/mL SXF doses respectively.

3.4 Hoechst-PI StainingHoechst-PI double staining showed a decrease in the rate of apoptosis with an increase in the concentration of SXF (Figure 3). The cells with blue and white fluorescence were undergo-

Figure 2. (i) Fluorescence microscopy images showing nuclear condensation in chondrocytes treated with (A) Control, (B) NO-control, (C) IM and (D), (E), (F) different concentrations of SXF (50, 100, 250 μg/mL) (Magnification= 20X and scale bar= 0.1 mm). (ii) Shows graphic representation of % apoptotic cells respective to their controls. The cells were counted manually under a fluorescence microscope in at least 10 random fields and percent apoptotic cells were calculated as detailed under materials and methods. Values were obtained from three independent experiments and expressed as mean and SEM. *P< 0.001 compared with control and #P< 0.05 compared to NO-control.

Figure 3. Fluorescence microscopy images showing the cells were treated with SXF extract and 25 μM of IM except control group. Pretreated cells were exposed to SNP for 24 h. Arrow marked the apoptotic cells with pink flouresence. (A) Control, (B) NO induced control without any treatment, (C) IM treated cells, (D), (E), (F) treated with 50, 100, 250 μg/mL of SFX extract. (Magnification: 20X; Scale bar: 0.1 mm).

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ing apoptosis and cells with pink fluorescence were dead. The NO-control group without any treatment shows maximum fluorescence. The degree of fluorescence was slightly reduced in IM treated group and 50 μg/mL SXF treated cells. However, the fluorescence was significantly reduced at 100 and 250 μg/mL concentration of SXF.

3.5 Inhibition of ROS FormationThe exposure of cells to hydrogen peroxide (H2O2) (10 μM stock solution) for 24 h, significantly reduces the number of chondrocytes. The microscopic examination from fluores-cence microscope shows that the intensity of fluorescence was decreased with increase in the concentration of dose of SXF

Figure 4. (i) Photomicrographs of chondrocytes showing antioxidant properties of SXF. Cells exposed to H2O2 for 24 h before treatment of IM and SXF extract (Magnification: 20X; Scale bar: 0.1mm). (A) Control, (B) H2O2 control, (C) IM, (D), (E), (F) at different concentration of extract (50, 100, 250 μg/mL). (ii) Graph representing percentage of ROS generating cells and calculated as DCF positive cells to total number of cells. Data are represented as mean and SEM. Non-parametric test one way ANOVA: *P< 0.001 versus control and #P< 0.05 versus H2O2 induced ROS control (ROS-Contr).

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i.e., from 50 to 250 μg/mL. The cells, which were exposed to IM shows significant decrease in fluorescence. Quantitative data analysis also demonstrates the significant increase (p<0.001) in intracellular ROS production when exposed to 10 μM H2O2 is 71.45%. However, when the cells were treated with IM, 50, 100 and 250 μg/mL of SXF, the production of intracellular ROS decreases about 63.41%, 64.63%, 58.70% and 39.10% respec-tively and thus increases the cell viability as shown in Figure 4.

4. DiscussionOA is a degenerative joint disease with several etiological risk factors.27Herbal plants produce safety profile compared to the NSAIDs28. From the phytochemical screening of SXF extract shows the presence of various phytochemicals viz. flavonoids, anthraquinone glycosides, steroids, alkaloids, terpenoids, tan-nins and saponins. The phytochemicals detected were known to have certain medicinal importance. Alkaloids derived from plants show an anti-inflammatory property29. Phenolic com-pounds have anti-oxidative, anti-inflammatory, anti-diabetic and anti-carcinogenic properties30. Saponin was also known to act as anti-oxidants having anti-inflammatory, weight loss ability and other pharmacological activities31. Plant steroids have cardiotonic activity and generally used in herbal medi-cine and cosmetics32. Tannins have astringency property i.e., faster healing up of a wound and mucous membrane33. The plant polyphenols have significance, as they are anti-oxidants and free radical scavengers. Polyphenolic compounds have aromatic benzene ring that substitutes hydroxyl radical and its functional derivatives. They can absorb free radicals and can chelate metal ions that catalyze the formation of ROS that pro-motes lipid peroxidation. Among the polyphenols, flavonoids help to fight against diseases. Its antioxidant potency depends on the molecular structure and position of hydroxyl group including other features in its structure34.

There is a reduction in the level of oxidative stress produced due to exposure of cells to H2O2 pretreated with SXF extract was observed that might be due to the presence of the various anti-oxidant, anti-inflammatory and anti-apoptotic components in the extract. A study supports our finding that antioxidant present in Sumac leaves induced chondrogenesis through pre-venting ROS generation35. In a study, Zhuang et al., 2016 also describes the inhibitory effect of Angelica sinensis that protect the chondrocyte from H2O2 induced apoptosis in rat chondrocytes

through its anti-inflammatory, antioxidant and anti-apoptotic properties36. The anti-apoptotic activities on chondrocytes were observed in cells pretreated with SXF extract for 24 h and then exposed to SNP-generated NO-induced cell death. There are many studies that provide the correlation between the level of NO production and chondrocyte apoptosis. The effect of SNP over chondrocytes was evaluated with MTT assay for cell viability, nuclear condensation assay and Hoechst-PI staining for apoptosis of cells. In a study, Lee et al., has demonstrated cilostazol protect the chondrocytes from nitric oxide induce apoptosis37. It was observed from statistical data and photomi-crography that SXF induces proliferation of cells and reduces the apoptosis of chondrocytes in dose dependent manner. The medicinal properties of SXF are due to the presence of various phytochemicals including various polyphenols like fla-vonoids. These flavonoids possess anti-oxidant properties due to the indigenous origin and strong tendency to scavenge free radicals38. Other antioxidants curcumin and quercetin inhibit inflammatory processes and protect chondrocytes39. The antioxidant resveratrol protect chondrocytes apoptosis via effects on mitochondrial polarization and ATP production. In a study Beecher et al., 2007 suggested that antioxidant blocks cyclic loading induced chondrocyte death40. The data from nuclear condensation suggest that SXF protects chondrocyte from apoptosis. As phytochemicals of SXF contains antioxi-dants therefore it might be prevent apoptosis.

5. ConclusionThe result demonstrates the presence of certain phyto constitu-ents in the ethanolic fruit extract of Solanum xanthocarpum collected from local area of Lucknow, India contains alkaloids, tannins, saponins, flavonoids, steroids and cardiac glycosides. Moreover, the ethanolic extract of SXF shows antioxidant activ-ity by reducing the ROS formation and inhibiting NO-induced cell death in primary chondrocytes.

6. AcknowledgementAuthor Neelam Shivnath is thankful to ICMR, New Delhi, India for the award of Senior Research Fellowship (No. 45/46/2014/BMS/TRM). The Departmental Equipment Facility, Department of Zoology, University of Lucknow, pro-vided by DST-FIST-PURSE is duly acknowledged.

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Association of Cytokine TNF-α in Development of Osteoarthritis: A Comprehensive Study

AmitKumar1, Md Arshad2, Ajai Singh3, Habiba Khan2 and Suchit Swaroop1*

1 Department of Zoology, Experimental and Public Health Laboratory, University of Lucnow, Lucknow − 226007, India 2Department of Zoology, Molecular Endocrinology Lab, University of Lucknow, Lucknow − 226007, India

3Department of Orthopaedics, King George Medical University, Lucknow − 226003, India; [email protected]

Keywords: Cytokine, Gene Variants, Osteoarthritis, Pathophysiology, TNF-α

AbstractOsteo-Arthritis (OA) is a disease of joints affecting the normal functions of joint and causes physical disability. Many factors are responsible for development of osteoarthritis including over age, obesity, gender, drug abuse, over load on joints and genetic factors. OA causes health problems and impairs the quality of life with increased economic burden across the world. Apart from the pro-inflammatory role of cytokine Interleukin-1 (IL-1) in osteoarthritis, Tumor necrosis factor-alpha (TNF- α) is also involved in the progression of osteoarthritis. Members of TNF family are secreted from lymphocytes and natural killer cells but in OA patients it is also secreted from chondrocyte cells to influence the catabolic processes in Extra Cellular Matrix (ECM) by inducing the activity of matrix metaloproteinases (MMPs). In promoter region, most of the Single nucleotide polymorphisms (SNPs) of TNF-α located on -863, -857, -308, and -238. These SNPs are involved in various diseases including OA, rheumatoid Arthritis, systemic lupus erythematosus etc. Significance association of SNP -G308A of the TNF-α gene in OA has been observed in various studies. Aim of this mini review is to conclude the fundamental roles of TNF-α cytokine in patients with OA.

1. IntroductionOsteoarthritis is age related joint disease that adversely affects the normal daily activities of life in elderly population. It is marked by continuous breakdown of articular cartilage and other components of joints1. Italso leads to development of osteophyte. OA may affect almost all joints (hip, hand, knee etc) whereas in an obese person knee OA is most common. Females have slightly higher prevalence of developing OA as compared to males, females aged fifty and above suffer mostly from hand OA17. Among pro-inflammatory cytokines, both IL-1β and TNF-αhave predominant roles in destruction of healthy cartilage by inducing some proteases20. Two important cells of joint viz. chondrocyte and synovial cell secretes these pro-inflammatory cytokines in extracellular spaces, where they induce inflammatory response18. Anti-inflammatory cytokines such as IL-4, IL-10, IL-13 inhibit the action of pro-inflamma-tory cytokines30. Developing OA is marked by over expression of degradative enzymes and pro-inflammatory cytokines. Most common enzymes in synovial fluid are Matrix Metallo-Proteinases (MMPs), aggrecanases37. It can be very difficult to understand about concentrations of pro-inflammatory cytokines because some patients show high concentrations whereas in others show low concentrations. Due to this reason, cytokines remain controversial.

Over the years, many studies have reviled that the genetic susceptibility strongly contributing in pathophysiology of OA development. High levels of pro-inflammatory cytokines in synovial fluid of OA patients indicate the breakdown of carti-lage and progression of disease18. Members of TNF-family are predominantly synthesised by immune cells (B and T lympho-cytes), NK-cells. It has been also reported that theses cytokines concentration also high in synovial fluid in OA patients (O’Rourke et al., 2008). TNF-α encoding gene is present at most polymorphic region of DNA, where the major histo-compatibility complex type-III (MHC-III) gene is located12. Number of gene variants of TNF-α increase the susceptibility in OA. Aim of this study to explore the impact of TNF-α cyto-kine and their gene variants in development of OA.

2. Role of TNF-α in Pathophysiology of Osteoarthritis

TNF-α is a pro-inflammatory cytokine which plays a role in pathophysiology of OA34. It is a 17 kDa secreted protein by activated chondrocyte, macrophage and other cells which affects the synthesis of onthercytokines such as IL-1 and IL-89.

TNF-α along with IL-1β involved in etiology of the disease. It is one of the 19 members of Tumor Necrosis Factor (TNF)




superfamily5. TNF-α is primarily composed of three identical transmembrane proteins Type II (mTNFα) and later TACE/ADAM17 metalloproteinase cause maturation of mTNFα into soluble free TNF-α (sTNF-α), which is secreted out by the cell11. Both IL-1β and TNF- α are secreted from same cell of the joint.

TNF-α is mainly synthesized in synovial chondrocyte cells. Elevated level of TNF-α was detected in synovial membrane, cartilage tissue, sub-chondral bone layer7. TNF-α has affinity to interact with two receptor isotypes (TNF-R1 and TNF-R2). These receptors are expressed on membrane of about all nucleated cells22. The receptor TNF-R1 can be stimulated by the soluble as well as membrane forms ligand whereas receptor TNF-R2 only recognises membrane form of ligand. Thus, the receptor TNF-R1 has higher impact on cartilage breakdown than of receptor TNF-R222. Elevated expression of TNF-R1 has also observed in Fibroblast-like synoviocyte25. Structurally TNF-R1 and TNF-R2 are unrelated with their carboxyl terminal domain. TNF-R1 has associated with Death Domain (DD) in their carboxyl terminal which lack in TNF-R211. Insight the intracellular dissimilarity between these two receptors, they relay different signal from outside of the cell to the nucleus. Signal transduction through TNF-R1 is involved by formation of two complexes. The complex-1 is inherent

in the expression of proteins responsible for prevention of apoptosis and secretion of inflammatory cytokine whereas second complex is directly involving in signal transduction and fragmentation of cell35. The binding of TNF-𝛼 to TNF-R1 leads to interaction of carboxyl terminus (Death Domain) with adapter proteins such as TRADD, TRAF, c-IAP1, c-IAP2 and RIP16. This interaction results in proteasomal degrada-tion of RIP1 protein and interaction with other proteins such as TAB1, TAB2 and TAK. Subsequently, the phosphorylation of IKK protein takes place which is associated with NEMO protein. IKK is responsible of activation of most important transcription factor NF-kB21. During activation of complex-1 other pathway also triggered involving JaNus Kinase (JNK), Extracellular Regulated Kinase (ERK) and Mitogen Activated Protein Kinase (MAPK)14,38. Activation of complex-2 through TNF-R1 results in endocytosis of receptor and interaction with FADD and subsequently activation of pro-caspases-8 into activated caspases-8. This pathway finished with cell death28. After the formation of complex of mTNF-α and TNF-R2, interaction of adopter proteins such as TRAF2, TRAF3, RIP1, c-IAP1 and c-IAP2 with receptor occur and proteoso-mal degradation of RIP1 takes place. End responses through this signal are the activation of transcription factor NF-kb and AP124 (Figure 1).

Figure 1. Intra cellular signaling pathway of TNF-α in OA


AmitKumar, Md Arshad, Ajai Singh, Habiba Khan and Suchit Swaroop

The final responses are the production of metalloprotein-ases (MMPs), ADAMTS4, ADAMTS-5, collagenases and inhibition of Extra Cellular Matrix (ECM) components pro-duction. Tumor necrosis factor-α receptor1 (TNF-αR1), Tumor necrosis factor-α receptor2 (TNF-αR2), Tumor necrosis factor receptor type 1 associated death domain protein (TRADD; Fas-Associated protein with Death Domain (FADD); TNF receptor-associated factor 6 (IRAF6); c-IAP also called BIRC3 (Baculovirus IAP repeat-containg protein3).Receptor-interacting protein kinase 1 (RIP1); TAB1 is also known as mitogen-activated protein kinase kinasekinase 7 inactivating protein 1(MAP3K7IP1) and TAB2 is also known as mitogen-activated protein kinase kinasekinase 7 inactivating protein 2(MAP3K7IP2); TAK is a mitogen-activated protein kinase kinasekinase 7(MAP3K7); NF-kB essential modulator/NF-kB inhibitor kinase1,2 (NEMO/IKK1,2); mitogen-activated pro-tein kinase (MAPK); basal transcription activating protein called activator protein-1 (AP-1).

The catabolic effect of TNF-α leads to cartilage breakdown and also induces the sensory neurons through the TNF-α receptor 1, 2 (TNFR1 and TNFR2). TNF-α induced neuro-pathic pain may be treated by application of anti-inflammatory medications. Ibuprofen and celecoxib are known to reduce the pain through their anti-inflammatory properties32. It causes initiation of events of cascade in inflammatory reactions with other pro-inflammatory cytokines such as IL-1β, IL-6 etc3. Both TNF-α and IL-1β are involved synergistically in in many cases during course of OA. These cytokines are sharing some cytosolic events and trigger the catabolic phenomenon and inflammation during development of OA16 TNF-α is affect-ing the function of chondrocyte, blocking the production of extracellular components such as collagen type 2, proteogly-can15. Activated chondrocytes are increasing the expression of extracellular components degradative enzymes include MMPs and ADAMTS-429. Chondrocyte apoptosis and migration of CPCs (Chondrogenic progenitor cells) will results in reduction of any chance of reappearing of normal articular cartilage10. Both TNF-α and IL-1β also disrupt the normal functioning of mitochondria and causes reduction in ATP synthesis hence, less energetic chondrocyte cell will also loss the mitochondrial membrane permeability23.

3. Gene Variants of TNF-α in Osteoarthritis

Gene for TNFα cytokine is located in core region of major his-tocompatibility complex. This gene encodes one of the most important proinflammatory cytokine is known as TNFα. This cytokine plays important role in cartilage damage of healthy joints especially in hand and knee. A study on Han Chinese population articulated that allele ‘A’ of TNF-α -308 variant has

an impact on risk of OA however,rs361525 SNP of TNF-α -238 has no impact on same population with OA4,. HLA (Human leucocyte antigen)-class II/III region and TNF-α gene, where some SNPs such as rs7775228 and rs10947262 were found to be associated with knee OA27,33.

Recent study in Finnish women population was suggested that TNFα gene variants play crucial function in etiological process of hand OA. Both haplotype and minor alleles poisoned at -1031 and -863 of the TNFα were involved independently in risk of hand OA31. Another study has been done in Egyptian female with severity of early onset knee OA suggested that TNF-α –G308A polymorphism involved in susceptibility to disease progression2.

Human Leucocyte Antigen (HLA) class-II and class-III con-sist of some variants of TNF-α gene which include rs7775228 and rs10947262. It has been observed that these variants are strongly associated with possibility to develop knee OA27,33. As per high level of expression of TNF-α with -308A allele and low expression of -308G allele, meta-analysis concluded that AA and AG genotypes influence the rate of risk of OA, whereas individuals with GG genotype might have lesser impact on developing of OA19. Among other variants of TNF-α, the minor allele of the “-308” locus is affecting the TNF-α protein expression under various stimuli36. Results of some previous studies unclear the association of gene variants of TNF-α with OA13. It was found that TNF-α can perform function indepen-dently in OA or in association with other cytokines such as IL-1β, IL-6 etc18. The locus “-1082” polymorphism of IL-10 inhibits the synthesis and production of TNF-α. IL-10 is an anti-inflammatory cytokine might be opposing the function of TNF-α in OA8.

4. ConclusionOA is considered as one of the most prevalent joint disorder in elderly population worldwide. It commonly affects knee and hand joints but other joints also affected with limited function. It affects the daily activity of an individual who are susceptible to OA. Among other risk factors, proinflammatory cytokines such as IL-1, TNF-α are involved in progression of disease. During onset of OA, elevated levels of these cytokines observed in synovial fluid.

The TNF-α along with IL-1β plays important role in car-tilage destruction in joints and leads to progression of OA. Remodelling of joint bone also influence by these cytokines. After binding of TNF-α with their receptors, it activates proin-flammatory signal cascade through different cytosolic kinases and transcription factors.

In continue, proinflammatory cytokines directly force the expression of proteolytic enzymes such as collagenases and others in the synovial fluid of joints. These enzymesare



responsible for breakdown of healthy cartilage and extra cellu-lar matrix components of joint and leads to narrowing of joint space37. This will result in friction of two bone terminus and inflammation.

The gene variants of TNF-α are located in MHC protein encoding region. Among other variants of TNF-α, the locus “-308” has been involved in susceptibility of disease4 that reported in many studies over decay.

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Journal of Ecophysiology and Occupational Health(A Multidisciplinary Reviewed International Research Journal

Concerned with Environmental Sciences, Toxicology and Occupational Health)

1. Instructions to Authors

Minimum Standards for Online SubmissionsPrepare your Manuscript for Online Submission as indicated below. These standards are necessary for peer-reviewing.

• Submitted papers should be in English Language• Manuscript should be submitted as a MS word Document• Manuscript should be prepared as per journal guidelines• All papers are subjected to scientific peer review.• All papers must be submitted online after registering as author

2. Standards for Submission

I. Covering LetterShould contain

• Name, Address, Email id, Mobile No. of the Author for Correspondence.• The letter should mention in brief what is already known about this subject and what new has added by the submitted work.• Title of the paper.• Authors’ initials and names.• An abstract (not exceeding 10 lines) which should be most informative, giving clear indications of the nature and range of the results

contained in the paper, and should not duplicate the conclusions.• 3 to 4 keywords.

II. LengthThere is no restriction on length of manuscript. However a concise manuscript with all information is always well appreciated.

III. AbbreviationsAbbreviations to be defined where they first appear in the text.

IV. NomenclaturePlease follow the correct and accepted Nomenclature

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Journal of Ecophysiology and Occupational Health(JEOH)


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The original research articles should be structured in the following manner.

1. TitleThe title should be concise and reflect the entire work of the submitted manuscript.

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• The purpose of the work• Methods used• Key findings and major conclusion drawn from the work in no more than 500 words• Use of abbreviations in abstract should be avoided however if essential should be expanded at its first appearance

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1.1 Model Description

Botanical Name : Ulam Herbs of Oenanthe javanica and Cosmos caudatus: an overview on their Medicinal Properties

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10. ConclusionGive the major conclusion from the present study. This section may stand alone or be clubbed together with discussion.

11. AcknowledgementsAcknowledge those persons who helped you in the present study by providing facilities, personal assistance and funding if any.

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a. In Text Referencing

• References within the text of the article should be represented by numbers and should be superscripted.• The number originally assigned to a reference should be re-used if that reference is cited again later in the text.• In case of multiple references, separate the numbers with a hyphen. E.g. 2-5, or commas in case of non-inclusive numbers E.g. 3, 9, 14.• As a general rule, reference numbers should be placed outside of full-stops and commas, and inside of colons and semicolons.

Eg. Hoppert M. Microscopic techniques in biotechnology. Weinheim: Wiley-VCH; 2003.



Gillespie NC, Lewis RJ, Pearn JH, Bourke ATC, Holmes MJ, Bourke JB, et al. Ciguatera in Australia: occurrence, clinical features, pathophysiology and management. Med J Aust. 1986;145:584-90.

b. Reference List

• References which are cited in the text should be mentioned here.• It should appear at the end of your text.• It should be arranged numerically by citation number.

c. Referencing Journal Articles

• Elements of the Citation: Author(s) – Family name and initials. Title of article. Title of journal – abbreviated. Publication year, month, day (month and day only if available); volume(issue):pages E.g. 10. Halpern HD, Yadav GA, Caplan KL. Treatment of human brucel-losis: systematic review and metaanalysis of randomised controlled trials. BMJ. 2005 Mar 24;336(7646):701-4.

d. Referencing Books

• Elements of the Citation: Author(s) – Family name and initials, Multiple authors separated by a comma. Title of book. Place of Publication: Publisher Name; Year of Publication. E.g. McKenna DJ, Jones K, Hughes K. Social care practice in rural communities. New York: Oxford University Press; 2000.

e. For Edited books

• Elements of the Citation: Author(s) – Family name and initials, Multiple authors separated by a comma. In name of book editors followed by a comma and editors. Title of book. Edition of book if later than 1st ed. Place of Publication: Publisher Name; Year of Publication. E.g. Rowlands TE, Haine LS. Acute limb ischaemia. In: Donnelly R, London NJM, editors. ABC of arterial and venous disease. 2nd ed. West Sussex. Blackwell Publishing; 2009.

f. Tables

• Tables should be numbered consecutively in accordance with their appearance in the text.• They should be embedded in appropriate locations as per the text along with the captions.• Place Legends for tables below the table body and indicate them with lowercase letters in superscript.• Avoid vertical rules and ensure that the data presented in tables do not duplicate results described elsewhere in the article.• Please note that tables embedded as Excel files within the manuscript are NOT accepted.• Tables in Excel should be copied and pasted into the manuscript Word file.

g. Figures

• These must be numbered and cited in the text.• Mark clearly in the margin of the manuscript where the figure is to be inserted and do not embed in the text.• All figures should be in TIFF.• Images provided should be with below mentioned resolutions:

Black & White Images - 900 dpiColoured Images - 600 dpiLine art- grey colour - 600 dpi.



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h. Overview of Production Process

Published Since 2010

Journal of Ecophysiology and Occupational Health (JEOH) is a Quarterly publication. The Journal is open to everyone who has a scienti�c, technological, practical intrest and inclination in the �eld of science and Technology, Environmental Pollution, Occupational Health and Toxicology.

₹ 2000$150

Volume - 18

Number - 3 & 4

December 2018

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