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Pyrrolizidine alkaloids: Chemical basis of toxicity
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Authors Cooper, Roland Arthur, 1963-
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PYRROLIZIDINE ALKALOIDS: CHEMICAL BASIS OF TOXICITY
by
Roland Arthur Cooper
A Dissertation Submitted to the Faculty of the
COMMITTEE ON PHARMACOLOGY AND TOXICOLOGY (GRADUATE)
In Partial Fulfillment of the Requirements For the Degree of
DOCTOR OF PHILOSOPHY
In the Graduate College
THE UNTVERSITY OF ARIZONA
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2
THE UNIVERSITY OF ARIZONA ® GRADUATE COLLEGE
As members of the Final Examination Committee, we certify that we have
read the dissertation prepared by Roland Arthur Cooper
entitled Pvrrolizidine Alkaloids: Giemical Basis of Toxicity
and recommend that it be accepted as fulfilling the dissertation
requirement for the Degree of Doctor of Philosophy
>-1 h -> JfG
~J ~A Date
Date
Date
Final approval and acceptance of this dissertation is contingent upon
the candidate's submission of the final copy of the dissertation to the
Graduate College.
I hereby certify that I have read this dissertation prepared under my
direction and recommend that it be accepted as fulfilling the dissertation
requii?ement.
Dissertation /Director
/ ^ / - 6 Date '
3
STATEMENT BY AUTHOR
This dissertation has been submitted in partial fulfillment of requirements for an advanced degree at The University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the Library.
Brief quotations from this dissertation are allowable without special permission, provided diat accurate acknowledgment of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when in his or her judgement the proposed use of the material is in die interests of scholarship. In all other instances, however, permission must be obtained by the author.
SIGNED: i
4
ACKNOWLEDGMENTS
This thesis is dedicated to my family, who have provided constant encouragement,
patience, guidance and love throughout my college years.
A special thanks goes to my advisor, Dr Ryan Huxtable, for his support,
friendship, inspiration, some fine dinners and a wonderful trip to Brazil. Also to the rest of
my graduate committee. Dr. Hugh Laird, Dr. Klaus Brendel, Dr. Dan Liebler, Dr. Jim
Halpert, and Dr. Ray Nagle.
I thank many individuals for their assistance with various matters: Dr. Glenn
Sipes, Dr. Frank Porreca, Dr. Ron Lukas, Richard Felger, Carla Hayes, Jane Ageton, and
Sandi Sledge. I also thank Dr. C.C. Yan for the valuable data from the isolated perfused
liver.
I have made many friends while at the University of Arizona who have helped
make my stay in Tucson a lot of fun, from the parties to the great fishing trips in the White
Mountains: Gina Escobar, Steve Barr, Brent Barber, Ed Bilsky, Mike Nichols, Steve
Stratton, Todd Vanderah, Di Bian, Carla Beckham, John Murphy, Rion Bowers, Pierre-
Louis Lieu, Bob Horvath, Sandy Wegert, C.J. Kovelowski, just to mention a few.
5
TABLE OF CONTENTS
LIST OF FIGURES 9
LIST OF TABLES 11
ABSTRACT 12
INTRODUCTION 14
Plant Sources of PAs 15
PA Exposure 15
PA Isolation, Separation and Puriflcation 18
Extraction from plant material 18
Spectrophotometric determination of PAs 18
Preparative chromatographic separation of PAs 19
High-speed counter-current chromatography 19
PA Structure 20
Chemical Properties of DHAs 24
PA Metabolism 25
N-Oxidation 26 Hydrolysis 26 Dehydrogenation to pyrroles 27
Toxic Effects of PAs 28
Hepatotoxicity 29 Pneumotoxicity 33 Neurotoxicity 34 Carcinogenicity and anti-tumor activity 35 Mutagenicity and cytotoxicity 35
Metabolites Responsible for Extrahepatic Toxicity 37
DHAs 37 Sulfur conjugated pyrroles: 7-GSDHP and 7-CYSDHP 38 DHP 39
Effect of PA Structure on Metabolism and Toxicity 40
Lipophilicity 40 Ester hydrolysis 40 Bioactivation to DHAs versus N-oxidation 41
6
TABLE OF CONTENTS - Cont.
DHA reactivity 42
Statement of the Research Problem and Hypothesis 43
METHODS 46
PA Isolation, Separation and IdentiHcation 46
Plant material 46 Alkaloid isolation 46 High-speed counter-current chromatography 47 Thin-layer chromatography 48 Gas chromatography electron impact-mass spectrometry, method 1 48 Gas chromatography electron impact-mass spectrometry, method II 49 Flow-injection atmospheric pressure chemical ionization mass spectrometry 49
Preparation of PA Metabolites and Derivatives 49
DMAs 49 7-CYSDHP (7-cysteinyl-6,7-dihydro-I-hydroxymethyl-5H-pyrrolizine) 50 7-GSDHP (7-glutathionyl-6,7-dihydro-I-hydroxymethyl-5H-pyrrolizine) 50 DHP ((±)-6,7-dihydro- 7-hydroxy-1 -hydroxymethyl-SH-pyrrolizine) 50 9-Ethoxy DHP (7-hydroxy-9-ethoxy-6,7-dihydro-5H-pyrrolizine) 51
7-Ethoxy DHP ((±}-7-ethoxy-I-hydroxymethyl-6,7-dihydro-5H-pyrrolizine).. .51
7,9-Diethoxy DHP ((±)-7-ethoxy-9-ethoxy-6,7-dihydro-5H-pyrrolizine) 51
Toxicity of Sulfur-Conjugated Metabolites of PAs, in vivo 51
7-CYSDHP 51
7-GSDHP 52
Chemistry of Sulfur-Bound Pyrroles 52
Preparation and activation ofThiopropyl-Sepharose 6B resin 52 Recirculating aqueous flow system 53
Identification ofThiopropyl-Sepharose resin-bound pyrroles 54 Identification of liver sulfur-bound pyrroles 54 Quantitation of DHA binding to Thiopropyl-Sepharose resin 55
Studies of DHA Reactivity 56
Alkylation ofDHAs by 4-(p-nitrobenzyl)pyridine 56 Potentiometric measurement of hydrolysis ofDHAs 56
Inhibition of Hexokinase, in vitro 57
Yeast hexokinase assay 57 Rat brain hexokinase assay 58
7
TABLE OF CONTENTS - Cont.
Gliicose-6-phosphate dehydrogenase assay 59
RESULTS 62
High-Speed Counter-Current Chromatography 62
Toxicity of Sulfur-Conjugated Pyrroles, in vivo 69
7-CYSDHP 70 7-GSDHP 70
Chemistry of Suifur-Bound Pyrroles 77
Identification ofThiopropyl-Sepaharose resin-bound pyrrole 77 Chemistry of liver sulfur-bound pyrroles 78 Quantitation ofDHA binding to Thiopropyl-Sepharose resin 78
Hydrolysis of DHAs 80
Alkylation of 4-(p-nitrobenzyl)pyridine by DHAs 83
Inhibition of Hexokinase by DHAs, in vitro 87
Yeast hexokinase 87 Rat brain hexokinase 88
Glucose-6-phosphate dehydrogenase 89
DISCUSSION 98
Counter-Current Chromatography 99
Toxicity of 7-CYSDHP and 7-GSDHP 104
Chemistry of Sulfur-Bound Pyrroles 107
Identification of Sulfur-Bound Pyrrole Ill
Relative Reactivity of DHAs Toward Thiopropyl-Sepharose 112
Hydrolysis of DHAs 113
Alkylation of 4-(p-nitrobenzyl)pyridine by DHAs 115
Inhibition of Yeast and Rat Brain Hexokinase by DHAs, in vitro 117
The Relationship Between the Physicochemical Properties of
DHAs and Their Further Metabolism and Toxicity 121
Macrocyclic diester PAs 121
Open ester DHAs 124
Conclusion 127
8
TABLE OF CONTENTS - Cont.
APPENDIX A 129
Electron Impact-Mass Spectra of PAs 129
REFERENCES 145
9
LIST OF FIGURES
! Structure and numbering system of macrocyciic diester PAs 21
2 Structure of open mono- and diester PAs 22
3 Structure of PA metabolites and derivatives 23
4 Pathways of PA metabolism 30
5 Mechanism of metabolic dehydrogenation of PAs to DHAs by
cytochrome P450 31
6 Proposed mechanism of alkylation by DHAs 32
7 Schematic of recirculating aqueous flow system for "trapping" of DHAs 61
8 Separation of PAs from Amsinckia tessellata using high-speed
counter-current chromatography 65
9 Separation of PAs from Symphytum spp. using high-speed
counter-current chromatography 66
10 Separation of alkaloids from Senecio douglasii var. longilobiis using
high-speed counter-current chromatography 67
11 Separation of PAs from Trichodesma incanum using high-speed
counter-current chromatogaphy 68
12 Representative potentiometric recording of the hydrolysis of
dehydroretrorsine and dehydrotrichodesmine 81
13 Rates of alkyaltion of macrocyciic DHAs by 4-(p-nitrobenzyl)pyridine 85
14 Rates of alkylation of open-ester DHAs by 4-(p-nitrobenzyl)pyridine 86
15 Concentration-effect curve of the inhibition of yeast hexokinase by dehydrotrichodesmine 91
16 Concentration-effect curve for the inhibition of yeast hexokinase by dehydrotrichodesmine and dehydrolycopsamine 92
17 Saturation curves for the inhibition of yeast hexokinase by dehydrotrichodesmine... .93
18 [Substrate/y versus [Substrate] plot of the inhibition of yeast hexokinase by
dehydrotrichodesmine 94
19 Effect of ATP and mannose on the inhibition of yeast hexokinase by dehydrotrichodesmine 95
20 Effect of DHAs on rat brain hexokinase, in vitro 96
21 Effect of dehydrotrichodesmine and dehydrolycopsamine on rat brain hexokinase,
in vitro 97
22 Proposed reaction of DHAs and Thiopropyl-Sepharose resin 109
23 A) Proposed mechanism of cleavage of sulfur-bound pyrroles by
mercuric chloride or silver nitrate. B) Structures of pyrrolic ethyl ethers
formed by solvolysis of sulfur-bound pyrroles by ethanolic silver nitrate 110
24 Proposed reaction between DHAs and 4-(/;-nitrobenzyl)pyridine 115
10
LIST OF FIGURES - Cont.
25 Structure of 2,3-dihydro-l^-pyrroIizine-l-oI and dehydrosupinidine 116
26 Schematic of the hexokinase assay 118
27 Electron impact-mass spectrum of monocrotaline 128
28 Electron impact-mass spectrum of trichodesmine 130
29 Electron impact-mass spectrum of incanine 131
30 Electron impact-mass spectrum of retrorsine 132
31 Electron impact-mass spectrum of seneciphylline 133
32 Electron impact-mass spectrum of senecionine 134
33 Electron impact-mass spectrum of florosenine 135
34 Electron impact-mass spectrum of lycopsamine/intermedine 136
35 Electron impact-mass spectrum of 3-acetyl-lycopsamine/-intermedine 137
36 Electron impact-mass spectrum of 7-acetyl-lycopsamine/-intermedine 138
37 Electron impact-mass spectrum of 3,7-diacetyl-lycopsamine/-intermedine 139
38 Electron impact-mass spectrum of symphytine and isomers 140
39 Electron impact-mass spectrum of 7-ethoxy DHP 141
40 Electron impact-mass spectrum of 9-ethoxy DHP 142
41 Electron impact-mass spectrum of 7,9-diethoxy DHP 143
42 Positive ion FAB mass spectrum of 7-CYSDHP 144
11
LIST OF TABLES
1 TLC Conditions for PAs and Pyrroiic Derivatives 60
2 Results of Separation of Pyrrolizidine Alkaloid Fractions Using
High-Speed Counter-Current Chromatography 64
3 Summary of Wet Organ Weight / Body Weight Ratios in Rats at 21 Days
Following a Single Injection (i.v.) of 7-CYSDHP 71
4 Summary of Dry Organ Weight / Body Weight Ratios in Rats at 21 Days
Following a Single Injection (i.v.) of 7-CYSDHP 72
5 Summary of Wet Organ Weight / Dry Organ Weight Ratios in Rats at 21
Days Following a Single Injection (i.v.) of 7-CYSDHP 73
6 Summary of Wet Organ / Body Weight Ratios in Rats at 21 Days
Following a Single Injection (i.v.) of 7-GSDHP or Monocrotaline 74
7 Summary of Dry Organ / Body Weight Ratios at in Rats at 21 Days
Following a Single Injection (i.v.) of 7-GSDHP or Monocrotaline 75
8 Summary of Wet Organ / Dry Organ Weight Ratios in Rats at 21 Days
Following a Single Injection (i.v.) of 7-GSDHP or Monocrotaline 76
9 Relative Quantity of Pyrrole Bound to Thiopropyl-Sepharose Resin After
Exposure to DHAs 79
10 Rate Constants and Half-Lives of Hydrolysis of DHAs 82
11 Rate Constants for the Alkylation of DHAs by 4-(p-nitrobenzyl)pyridine 84
12 Effect of Dehydrotrichodesmine on Yeast Hexokinase Phosphotransferase
Activity in the Presence of Increasing Substrate Concentrations 90
13 Effect of Mannose (10 mM) and ATP (10 mM) Pretreatment on the Inhibition
of Yeast Hexokinase by Dehydrotrichodesmine 90
14 Relationship Between Pattern of Metabolism of PAs in the Isolated Perfused
Rat Liver and DHA Half-Life 125
15 Tissue-Bound Pyrroiic Metabolites 24 hr After i.p. Injection of PAs in Rats 126
12
ABSTRACT
In humans, livestock and experimental animals, pyrrolizidine alkaloids (PAs) are
toxic as a consequence of their hepatic metabolism to reactive pyrrolic esters, or
dehydroalkaloids (DHAs). Despite their similarity in structures, PAs often vary markedly
in their lethality (LDjgS) and in the organs in which toxicity is expressed. We have
examined whether there are differences in the physicochemical properties of certain DHAs
which are associated with differences in patterns of metabolism and toxicity produced by
the parent PA. Using a potentiometric method to measure hydrolysis, it was determined
that the half-lives of the corresponding DHAs of retrorsine, seneciphylline, monocrotaline
and trichodesmine were 1.06, 1.60, 3.39 and 5.36 sec, respectively. These values were
supported by similar results from experiments measuring reactivity of DHAs toward 4-{p-
nitrobenzyOpyridine. Studies from the isolated rat liver perfused with PAs show that DHA
stability is related to patterns of metabolism and toxicity. Perfusion of the primarily
hepatotoxic retrorsine and seneciphylline is associated with a greater proportion of
metabolite released as non-toxic 7-glutathionyl-6,7-dihydro-l-hydroxymethyl-5/f-
pyrrolizine (7-GSDHP), a greater proportion alkylating liver macromolecules, and a lower
proportion released as DHA into the circulation. Perfusion with monocrotaline and
trichodesmine, PAs producing extrahepatic toxicity, produced lower proportions of 7-
GSDHP release and liver alkylation, and higher proportions of DHA released into the
circulation.
Other studies characterizing DHAs included the use of an in vitro enzyme assay in
which DHAs were shown to inhibit the phosphotransferase activity of yeast and rat brain
hexokinase. Parent PAs, and the hydrolysis product of DHAs, (±)-6,7dihydro-I-
hydroxymethyl-5f/-pyrrolizine (DHP) did not affect enzyme activity. In vivo smdies in
rats have established that glutathione and cysteine-conjugated pyrrolic metabolites of PAs
13
likely represent detoxication pathways, providing further support for DHAs as the primary
toxic metabolite. We have examined the chemical form of suifur-bound pyrroles to
establish the importance of the 7-ester position in PA toxicity. Additionally, we have
developed an efficient technique for the rapid separation and purification of large quantities
of PAs using high-speed counter-current chromatography.
14
INTRODUCTION
Pyrrolizidine alkaloids (PAs) are a large and structurally similar group of plant
compounds, most of which are toxic to animals, including man. PAs are fascinating tools
for scientific study for many reasons. PAs are important in plant chemosystematics (Kelley
and Seiber, 1992) and they are studied for their role in insect chemical defense and as
precursors to the biosynthesis of pheromones in moths and butterflies (Boppre, 1986). To
the toxicologist, PAs are of special interest because they require hepatic metabolism to
produce toxicity. Additionally, they are ideal for structure-activity studies because despite
similar structures, they sometimes produce markedly different toxicities.
PAs represent a significant world-wide public health risk. Human exposure results
from the use of PA-containing plants as food, herbal medicines or in beverages (Huxtable,
1989). Liver disease is the most common effect produced in humans, but extrahepatic
effects are observed in experimental animals and livestock. Human poisoning epidemics
have occurred in several countries due to contamination of cereal grains with PA-containing
plants. PAs are probably the most significant plant toxins in terms of their ill effects to the
health of man and animals, and their associated economic costs (Huxtable, 1989). The
continued study and dissemination of knowledge concerning PAs is an important step
towards increasing awareness and subsequent reduction of PA poisonings.
Toxicity of PAs is related to their metabolism to reactive pyrrole intermediates,
which can bind to cellular constituents (Mattocks, 1986; Glowaz et al., 1992). The
relationship between PA structure and toxicity is both complicated and poorly understood.
Some PAs such as retrorsine are generally hepatotoxic, while monocrotaline is
pneumotoxic as well. Trichodesmine is a potent neurotoxin. How structurally similar
alkaloids produce such different toxicities is unclear. We have addressed this question by
investigating the chemical properties of a series of PA metabolites, and how their respective
15
structures influence their stability, patterns of metabolism and toxicity. The results provide
further information on the relationship of hepatic metabolism of xenobiotics and tlie
extrahepatic toxicity they produce.
Plant Sources of PAs
More than 350 PAs have been identified, from over 300 plant species (Huxtable,
1989; Logie et al., 1994). This represents approximately 3% of the worlds flowering plant
species. PA containing genera have worldwide distribution. Some important sources are
the genus Crotalaria (Leguminosae), the tribe Senecionae (Asteraceae), and the
Boraginaceous genera. Amsinckia, Heliotropium, Symphytum and Trichodesmine (Smith
and Culvenor, 1981; Mattocks, 1986). Many plants contain both PAs and a variable
proportion of the corresponding A'-oxide. Quantitative and qualitative PA content of a
particular plant may be influenced by its location, time of year, or other environmental
factors.
PA Exposure
Humans are exposed to PAs because of either intentional or unintentional
consumption of plant products containing them. These products may be consumed in the
form of herbal medications or teas, as green vegetables, or as grain contaminated with PA
containing seeds. PAs have been found in honey produced by bees feeding on the nectar of
plants (Deinzer et al., 1977; Culvenor et al., 1981) containing them, and in the milk of
goats and dairy cows fed on ragwort {Senecio jacobaea) (Dickinson et al., 1976;
Dickinson, 1980). However, there are no known cases of human toxicity resulting from
the consumption of contaminated milk or honey (WHO, 1988). Chronic and heavy herb
users, young children, elderly, and sick individuals are particularly susceptible to
intoxication (Huxtable, 1990b). Additionally, certain ethnic groups such as Mexican-
16
Americans and Native Americans are at risk to PA exposure because of extensive traditional
use of herbal remedies. Prevention of poisoning lo humans can be achieved by controlling
PA containing plants in agricultural areas, by educating populations at risk, and by banning
the sale of PA containing herbal products.
Exposure to PAs by the use of herbal products in the form of infusions or medicinal
remedies occurs for several reasons. Ingestion may be intentional, as in the case of comfrey
(Symphytum) preparations, or accidental, such as when an herbal product is incorrectly
identified. Medicinal use of herbs in traditional medicine has long been practiced in many
countries including China, Greece, India and the United States (WHO, 1988). Many cases
of suspected PA poisonings have occurred in the United States and other countries as result
of such use (Huxtable, 1989). The incidences of these poisonings likely will continue in
light of the growing interest in herbal products as food and home remedies in the
developing world (WHO, 1988; Sperl etal., 1995).
PA-induced veno-occlusive disease of the liver was endemic in Jamaica and other
West Indian countries for many years (Hill and Rhodes, 1953; Bras et al., 1954; Stuart and
Bras, 1956; Stuart and Bras, 1957). This illness was associated with the consumption of
herbal or "bush" teas which often contained Crotalaria, in particular C.fidva, although
Senecio has been implicated as well (Bras et al., 1957). Herbal teas were frequently used
as infant remedies, and many children died of massive ascites associated with veno-
occlusive disease of the liver. The morphological changes to the liver brought about by this
disease were first recognized and described by Bras et al. (1954).
Comfrey (Symphytum spp.) is cultivated in many areas of the world for use as an
herbal tea, digestive-aid and cosmetic. These preparations are known to contain PAs and
are implicated in cases of PA poisoning (Ridker er a/., 1985; Huxtable, 1989;. PA levels in
comfrey products vary considerably, depending on the species and strain of Symphytum
and the plant part utilized. Comfrey root, which is frequently used for herbal teas, contains
17
the highest concentrations of alkaloid. Long-term use of comfrey products is common
(Huxtabie eiai., 1986); thus effects of toxicity may appear after chronic use. In addition to
being hepatotoxic, some of the alkaloids from Symphytum are known to be carcinogenic in
rats (Hirono et ai, 1978, 1979). Comfrey products are still widely available in the United
States.
Humans may accidentally be poisoned by contamination of food grains with PA-
containing plant material, particularly seeds. In 1950, an outbreak of Trichodesma
poisoning occurred in the Samarkand region of Uzebekistan, killing at least 44 people
(Ismailov et al., 1970). More than 200 individuals were affected, due to the consumption
of bread prepared from grains contaminated with the seeds of T. incanum. Deaths were
attributed to central and peripheral neuropathies. Until recently, PA poisoning in India,
Afghanistan and the former USSR was endemic (Huxtabie, 1989) due to contamination of
grains with Heliotropium species. These outbreaks of PA poisoning are among the largest
known, with some 35.000 people exposed in one epidemic of liver disease in Afghanistan
(Mohabbat erfl/., 1976; Tandon era/., 1978a, 1978b).
PA poisoning of livestock is a serious economic problem in some areas of the
world (Mattocks, 1986). Many PA containing plants, such as Senecio jacobaea (ragwort)
and various Crotalaria thrive during drought or in pastures of poor condition. Thus they
may be eaten by stock during times of hardship. At one time, S. jacobaea was said to cause
more cattle losses than all other poisonous plants combined (Mattocks, 1986). Cattle
feeding on Senecio typically display liver toxicity characterized by hepatic lesions and
veno-occlusive disease (Thorpe and Ford, 1968; Johnson et al., 1985; Mattocks, 1986).
Livestock which have been grazing on Crotalaria frequently display extrahepatic
symptoms, especially to the lungs and kidneys (Mattocks, 1986).
18
PA Isolation, Separation and Puriflcation
Extraction from plant material
PAs are extracted from either fresh or dried plant material in the form of ground
leaves, stems, seeds, etc. Plant material is generally extracted with hot alcohol in a Soxhlet
apparatus. The alcohol is evaporated and the residue is resuspended in dilute acid. This
solution is extracted with an organic solvent to remove chlorophylls and lipids. Zinc dust is
added to the acidic aqueous portion to reduce A/^oxides to their respective tert-hases. The
solution is made basic with ammonia and extracted with chloroform, yielding the alkaloidal
fraction. Extracts containing polar alkaloids such as lycopsamine are saturated with NaCl to
increase alkaloid extraction (Culvenor and Smith, 1966). If the fraction contains only one
PA, it may often be obtained pure by recrystallization from alcohol. Other alkaloidal
fractions are obtained crystalline, but are comprised of several PAs which must be
separated chromatographically (Adams and Govidachari, 1949). Fractions homAmsinckia
or Symphytum are typically isolated as brown gums (Culvenor and Smith, 1966; Roitman,
1983; Cooper et al., 1996), resisting all efforts at crystallization. Most fractions are isolated
as PA mixtures, often in the form of stereoisomers.
Spectrophotometric determination of PAs
Unsaturated PAs may be quantified spectrophotometrically by reacting with Ehrlich
Reagent after the PA has been converted to a pyrrole (Mattocks, 1967, 1968a). Initially, the
PA is oxidized with hydrogen peroxide to its respective A^-oxide which is then converted to
a pyrrole by acetic anhydride. Pyrroles react strongly with modified Ehrlich Reagent (4-
dimethylaminobenzaldehyde in acidified ethanol) (Figure 3) to produce a magenta solution
with a at 562 nm. This method of detection is sensitive (below 5 |ig for some PAs)
and reproducible (Mattocks, 1968a). PAs may be visualized on TLC plates by first
19
spraying with methanolic o-chloranil, followed by Ehrlich Reagent. Pyrroles may be
visualized by Ehrlich Reagent alone. Both yield magenta colored spots when treated in this
manner.
Preparative chromatographic separation ofPAs
As only monocrotaline has been available commercially, it is often necessary to
isolate and purify large amounts of PAs for toxicological or other studies. Mixmres of PAs
have been separated and purified with varying degrees of success using column
chromatography with different types of adsorptive media. Typical matrices have been
alumina and silica (Segall, 1984; Mattocks, 1986). However, column chromatography is
slow, and repeated runs are often necessary for complete or partial resolution of alkaloids
(Adams and Govindachari, 1949). Preparative HPLC has recently been used for separation
of PAs (Segall, 1984). Columns employed are reversed phase C-18 or PRP types.
Separations are rapid, with good resolution, but columns are expensive and cannot separate
large quantities. PAs have been separated with droplet counter-current chromatography
(DCCC) (Zalkow era/., 1988; Asibal era/., 1989a, 1989b; Culvenorera/., 1980) with
excellent results, but the technique is slow and has generally been replaced by the newer
high-speed method described below.
High-speed counter-current chromatography
Counter-current chromatography (CCC) is a method of liquid-liquid partition
chromatography that does not employ a solid matrix. Ito et al. (1966) observed that two
immiscible liquids will become uniformly segmented in the coils of a helical column when
the latter is rotated. The liquid designated as the stationary phase is retained by centrifugal
force, while the mobile phase is pumped through die rotating column. Advantages of high
20
speed CCC include total sample recovery, wide choice of solvent systems, rapid separation
Limes, good resolution (350-1000 plates) and high reproducibility. Since there is no solid
support mechanism within die column, sample contamination, adsorption, and denaturation
are normally eliminated (Conway, 1990). A recently developed instrument for high-speed
CCC, the multilayer coil planet centrifuge (Ito et al., 1982), is well suited for the rapid
separadon and purification of natural products on a preparative scale (Conway, 1990;
Fischer a/., 1991; Zhang era/., 1988; Marston and Hostettman, 1994).
PA Structure
PAs are generally derivadves of 1-methylpyrrolizidine (Mattocks, 1986), consisting
of two fused five-membered rings sharing a common nitrogen at the 4 position (Fig. 3).
When hydroxylated, this structure is referred to as a necine. Necine bases may also be M-
methylated, forming otonecine. Most PAs have an acid moiety, termed a necic acid,
esterifying either or both the 7 or 9-hydroxyl position of necine. Hepatotoxic PAs are esters
of unsaturated necines, containing a double bond in the 1:2 position of the pyrrolizidine
ring. PAs may exist as monoesters, diesters, or as macrocyclic diesters. Figures 1 and 2
shows the structures and numbering system of PAs used in this dissertation. Many necic
acids can comprise the ester portion of PAs. These often differ slightly, and many exist as
stereoisomers. Differences in the acid structure can cause marked differences in toxicity
produced by the parent PA. The macrocyclic diesters such as retrorsine are generally the
most toxic. Open diester PAs such as symphytine or lasiocarpine may be as hepatotoxic as
some macrocyclics, but others are much less so (Mattocks, 1986). Monoester PAs such as
lycopsamine and indicine are weakly hepatotoxic in comparison to diester PAs (Fowler and
Schoental, 1967; Schoental, 1968).
21
CH
3 5
R=H; Senecionine R=OH; Retrorsine
CH
Seneciphylline
18
R'=Me, R"=OH; Monocrotaline
R'=CH(CH3)2, R-=H; Incanine
R'=CH(CH3)2, R"= OH; Trichodesmine
O II
.CH
CH
Florosenine
Figure 1. Structure and numbering system of macrocyclic diester PAs. All alkaloids are retronecine based, except for florosenine, which contains an otonecine base. Hepatotoxic PAs are esters of unsaturated necines, containing a 1-2 double bond.
22
I JZHs
O CH
^0-C-C-«OH H J I
\ R3.-C-CH:
V 'r-
R'=Me, R^=H, R^=H, R'^=OH; Symphytine
R'=H, R"=Me, R^=H, R'^=OH; Symlandine
R'=Me, R~=H, R^=OH, R'^=H; Myoscorpine
R'=:H, R~=Me, R^=OH, R'^=H; Echiumine
R'=H, R"=H, R^=0H; Lycopsamine
R'=H, R^=0H, R^=H; Intermedine
R'=H, R"=H, R^=AC; 3-Acetyl-lycopsamine
R'=H, R"=AC, R^=H; 3-AcetyI-intermedine
R'=AC, R-=H, R^=0H; 7-Acetyl-lycopsamine
R'=AC, R^=0H, R^=H; 7-Acetyl-intemiedine
R'=AC, R^=H, R^=AC; 3,7-Diacetyl-lycopsamine
R'=AC, R^=AC, R^=H; 3,7-Diacetyl-intermedine
HjC CH3 \ /
O CH
Figure 2. Structure of open mono- and diester PAs.
CH
I -methylpyrrolizidine
R' = H, R~ = OH; Dehydroretronecine
R' = OH, R" = H; Dehydroheliotridine
COtH O O I ' II II I CH^NHC- CH- NHC-CH2CH2CHNH0
I
OH
7-GSDHP
OH HO
DHP
ROCO OCOR'
NMe CH
Ehrlich Adduct, 562 nm
O II
H-,NCHCOH
CH
OH
7-CYSDHP
Figure 3. Structure of PA metabolites and derivatives. PAs are derivatives of 1-methylpyrrolizidine. DHP is a racemic mixture of dehydroretronecine and dehydroheliotridine.
24
Chemical Properties of DHAs
DHAs are formed in the iiver by the metabolic dehydrogenation of unsaturated PAs,
catalyzed by cytochrome P-450. The DHAs are also easily prepared chemically from PAs
by oxidation with the quinones o-bromanil or o-chloranil (Mattocks etal., 1990). The
subsequent pyrrolic product differs from the PA in that it contains an additional double
bond in the necine ring (Figure 4). Because the DHA is much more reactive than the parent
PA, they are characterized by their susceptibility to hydrolysis and polymerization under
acidic conditions and rapid reactivity toward nucleophiles (Culvenor etal., 1970). This
makes DHAs difficult to work with since special handling is required. Their inherent
reactivity is also why they have never been isolated intact from animals exposed to PAs or
from in vitro systems such as liver microsomes. DHAs are reactive because the conjugated
double bonds within the pyrrole ring leads to a dispersion of charge, made possible by the
resonance donation of pi electrons from the nitrogen atom. This conjugation makes the
esters linked to the pyrrole at C7 and C9 labile, hence they become good leaving groups. In
an aqueous environment, this instability leads to rapid hydrolysis and formation of a more
stable pyrrolic alcohol, (±)-6,7-dihydro-7-hydroxy-I-hydroxymethyl-5//-pyrrolizine
(DHP) (Kedzierski and Buhler, 1986). The extent of racemization at C7 when DHAs react
with alcohols or water indicates hydrolysis proceeds through an S^l mechanism, via
formation of a pyrrole-stabilized carbonium ion (Figure 5) (Mattocks, 1986; Kedzierski and
Buhler, 1986). Thus DHP is a racemic mixture of dehydroheliotridine and its enantiomer,
dehydroretronecine. Diester PAs are potentially bifunctional alkylating agents because they
contain a ester at both C7 and C9. The C7 carbonium ion is more reactive than C9 as it
forms a more stable secondary carbonium ion as opposed to a primary carbonium ion
formed at C9. Thus, once substitution has occurred at C7, a similar reaction can occur at
C9. Formation of stabilized carbonium ions allows covalent reactions with cellular
nucleophiles such as glutathione or macromolecules (Figure 5). The macrocyclic DHAs
25
have been shown to cross-link DNA (Petry et al., 1984; Hincks et al., 1991), presumably
due to their bifunctionality. DHP is an amino alcohol and is considerably more stable than
DHAs. This is because hydroxyl functions do not make good leaving groups, despite the
resonance stabilization of the pyrrole ring. Under acidic conditions however, DHP can
react with nucleophiles (Mattocks and Bird, 1983b) and can alkylate DNA (Petry etai,
1984; Hincks era/., 1991).
PA Metabolism
PA must be metabolized to produce toxicity. Only the liver appears capable of
bioactivation of PAs to toxic metabolites. Several lines of evidence, reviewed by Mattocks
(1986), support the idea that PA metabolites, and not the parent PAs, are responsible for
toxicity: (1) PAs are not toxic at injection sites, nor are they toxic to the skin; (2) the liver
is the main target organ of toxicity, regardless of route of administration; (3) drug
treatments which modify metabolism influence the toxicity of PAs; 4) animals vary greatly
in susceptibility to PAs; 5) PAs are rather inert compounds, and they would not be
expected to react with cellular constituents under physiological conditions; 6) many insects
accumulate large amounts of PAs in their tissues without harm.
In the laboratory animal, the main routes of metabolism for PAs are ester
hydrolysis, yV-oxidation and dehydrogenation to pyrrolic products (Figure 4). The first two
pathways represent detoxication mechanisms, while the latter is associated with toxicity.
Additionally, much alkaloid is excreted unchanged, principally in the urine (Eastman et al.,
1982). The balance of these pathways will determine the eventual biological effect
produced by a PA in a particular animal (Mattocks, 1986). The toxic properties of pyrrolic
metabolites are consistent with the cytotoxic action of PAs, and it is improbable that other
important routes of bioactivation will be discovered (Mattocks, 1986).
26
N-Oxidation
Of the two major oxidation products formed by the hepatic metabolism of PAs,
A^-oxidation represent a detoxication pathway. Many species metabolize PAs to their
corresponding N-oxides, including rats, mice, hamsters, guinea pig, sheep, quail, fowl,
and humans (Mattocks, 1968; White et al., 1973; Williams et al., 1989). PAs may be
converted to ^/-oxides by both P450 and flavin-containing monooxygenase (FMO), with
the requirement for and NADPH. The relative contribution by either enzyme is species
and tissue dependent. Purified pig liver FMO has been shown to oxidize senecionine to its
A/^oxide, while in rat liver, P450 makes the most significant contribution (Williams et ai.
1989). In humans, P4503A4 is the major catalyst for the conversion of senecionine to its
A^-oxide (Miranda et al., 1991). Guinea pig liver, lung, and kidney microsomes convert
senecionine primarily to A'-oxide via FMO, with little pyrrole activation observed (Miranda
et al., 1991). This is in keeping with the resistance to PA intoxication by guinea pigs. The
A/-oxides are water soluble and are rapidly excreted in the urine (Mattocks, 1968a).
Additionally, they do not appear to be a substrate for pyrrole formation (Jago et al., 1970:
Mattocks and White, 1971). Although A/-oxides may be reduced to pyrrolizidine bases in
the liver, this appears to be a minor pathway (Powis and Wincentsen, 1980). A/^oxides are
extensively reduced in the gut however, and the alkaloid base is reabsorbed. Rats, sheep
and rabbits all have the capacity to reduce A^-oxide PAs (Mattocks, 1971; Powis et ai,
1979). The reduction of A^-oxides to tertiary bases in the gut is made evident by their higher
toxicity when administered orally rather than i.p. (Mattocks, 1972).
Hydrolysis
PAs are subject to enzymatic hydrolysis by hepatic esterases (Mattocks, 1972,
1982; Dueker et al., 1992, 1995; Chung and Buhler, 1995). This results in the formation
27
of a necine base and necic acid, neither of which are known to be toxic (Culvenor et al.,
1976). Hydrolysis is tlius considered a detoxication pathway for PAs. Hydrolysis of PAs
is markedly reduced in rats pretreated with tri-orthocresylphosphate, an esterase inhibitor,
and subsequently formation of toxic pyrrolic metabolites is increased (Mattocks, 1981;
Mattocks, 1986). Both chemical (base hydrolysis) and enzymatic hydrolysis (rat liver
homogenates) of natural PAs and synthetic derivatives are strongly influenced by steric
hindrance from the acid substitution in the vicinity of the ester groups (Mattocks, 1982).
PAs containing esters with short unbranched chains such as acetate and propionate are
subject to rapid hydrolysis both chemically and enzymatically. Open branched diesters,
such as retronecine ditiglate, or macrocyclics like monocrotaline and retrorsine are much
more resistant to hydrolysis. Hepatic esterase hydrolysis of PAs may be a relatively minor
metabolic pathway in rats, but appears be significant in guinea pigs (Chung and Buhler,
1995). The contribution of esterases in blood and other tissues toward PA hydrolysis is
unknown. Guinea pigs are especially resistant to PA toxicity (White et al., 1973; Mattocks,
1986; Miranda et al., 1991). However, they are particularly sensitive to the PA jacobine
(Swick et al., 1982). Recent studies have shown that purified guinea pig carboxylesterases
GPHl and GPLl are weakly active toward jacobine in comparison to other PAs (Chung
and Buhler, 1995; Dueker et al., 1995). In the whole animal, more jacobine may be
available for bioactivation to DHA, since hydrolysis of the alkaloid appears limited.
Dehydrogenation to pyrroles
PAs are metabolized in the liver to pyrroles by the cytochrome P450s, an enzyme
system responsible for the detoxication of many foreign compounds. These heme-
containing enzymes catalyze oxidative metabolism by the transfer of an oxygen atom or
oxidative equivalent into a variety of xenobiotics. Occasionally, this metabolism results in
28
the generation of reactive, electrophilic compounds, a process termed "bioactivation"
(Vamvakas and Anders, 1990). Tne toxicity of many drugs, chemicals and pollutants
results from the formation of reactive intermediates during their metabolism.
The P450s exist as isozymes in a given animal species. In rats and man, the
P4503A isoform is responsible for the majority of pyrrole formation (Williams et al., 1989:
Miranda et al., 1991), while P4502B appears to be the most significant isoform in the
guinea pig (Chung et al., 1995). Pyrrole formation is associated with the cytotoxicity of
PAs (Mattocks, 1968; Mattocks and White, 1971; White et al., 1973). As proposed by
Mattocks, (1968) PAs are initially dehydrogenated to pyrrolic esters, or DHAs (Figure 6).
Dehydrogenation is believed to result from hydroxylation at C8 on the unsaturated necine
moiety (Mattocks and White, 1971; Mattocks and Bird, 1983), forming a chemically
unstable carbinolamine. This species would spontaneously decompose, losing first OH"
and then H"", to yield the DHA (Figure 6) (Mattocks, 1986). Once a DHA has formed, it
can alkylate macromolecules and soluble nucleophiles such as glutathione or rapidly
hydrolyze to DHP. This compound was identified as the major urinary metabolite in rats
given i.p. monocrotaline (Hsu etal., 1973), providing indirect evidence for the formation
of dehydromonocrotaline. DHP is also produced from PAs using in vitro metabolism
systems (Mattocks, 1968; Jago etal., 1970; Glowaz etal., 1992). Relative pyrrole levels in
the liver following exposure to different PAs correlate well with the acute hepatotoxicity of
the parent alkaloids (Mattocks, 1972). The toxicity of the DHAs, and their hydrolysis
product, DHP, is discussed below.
Toxic Effects of PAs
PA toxicity is a consequence of their hepatic metabolism to pyrroles. These
metabolites are electrophilic and bind covalently to tissue nucleophiles, either in the liver or
29
at more distant sites sucli as the lungs. The covalent modification of critical cellular
macromolecuies, including proteins and nucieic acids, presumably iniuates ceil damage.
Hepatotoxicity
The primary acute action of PAs in laboratory animals and humans is
hepatotoxicity, producing a pathognomonic syndrome known as veno-occlusive disease of
the liver (Bras et ai, 1954). Acute poisoning in animals leads to zonal hemorrhagic
necrosis followed by a narrowing of the endothelium of the small hepatic veins leading to
occlusion. Hepatic sinusoids may become dilated and pooled with blood, a finding
resembling the human condition. HepatocytomegaJy and fibrotic lesions are characteristic
pathologies observed in animals with chronic PA exposure. Megalocytes are produced as a
result of the antimitotic activity of PA metabolites on parenchymal cells. Interestingly,
megalocytes have not been observed from the liver of humans exposed to PAs (Mattocks et
ai, 1986). Hepatic veno-occlusive disease in man due to PA consumption has been well
documented (Hill and Rhodes, 1953; Bras et al., 1954; Bras and Hill, 1956; Stuart and
Bras, 1957). The acute human condition is characterized by portal hypertension with
massive ascites attributable to occlusion of centrilobular or sublobular hepatic veins (Sped
et al., 1995). The patient (most often a child) experiences rapid onset of abdominal
discomfort, with a smooth and firm hepatomegaly (Stuart and Bras, 1957). Abdominal
swelling due to ascites and dilated veins in the abdominal wall are observed. Death may
shortly ensue, but veno-occlusive disease is often reversible if PA exposure is stopped and
the patient given conservative therapy (Bras and Hill, 1956, Lyford et al., 1976; Sperl et
al., 1995). The chronic condition is characterized by cirrhosis, which is clinically
indistinguishable from other forms of cirrhosis. Jaundice may be observed and there is
biochemical evidence of liver dysfunction.
30
ROCO ROCO HO OH
Hydrolysis
P-450, FMO Esterases
PA
PA A'-oxide
Dehydrogenation P-450
Retronecine
HO ROCO OH GS OH
Hydrolysis GSH •Nv
DHP
-N^
DHA
I GSH \
7-GSDHP
Alkylation of cellular nucleophiles
HEPATIC AND EXTRAHEPATIC TOXICITY
Figure 4. Pathway^f PA metabolism. Cytochrome P-450s, flavin-containine esterases catalyze the metabolism of PAs Toxicity is a result
of dehydrogenation by cytochrome P-450 to a reactive pyrrole intermediate, th^DF^
31
OCOR"
P-450
Liver
OCOR
-OH
OCOR" RO OCOR" RO
H
Figure 5. Mechanism of metabolic dehydrogenation of PAs to DHAs by hepatic cytochrome P-450, as proposed by Mattocks and Bird (1983). The first step in bioactivation is the hydroxylation of the pyrrolizidine ring at C8. This results in an unstable carbinolamine which decomposes to a dihydropyrrolizine (DHA) by losing first OH" followed by H"^.
32
CHiO- C- R-R ' — C - 0 Nu CH^O-C- R-
Nu
O. 11^ , CH2O- C- R- Nu Nu Nu
Nu
Nu = nucleophile
Figure 6. Proposed mechanism of alkylation by DHAs. Esters linked to the pyrrole ring are made labile by their proximity to nitrogen. This structure favors the formation of pyrrole stabilized carbonium ions at C7 and C9, which proceeds through an Sjsjl mechanism. The positively charged pyrrole nucleus can then react with cellular nucleophiles, likely producing the toxicity associated with PAs. From Huxtable (1990).
33
Pneiimotoxicity
Many PAs effect the lungs of certain animals, producing pulmonary arterial
hypertension and right ventricular hypertrophy. This subject has been reviewed by
Huxtable (1990). The 11-membered macrocyclic diesters such as fulvine and
monocrotaline are the most active in producing lung damage, although the Senecio alkaloid
seneciphylline was reported to be pneumotoxic in one instance (Ohtsubo et al., 1977).
Monocrotaline is often used to produced the syndrome in animal models due to the
similarity to the human condition (Kay and Heath, 1969; LaFranconi and Huxtable, 1981).
In the rat, the appearance of pulmonary hypertension following exposure to a single dose
of monocrotaline is delayed for up to 2 weeks despite the appearance of liver damage. The
pneumotoxic process is initiated upon exposure to the alkaloid, which is largely excreted
and metabolized by 24 hours (Candrian et al., 1985; Huxtable, 1990). Following exposure
to the pneumotoxic PA, a remodeling of the pulmonary vasculature occurs associated with
endothelial hyperplasia, arterial medial thickening, spreading of arterial smooth muscle into
unmuscularized areas, smooth muscle hypertrophy and increased lung mass. Platelet
accumulation initiates the formation of microdirombi in the capillaries, which leads to
capillary occlusion and edema. Row resistance and thus arterial pressure is increased,
forcing a greater workload upon the right heart. This in turn produces right heart
hypertrophy and cor pulmonale. The degree of pulmonary arterial hypertension produced
by monocrotaline strongly correlates with the degree of right heart hypertrophy (Kay and
Heath, 1969; Huxtable etal., 1978; Huxtable, 1990). Many complex biochemical changes
occur during PA-induced pulmonary hypertension, including alterations in the metabolism
of the vasoactive substances serotonin and angiotensin (Huxtable etal., 1978; Huxtable.
1979; LaFranconi and Huxtable, 1983). Increases in the synthesis of protein and RNA
occur both in the right heart and the lung (LaFranconi et al., 1984).
34
Neurotoxicity
Neurotoxicity is observed in some instances of PA poisoning of livestock,
especially in horses (Rose et ai, 1957a, 1957b). The non-specific effects reported were
attributed to a hyper-ammonemia following liver disease. Spongy degenerations in the
central nervous systems in cattle, sheep and pigs have been reported in animals exposed to
PAs (Hooper ef a/., 1974; Hooper, 1975a, 1975b).
The Trichodesma alkaloids produce neurotoxicity as their primary action (WHO,
1988; Huxtable, 1989), as opposed to the hepatotoxicity associated with other PAs. The
neurotoxicity of Trichodesma in dogs, rabbits and mice has been reviewed in the Russian
literature (Shtenberg et al., 1955; Ismailov et al., 1970). Mice treated with Trichodesma
alkaloids develop hind limb paralysis, opisthonus and clonic convulsions. An equine
disease known as "suiljuk" is associated with consumption of T. incanum by horses
(Smimov etal., 1959a, 1959b, Smimov and Stoljarova, 1959). Degenerative changes in
liver, lung, kidneys and heart were reported. Cows are similarly effected (Smimov et al.,
1959b). An outbreak of human PA poisoning due to T. incanum contaminated grain
occurred in the Samarkand region of Uzebekistan from 1942-1951 (Ismailov etal, 1970).
The disease, known as "Ozhalanger encephalitis", affected over 200 adults. Curiously,
children under the age 10 were not affected. Observed symptoms, appearing after a 10 day
latency, included nausea and vomiting, malaise, headaches, delirium, loss of
consciousness, pathological reflexes and paresis of the extremities and facial nerves. Forty-
four of the affected individuals died. T. incanum contains the Crotalaria type PAs incanine
and trichodesmine (Yunosov and Plekhanova, 1957a, 1957b, 1959), which are structurally
similar to monocrotaline (Figure 1).
35
Carcinogenicity and anti-tiimor activity
PA are among the few compounds found in plants that are carcinogenic. This is not
surprising in light of the powerful in vitro antimitotic and mutagenic effects of many PAs
on cells. While increased incidence of cancer or mmors in humans during PA poisoning
outbreaks have not been reported, PAs are moderately carcinogenic in experimental
animals (Mattocks, 1986). A variety of unsaturated PAs have been examined for
carcinogenicity in rats. Regardless of the dosing schedule (continuous or intermittent) and
route of administration, tumors occurred most commonly in the liver (Mattocks, 1986).
Thus it is likely that hepatic metabolism plays a key role in carcinogenicity. Both
macrocyclic PAs {Senecio type) (Harris and Chen, 1970) and open esters (Hirono et al.,
1978) are hepatocarcinogens. DHP is also carcinogenic, but less so than monocrotaline
(Mattocks, 1986), indicating that other PA metabolites, such as the DHAs, likely play a
significant role in carcinogenicity.
Some PAs and PA A^-oxides have been shown to posses antitumor activity.
Indicine A'^oxide has demonstrated antitumor activity in mice against Walker 256
carinosarcoma (Kugelman etal., 1976). This compound has undergone clinical trials in
humans against childhood leukemia (Miser et al., 1992) and advanced solid tumors
(Kovach etal., 1979). Remissions were achieved in several cases of the leukemias, but the
hepatotoxicity produced as a side effect precludes the continued pursuit of indicine AZ-oxide
as an anticancer drug.
Mutagenicity and cytotoxicity
PAs possessing the structural features necessary to cause hepatotoxicity also can
alkylate DNA. The pyrrolic metabolites dehydroheliotridine and dehydroretronecine can
alkylate DNA as well (Black and Jago, 1970; Robertson, 1982). Robertson (1982) has
36
identified a 7-adduct of dehydroretronecine bound to the N2 position of deoxyguanosine.
Tlie DHAs, dehydromonocrotaline and dehydroretrorsine, were much more active cross-
linkers of DNA than dehydroheliotridine (White and Mattocks, 1972). This is in keeping
with the much greater electrophilic reactivity of the former.
In mutagenicity assays, metabolic activation of PAs is necessary to produce
maximum effect, as DNA damage is probably caused primarily by the DHA. Bifunctional
alkylating PAs (diesters) are more effective mutagens, and can cross-link DNA (Petry et
al., 1984; Hincks et al., 1991). Metabolically generated DHAs containing an a, p-
unsaturation, such as dehydrosenecionine, were more active at cross-linking DNA in
cultured bovine kidney epithelial cells than the cyclic diester dehydromonocrotaline or open
ester DHAs (Kim et al., 1993). This may be due to extent of metabolic activation of
different PAs rather than alkylating ability of the reactive species. PAs are mutagenic in
Drosophila melanogaster. Salmonella typhimuriiim and CHO cells (Ord et al., 1985;
Mattocks, 1986; Carballo er a/., 1992; Frei etai, 1992). Mutagenicity in Drosophila is well
correlated with hepatotoxicity of PAs in rodents. Frei et al. (1992) have shown that
macrocyclic diester PAs were the most genotoxic to Drosophila, followed by open diesters,
and open monoesters. The C9 monoester supinine, which lacks an alkylating function at
C7, was not genotoxic.
Most hepatotoxic PAs, and the metabolite DHP, produce a powerful antimitotic
effect on liver parenchymal resulting in characteristic enlarged hepatic parenchymal
(megalocytes) cells (Peterson, 1965; Jago, 1969; Mattocks and Legg, 1980). Mitosis is
persistently inhibited during the S or early G phase of the cell cycle, possibly by inhibiting
the synthesis of specific proteins required for cells to progress from G, to mitosis (Jago
and Samuel, 1975). Mattocks and Legg (1980) observed a similar effect in vitro using a rat
liver parenchymal cell line with a series of pyrrolic alcohols, including DHP. In the
presence of rat liver S9, PAs with an a, [B-unsamration {Senecio alkaloids) were the most
37
effective in producing megalocytosis in bovine kidney epithelial cells (Kim etal., 1993).
Monocrotaline, and open diesters produced only a weak response.
Metabolites Responsible for Extrahepatic Toxicity
Extrahepatic toxicity is likely a result of hepatic metabolism, as other tissues,
including the lungs, appear incapable of PA bioactivation (Mattocks, 1986; Huxtable,
1990; Miranda et al., 1991). The reason that some PAs cause extensive extrahepatic
toxicity is still poorly understood. Whether extrahepatic toxicity is due to the highly reactive
DHA or a stabilized secondary pyrrolic metabolite is uncertain.
DHAs
DHAs are the primary pyrrolic metabolites produced in by liver from PAs. DHAs
are highly reactive, alkylating a variety of cell nucleophiles, including proteins and DNA
(Petry etal., 1984; Hincks etal., 1991; Kim etal., 1995), and are likely responsible for
hepatic toxicity. DHAs injected into mesenteric veins produce hepatotoxicity similar to the
parent alkaloids (Mattocks, 1986), but much lower doses of the former are required.
Dehydromonocrotaline, given via tail vein to rats in a non-protic solvent, produces
pneumotoxicity consistent with the parent alkaloid (Butler et al., 1970; Plestina and Stoner,
1972; Bruner et al., 1983). Doses as low as I mg/kg of the former are required compared
to approximately 60 mg/kg for monocrotaline (Pan et al., 1993). However, the role of
DHAs in extrahepatic toxicity has remained unclear. They are extremely reactive, having
aqueous half-lives of only a few seconds (Mattocks and Jukes, 1990; Cooper and
Huxtable, 1996). It has thus been much debated whether DHA produced in the liver could
survive transport to the lungs, or organs even further downstream (Huxtable, 1990; Pan et
al.. 1993).
38
Sulfur conjugated pyrroles: 7-GSDHP and 7-CYSDHP
Phase n conjugation with the tripeptide glutathione (y-gltamyl-L-cysteinyl-glycine)
is generally known as a detoxication pathway, although several classes of xenobiotics form
reactive intermediates by this route (Anders etal., 1988; Vamvakas and Anders, 1990;
Koob and Dekant, 1991). Large quantities of sulfiar-conjugated pyrroles are released into
the bile from isolated perfused rat livers exposed to PAs (LaFranconi et al., 1985; Mattocks
et al., 1991; Yan and Huxtable, 1994). These pyrroles have been identified as 7-GSDHP
and its breakdown products (Lame etaL, 1990; Mattocks etal., 1991; Lame etal., 1995).
The presence of sulfur-conjugated pyrroles in the urine has also been demonstrated,
including a A^-acetyl-cysteine adduct (Estep et al., 1990; Mattocks and Jukes. 1992).
Conjugation widi glutathione by a pyrrolic metabolite of senecionine has been demonstrated
in vitro with hepatic microsome preparations (Buhler et al., 1990; Reed etal., 1992). It is
not known whedier GSH conjugation with DHAs is enzymatically or chemically mediated.
However, this reaction occurs rapidly in vitro without the presence of enzymes (Mattocks
and Jukes, 1990a, 1992b). Although a cysteine conjugate of DHP has not been identified,
its formation from the glutathione conjugate could be catalyzed by y-glutamyl
transpeptidase and dipeptidases localized in the kidney. Further metabolism of a cysteine
conjugate by (S-lyases could produce an unstable thiol which yields electrophilic products
(Vamvakas and Anders, 1990).
The role of 7-GSDEIP and the further putative metabolite 7-CYSDHP in
extrahepatic toxicity of PAs was a debated question for some years, with studies yielding
conflicting results (Huxtable et al., 1990; Cooper et al., 1992; Pan etal., 1993). It was
shown that 7-GSDHP and 7-CYSDHP can produce pneumotoxicity as measured by the
reduction of serotonin transport in the isolated perfused rat lung (LaFranconi et al., 1985)
39
and produce pulmonary hyperplasia in rats in vivo (Huxtable et al., 1990), suggesting a
possible roie for these compounds in the pneumotoxicity of monocrotaline.
DHP
The pyrrolic metabolite DHP is formed by the hydrolysis of the DHAs. Hsu et al.
(1973) studied the distribution and toxicity of [^H]DHP, finding that the compound quickly
distributes throughout the body. The major accumulation and subsequent site of toxicity
was found in the glandular portion of the stomach. Mattocks (1986) suggested that this is
due to the acidity of this region, which causes an increase in the reactivity of this
compound. Thus local polymerization and alkylation by DHP is observed. DHP causes
pneumotoxicity (Huxtable et al., 1978) in rats, but doses of 100 mg/kg are needed,
compared to 60 mg/kg for monocrotaline. There are also qualitative differences in the
pneumotoxicity produced by DHP and monocrotaline, suggesting that DHP is not the sole
pneumotoxic metabolite. DHP has also been shown to be hepatotoxic, antimitotic,
immunosuppressive, teratogenic and carcinogenic (Peterson et al., 1972; Samuel and Jago,
1975; Peterson and Jago, 1980; Percy and Pierce, 1971; Peterson et al., 1983). Many of
the toxic effects of DHP are similar to that produced by the parent alkaloids, but higher
doses of the pyrrole are required. This is because DHP, a pyrrolic alcohol, is much less
reactive and more water-soluble than the corresponding DHA, an ester pyrrole. However,
because of the additional stability of DHP compared to the DHAs, it may travel to sites not
normally exposed to the DHAs. The lower reactivity and hence weaker alkylating activity
militate against DHP as the primary extrahepatic toxin, but it may be an important
component in the overall chronic toxicity expressed by a particular alkaloid.
40
Effect of PA Structure on Metabolism and Toxicity
Tne cytotoxicity of PAs is associated witii their hepatic metabolism to pyrroie
derivatives. Several structural factors determine the extent of conversion of PAs to toxic
pyrroles in relation to non-toxic metabolites. Structures which impart resistance to ester
hydrolysis, increase lipophilicity and favor metabolic dehydration rather than A/^oxidation
tend to increase the relative toxicity of a PA (Mattocks, 1986). The relationship between
these factors effecting pyrrole production in an animal is very complex, and any one factor
is insufficient to explain the actions of a particular PA. Toxicity will also be affected by the
nature of the pyrrole metabolites produced. Primary pyrroles differ greatly in their stability,
which causes differences in their abilities to react with tissue nucleophiles, rates of
detoxication, or which organs they will be exposed to.
Lipophilicity
In general, the more lipophilic a PA, the more susceptible it is to bioactivation by
hepatic P-450s (Mattocks, 1981; Mattocks and Bird, 1983; Mattocks, 1986). Additionally,
a lipophilic DHA metabolite would be expected to cross the cell membrane of a target organ
easier than a more water soluble DHA. The hydrophilic PAs, such as the 9-monoesters
lycopsamine or indicine, are less extensively metabolized to pyrroles, are rapidly excreted,
and are weakly toxic. However, some highly lipophilic PAs or semi-synthetic derivatives
produce unexpectedly less pyrroles in animals because they are subject to extensive
enzymatic hydrolysis (Mattocks, 1981).
Ester hydrolysis
The hydrolysis of PAs by hepatic esterases is a detoxication pathway. PAs which
are normally innocuous become distinctly hepatotoxic in animals which have been treated
41
with compounds (e.g. tri-orthocresylphosphate) to irreversibly inactivate esterase activity
(MaLlocks, 1981; Mallocks, 1986). This is because more PA becomes available for
bioactivation to pyrroles. Mattocks (1982) examined both the base and enzyme catalyzed
rates of hydrolysis of a series of PAs in vitro. Rates of hydrolysis under both conditions
were dependent on steric hindrance to the ester groups. Open ester PAs with simple,
unbranched acids were extremely sensitive to hydrolysis. Those with bulkier or branched
acid substitutions hydrolyzed much slower. Despite the rigidity of the ester groups,
macrocyclic PAs are less susceptible to hydrolysis than open type PAs. Again, those which
contain hindering groups near the ester portion are less prone to hydrolysis. The PA
jacobine contains an epoxide joining CI5 and C20 in the acid moiety, which is near the C7
ester. This substitution significantly inhibits hydrolysis by guinea pig carboxylesterases
compared to senecionine, which differs only in the lack of the epoxide function (Dueker et
al., 1995; Chung and Buhler, 1995). This may partially explain the increased susceptibility
of the guinea pig to jacobine, since more alkaloid would be available for bioactivation to
toxic pyrroles.
Bioactivation to DHAs versus N-oxidation
N^-oxidation represents a major detoxication pathway for PAs in many animals. The
balance between this pathway and dehydrogenation to pyrroles is important in the
expression of toxicity. Aside from lipophilicity, there are no clear-cut structural features
which are known to favor one pathway or another. Metabolic dehydrogenation is believed
to occur by an initial microsomal hydroxylation at C8 of the pyrrolizidine ring (Mattocks
and Bird, 1983) (Figure 5). For optimal pyrrole production, a minimum of steric hindrance
in the vicinity of C8 may be an important factor, although this would be expected to allow
increased access for hydrolysis. Macrocyclic diesters produce the greatest amounts of
42
pyrrole metabolites compared to A/-oxides (Mattocks and Bird, 1983), in part due to their
high lipophiliciiy. Tne macrocyciic structure may favor pynole fomiation because tlie ring
is relatively rigid, being held away from the "top" of the necine, allowing access to C8 by
P-450 enzymes (Mattocks, 1986). The pyrrole to N-oxide ratio is highest in macrocyciic
diesters and open monoesters. This ratio is lowest from open diesters, which have the most
hindrance around C8 due to two flexible acid substitutions.
Little effect is observed between metabolism of heliotridine (hydroxyl at C7 is a)
based or retronecine (hydroxyl at C7 is |3) based semisynthetic PAs with simple ester
groups (Mattocks and Bird, 1983). This implies the conformation at C7 does not influence
microsomal metabolism. However, the natural PA heliotrine is consistently metabolized to
pyrrole to a greater extent than its isomer indicine both in vivo and in vitro, and is also
more toxic in vivo (Bull etal., 1958; Schoental, 1968; Mattocks, 1972). Thai the natural
heliotridine based PAs are frequently more toxic than retronecine based PAs may be due to
increased resistance to hydrolysis by the former (Mattocks, 1981).
DHA reactivity
While it is clear that PA structure affects the rate and extent of metabolism, structure
is also critical in determining the fate of the reactive metabolite once it has formed. These
compounds are highly reactive, with half-lives in water of a few seconds (Mattocks and
Jukes, 1990). The DHAs differ significantly with respect to alkylation reaction kinetics
(Karchesy and Deinzer, 1987) and rates of hydrolysis. Once formed, an extremely reactive
DHA may hydrolyze so fast that it does not have time to react with vital cellular
nucleophiles. If the metabolite has a slightly longer half-life, it may react with cellular
components in the vicinity of its formation, thus causing hepatotoxicity. A portion of
metabolites with longer half-lives may survive transport to organs downstream from the
43
liver, particularly the lungs. Some DHA could conceivably survive long enough to cause
other symptoms such as neurotoxicity. On die other hand, if a DHA was sufficiently
unreactive, it may be excreted or largely dispersed before it can react with a macromolecule
(Mattocks, 1986).
In addition to stability, the number and posidon of reactive centers of the
dehydronecine moiety may influence toxicity of a PA. Diester PAs are believed to form
bifunctional alkyladng pyrroles, while the weakly toxic 9-monoester pyrroles only have a
single reactive center (Karchesy and Deinzer, 1987; Mattocks and Jukes, 1990a, 1990b).
The theory of bifunctionality is indirectly supported by studies which indicate that PA
metabolites cross-link DNA (Petry etai, 1984; Hincks etal., 1991). Additionally, only
macrocyclic diester PAs are known to produce pneumotoxicity. Driver and Mattocks
(1984) have demonstrated that synthetic derivatives of pyrrolic esters, "synthanecines", are
pneumotoxic only when diey posses two labile ester functions. Monoester synthanecines
(like monoester PAs) produced only hepatotoxicity. Whether this is attributable to cross-
linking of tissue nucleophiles by pyrroles is unknown.
Statement of the Research Problem and Hypothesis
PAs containing a I -2 double bond are hepatotoxic. Certain PAs, particularly some
of the macrocyclic diesters, produce toxicities in organs in addition to the liver. Of the PAs
used in this dissertation, monocrotaline is pneumotoxic, causing pulmonary hypertension
and right heart hypertrophy (Huxtable, 1990). Trichodesmine is a potent neurotoxin
(Shtenberg etal., 1955; Ismailov etai, 1970), while seneciphylline and retrorsine, except
under unusual circumstances (Mattocks and White, 1973), are hepatotoxic. The two open
ester PAs studied in this dissertation, lycopsamine and 7-acetyl-lycopsamine are known
solely as hepatotoxins. Bioactivation by the liver is a requirement for the expression of PA
toxicity. Mattocks (1968a) proposed that the primary metabolite of PAs were the
44
corresponding DHAs; electrophilic compounds which, by covalently binding to important
tissue nucleophiies, were responsible for toxicity of the parent aikaioids. Subsequent work
has established that DHAs are indeed produced by the liver (Mattocks etal., 1990; Glowaz
et al., 1992). Why structurally similar PAs often produce markedly different toxicities is
poorly understood. Additionally, the nature of the extrahepatic metabolite(s) is still unclear.
Whether the DHAs are sufficiently stable to reach sites away from the liver has been an
ongoing question. The hypotheses of this dissertation are that (1) DHAs are responsible for
the extrahepatic toxicity observed with some PAs, (2) stmctural differences in the ester
portion of DHAs cause significant differences in their physicochemistry (e.g. lipophilicity,
stability) and (3) these physicochemical differences are partially responsible for differences
in patterns of their further metabolism and observed toxicity. To address these problems we
have investigated the in vivo toxicity of the recently discovered PA metabolite 7-GSDHP
and the further putative metabolite 7-CYSDHP, to determine their role as
extrahepatotoxins. The results of this study will provide support for or against the DHAs as
the proximate extrahepatic toxin. Secondly, to establish the comparative stability of a series
of structurally similar DHAs, we have examined their reactivity toward a variety of
nucleophiies. These studies include the potentiometric determination of DHA half-lives of
hydrolysis, rates of DHA alkylation by 4-(/?-nitrobenzyl)pyridine, a nitrogen nucleophile,
and their relative reactivity toward a large molecular weight sulfhydryl in the form of
Thiopropyl-Sepharose® resin. The relationship between relative stabilities of the DHAs
and metabolism and toxicity of PAs will be examined. We have also determined the
chemical structure of sulfur-bound pyrroles formed both in vitro and by the in vivo
metabolism of PAs. These experiments will determine the position of the reactive center of
the various DHAs, providing information on how structure affects the namre of alkylation.
To examine the effects of DHAs on a model cellular target, the inhibition by DHAs of yeast
and rat brain hexokinase was studied in vitro. Hexokinase, a critical enzyme in cellular
45
energy production, is sulfhydryl rich and known to be inhibited by compounds which
alkylate these substitutions (Wilson, 1995). Finally, because monocrotaline is the only
commercially available PA, we have developed an efficient means of preparative separation
of PAs using high-speed counter-current chromatography. This has allowed us to utilize
locally and internationally collected plants as sources of structurally varied PAs. The
chromatographic technique enables the rapid separation and purification of quantities PAs
sufficient for these studies.
46
METHODS
PA Isolation, Separation and Identincation
Plant material
Amsinckia tessellata A. Grey was collected near Tucson, Arizona, in the spring of
1994 and 1995. Senecio doiiglasii DC. var. longilobus (Benth.) L. Benson was collected
in the spring of 1995 from Sonoita, Arizona. Seeds from Trichodesma incaniim Alph. DC.
were collected from Uzebekistan in the summer of 1990. Symphytum spp., sold
commercially as comfrey root, was purchased from local supermarkets. Monocrotaline was
purchased commercially or isolated from seeds of Crotalaria spectabilis Roth, collected in
Georgia, and from C. retiisa L. collected in Natal, Brazil, in September of 1992.
Alkaloid isolation
PAs were isolated from plant material according to die method of Adams and
Rogers (1939). Prior to extraction, Symphytum root, and seeds from Crotalaria and
Trichodesma were finely ground in an electric coffee mill. Plant material was continuously
extracted in a Soxhlet apparatus with EtOH for 24 hr. Dried whole plant material from
Senecio and Amsinckia was ground in a commercial hammer mill through 3 mm mesh,
followed by soaking in EtOH for 4 days in a commercial extractor. All extracts were then
reduced under vacuum using a rotary evaporator. The residue was resuspended in HiO and
made acidic (pH 3-4) with citric acid and reduced with Zn dust overnight. The aqueous
solution was filtered, and extracted with equal volumes of CHCI3 until the organic phase
was clear. The aqueous portion was then made basic (pH 8-9) with 30% NH4OH and
extracted 3X with equal volumes of CHCI3. Aqueous phases from Amsinckia and
Symphytum extractions were saturated with NaCl to increase extraction of the polar
47
alkaloids lycopsamine and intermedine (Culvenor er a/., 1980; Roitman, 1983). The
organic portions were combined, dried over anhydrous Na2S04, filtered and evaporated to
dryness, yielding the alkaloid fraction. Content of the alkaloid fractions was determined by
GC-electron impact MS (Method H) and TLC. Monocrotaline was obtained pure by
recrystallization from EtOH, as it was the only PA detected in the Crotalaria fractions.
Mixed PA fractions were separated and purified by counter-current chromatography as
described below. Alkaloids obtained (Figures 1 and 2), and their plant sources are listed in
Table 2.
High-speed counter-current chromatography
An Ito multilayer coil counter-current chromatograph equipped with a #10 coil
(PTFE tubing, 2.6 mm i.d., 380 ml capacity) was used for separations of alkaloidal
fractions isolated from Trichodesma, Senecio, Amsinckia and Symphytum. Potassium
phosphate buffer, 0.2 M, at an appropriate pH was used as the stationary phase. Mobile
phase was CHCI3, driven by a metering pump at a flow rate of 210 ml/hr in a head-to-tail
fashion. Prior to use, the two solvent phases were equilibrated by shaking together in a
separatory funnel, and then allowed to separate. The column was initially filled with
stationary phase, then rotated at 800 rpm while mobile phase was loaded. The column was
considered in equilibrium when only CHCI3 eluted from the tail end. At this point, alkaloid
samples, either solid or non-solid, 200-800 mg, were dissolved in 3-5 ml of 1:1 mobile
phaserstationary phase and injected into the column. Mobile phase was collected by a
fraction collector at 1.7 min intervals. Alkaloid remaining in the column after separation
was collected by purging the stationary phase with Ni, followed by basifying and CHCI3
extraction. Chromatograms were constructed by measuring relative PA content of each
fraction colorimetrically with Ehrlich reagent (2% boron trifluoride etherate, v/v and 2%
48
/7-dimethylaminobenzaldehyde, w/v, in EtOH) on a spectrophotometer at 562 nm
(Maltocks, 1968). Fractions containing llie same alkaloid were combined, filtered, dried
under vacuum and if possible, recrystallized from EtOH or acetone. Corrected retention
time of solute peaks, r'R, was found by subtracting time of peak maximum of the solvent
front from time of peak maximum of the solute. Partition coefficients, K, defined as solute
concentration in the mobile phase divided by solute concentration in the stationary phase,
were estimated by the method of Zhang et al. (1988). All peaks were analyzed on TLC and
GC-MS (Method H) for identification and purity. Where electron impact-MS failed to
produce a molecular ion, flow injection-atmospheric pressure chemical ionization (APCI)
MS was used.
Thin-layer chromatography
PAs, pyrrolic metabolites and derivatives were analyzed by TLC as listed in Table
1. Plates spotted with PAs were developed by spraying with methanolic o-chloranil (0.5%
W/V), followed by Ehrlich reagent. Pyrroles were visualized by Ehrlich reagent.
Gas chromatography electron impact-mass spectrometry, method I
GC EI-MS was performed by the Mass Spectrometry Facility, Department of
Chemistry, University of Arizona, on a Hewlett Packard 5890 system using an HP-5 fused
silica capillary column consisting of 5% phenyl-methyl silicone, 25 m x 0.2 mm with He
carrier gas at 23 psig. Conditions: injector temperamre 250 °C; starting temperature
programmed 70°, held 1 min, to 300° at 20° min"', held 7 min; split inj. ratio 70:1, vol. I ftl
(CH2CI2). The column was connected directly to an HP 5970 mass selective detector. Mass
spectra were recorded at 70 eV.
49
Gas chromatography electron impact-mass spectrometry, method II
Performed at the Southwest Environmental Health Sciences Center, University of
Arizona, on a Fisons MD800 using a DB-5, 15 m x 0.25 mm column with He carrier gas at
5 psig. Conditions: temperature program same as method I; spiitless injection volume 1 |il
(CH2CI2). The column was connected directly to a mass selective detector. Mass spectra
were recorded at 70 eV.
Flow-injection atmospheric pressure chemical ionization mass spectrometry
Flow-injection APCI MS was conducted on a Finnigan TSQ 7000 triple quadrupole
mass spectrometer at same location as GC-MS method H. Conditions: mobile phase MeOH
0.5 ml min ', samples introduced into the ion-source via direct inj., 50 |j.l inj. vol (MeOH),
10 eV offset added to the region between sample introduction and mass analysis to increase
fragmentation (in-source collision-induced decomposition). The TSQ was equipped with an
atmospheric chemical ionization source and was scanned 100 to 700 amu at I scan sec '.
Preparation of PA Metabolites and Derivatives
DMAs
DHAs were prepared from parent alkaloids according to Mattocks and Jukes
(1989). PAs (100 mg) were dissolved in CHCI3 (20 ml), added to a solution of o-bromanil
(125 mg) in CHCI3 (20 ml) and allowed to stand at room temperature for 2 min. This
solution was extracted with 10 ml of an ice-cold aqueous solution of sodium borohydride
(200 mg) and potassium hydroxide (5 g) by \'igorously shaking in a separatory funnel for
20 sec. The CHCI3 layer was quickly removed and dried over anhydrous Na2S04, filtered
and evaporated under vacuum with a rotary evaporator. TLC analysis indicated that parent
alkaloid or DHP was not present in the reaction product. DHAs were stored at -20 °C under
50
nitrogen. DEIA samples were disgarded if polymerization was apparent, as indicated by the
presence of a reddish-brown color.
7-CYSDHP (7-cysteinyl-6,7-dihydro-1-hydroxymethyl-SH-pyrrolizine)
7-CYSDHP was synthesized as described by Mattocks and Jukes (1990).
Dehydromonocrotaline (100-500 mg) was dissolved in 1 ml of ethylene glycol dimethyl
ether, and added dropwise over 5 minutes to 4 ml of a stirred solution of L-cysteine
(equimolar to dehydromonocrotaline) in ammonium acetate buffer (1.0 M, pH 7.5). The
reaction was carried out on ice, under N2and monitored by a pH meter. A 10% solution of
NH4OH was used to maintain a constant pH of 7.5-7.6 during the course of mixing by
adding dropwise when the pH of the reaction mixture began to fall. The resultant solution
was allowed to remain on ice for an additional 10 min. Precipitates were removed by
centrifiigation. The supernatant was lyophilized, leaving a pinkish-white solid material.
Positive ion FAB-MS showed an (M+H)"^ ion, m/z 257 (Figure 42). TLC indicated the
presence of 2 Ehrlich positive spots, which were also ninhydrin positive. FAB-MS was
conducted by the Midwest Center for Mass Spectrometry, University of Nebraska, Linoln,
Nebraska.
7-GSDHP (7-glutathionyl-6,7-dihydro-1-hydroxymethyl-SH-pyrrolizine)
7-GSDHP was synthesized in an analogous manner to 7-CYSDHP. Positive ion
FAB-MS indicated an (M+H)"^ ion, m/z 443.
DHP ((±)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-SH-pyrrolizine).
DHP, a racemic mixture of dehydroretronecine and dehydroheliotridine, was
prepared by the method of Mattocks and Jukes (1989).
51
9-Ethoxy DHP (7-hydroxy-9-ethoxy-6,7-dihydro-5H-pyrrolizine)
Ethoxy pyrroles were synthesized according to Mattocks and Jukes (1990a, 1992).
A solution of dehydrolycopsamineZ-intermedine (10 mg), was allowed to stand overnight in
5 ml of EtOH containing 0.2 ml of 1 M NaOH. Aqueous K2CO3 (5% w/v, 2 ml) was
added and mixed for 5 min. The ethanol was removed by reduced pressure. The aqueous
portion was then extracted with 3X with CHCI3 and dried over anhydrous Na2S04. The
combined organic extracts were combined, filtered and dried under vacuum to yield 9-
ethoxy DHP. Structures of all pyrrolic ethyl ethers were confirmed by TLC and GC-MS
(Method I).
7-Ethoxy DHP ((±)-7-ethoxy-1-hydroxymethyl-6,7-dihydro-5H-pyrrolizine)
Dehydromonocrotaline (5-10 mg) was dissolved in 5 ml of EtOH containing 0.2 ml
of 1 M NaOH. Isolation of the products proceeded as above. Product was primarily
7-ethoxy DEff. with a small amount of the diether present.
7,9-Diethoxy DHP ((±)-7-ethoxy-9-ethoxy-6,7-dihydro-5H-pyrrolizine)
The diethoxy pyrrole was produced as above using dehydromonocrotaline (5-10
mg) as the starting material and unmodified ethanol (5 ml) as the solvent. Workup and
isolation was identical to the monoethers.
Toxicity of Sulfur-Conjugated Metabolites of PAs, in vivo
7-CYSDHP
Rats were obtained from the University of Arizona Division of Animal Resources
and were housed four to a cage in 12 hr cycles of light and dark. They were allowed food
and tap water ad libitum. Rats (60-70 g) were randomly divided into three groups of six
52
animals. 7-CYSDHP, dissolved in normal saline, was administered via a single
intravenous tail vein injection (0.1 - 0.3 nil), at 300 and 600 p.niol/k.g. Control rats were
injected with normal saline. Body weight and overall health of the animals were recorded
daily. At 21 days, rats were weighed, killed and necropsied. Liver, lung, heart, thymus,
spleen and kidneys were removed for determination of tissue to body weight ratios. Wet
weights were immediately recorded, after which organs were dried at 50 °C for 48 hr prior
to obtaining dry weight. In order to detect evidence of pneumotoxicity and subsequent right
heart hypertrophy, the right heart to left heart plus septum weight ratio (RV/LV + S) was
determined. This was performed by dissecting the right atrium free from the left heart and
septum prior to weighing. Differences in means between treatment groups was determined
by ANOVA with the aid of a computer program (Pharm-Calc; PCS, Phildelphia, PA).
Group means were considered statistically different at /? < 0.05.
7-GSDHP
Rats weighing 160-190 g were randomly divided into three groups of six animals.
Groups were injected via tail vein with either 7-GSDHP in saline at 600 |i,moi/kg,
monocrotaline at 185 (imol/kg as a positive control, or saline. Animals and data collection
were treated as above for the remainder of the study.
Chemistry of Sulfur-Bound Pyrroles
Preparation and activation ofThiopropyl-Sepharose 6B resin
Desiccated resin (2 g) was suspended in 100 ml potassium phosphate buffer (pH
12, 0.2 M) and allowed to swell for 60 min with occasional stirring. The suspension was
centrifuged, the buffer decanted and the procedure repeated until resin had been washed
with 500 ml of buffer. When the buffer was decanted a final time, the resin had swelled to
53
a total volume of 6 ml. Buffer was added (14 ml) so that each ml of suspension contained
100 mg di7 weight of resin. Tlie resin was activated by adding 5 of p-mercaptoellianoi Lo
resin aliquots (2 ml suspension, containing 200 mg resin dry wt, approximately 22 nmol
thiol groups), and allowing them to stand for 2 hr at room temperature. Prior to use, the
resin was rinsed with buffer in a small glass column connected to a vacuum. The resin was
rinsed until |3-mercaptoethanol could not be detected (by the absence of yellow color) in the
eluent using Ellman's Reagent (1% 5,5'-dithiobis(2-nitrobenzoic acid) in acetone). Fully
activated resin contains approximately 105 ^mol of suifhydryls per g dry wt.
Recirculating aqueous flow system
Covalent binding of DHAs to immobilized thiols was studied using an aqueous
flow system described by Mattocks and Juices (1990b) (Figure 7). This system was
designed to mimic the slow release of DHA into the circulation from the liver. A small
quantity of DHA dissolved in a non-protic solvent was continuously injected by a syringe
pump over 1 hr into a fast moving stream of buffer (potassium phosphate, pH 7.2, 37 °C.
7.5 ml/min), rapidly mixed, and was passed over a bed of activated Thiopropyl-Sephorose
resin (200 mg dry wt containing 20 |j.mol of sulfhydryl groups) contained in a small glass
column. The buffer was recirculated from a reservoir via a peristaltic pump. The amount of
time between DHA injection and exposure to the resin was determined by the length of
tubing (polyethylene, 1 mm i.d.) through which the mixture must travel prior to passing
through the resin bed. This was determined by recording the amount of time for a small air
bubble to pass through the length of tubing. Dye was injected into the system via the
syringe pump to visually confirm that mixing was instantaneous and complete. At the
conclusion of each run, the resin was examined for quantity of bound pyrrole or the
position of sulfur binding as described below.
54
Identification ofThiopropyl-Sepharose resin-bound pyrroles
Thiopropyl-Sepharose resin was exposed to various DHAs using the recirculating
system described above. Upon injection, each DHA (10 i^mol) was exposed to the buffer
for either 1.5 or 30 sec prior to passing through the resin (5 and 400 cm tubing,
respectively). Following the 1 hr run, the resin was rinsed with water, then with EtOH
sufficient to remove all water. The resin was removed from the column and suspended in 5
ml of EtOH and stirred. An aqueous solution of Tris-buffered silver nitrate (1 ml) was
added dropwise (silver nitrate (400 mg) was mixed with aqueous (2 ml) tris-
(hydroxymethyl)mediylamine (560 mg) and neutralized with dilute HNO3 (Mattocks and
Jukes (1990a)). The solution was allowed to stir for 30 min, followed by addition of 10%
aqueous potassium carbonate (2 ml). The solution was allowed to stir for an additional 5
min followed by centrifugation to pellet solids. The supernatant was retained and the
ethanolic portion removed under reduced pressure. The remaining aqueous portion was
brought to 5 ml with HiO, then extracted with CHCI3. The organic layer was dried over
anhydrous Na2S04 and evaporated under reduced pressure. The resultant pyrrolic ethyl
ether was identified by comparing with standards on TLC and by GC-MS (Method I)-
Identification of liver sulfiir-bound pyrroles
Pyrrole metabolites covalently bound to liver and bile of PA treated rats were
released in the form of soluble pyrrolic ethyl ethers (Mattocks and Jukes, 1990). Male
Sprague-Dawley rats (120-130 g) were given a single i.p. injection of either monocrotaline
(3(X) |imol/kg), retrorsine (75 |imol/kg), seneciphylline (150 |imol/kg), trichodesmine (50
nmol/kg) or lycopsamine/intermedine (500 ^mol/kg). Animals were sacrificed after 24 hr.
Livers were removed and portions (5 g) were homogenized in EtOH and centrifuged 5 min
at 2700 rpm. Pellets were resuspended in EtOH (20 ml) and rapidly stirred. Aqueous Tris-
55
buffered silver nitrate (4 ml) was added dropwise and stirring continued for 30 min.
Workup of Ihe homogenale proceeded identically as with the resin. Pyrrolic ethyl ethers
were identified by TLC and GC-MS.
Bile samples (200-500 ̂ 1), collected from isolated rat livers perfused with PAs
from earlier experiments (Yan et al., 1995), were immediately frozen. Thawed bile samples
were treated with Tris-buffered ethanolic silver nitrate in an analogous fashion to liver
homogenates and resin.
Quantitation ofDHA binding to Thiopropyl-Sepharose resin
Relative pyrrole binding to activated Thiopropyl-Sepharose resin was measured
following its exposure to various DHAs using the recirculating aqueous flow system
described above. The amount of time between DHA mixing and exposure to the resin was
kept as short as possible (1.5 sec; 5 cm tubing) to minimize loss of DHA to hydrolysis.
DHAs (6 |j.mol in I ml dimethylformamide) were injected over one hour. Following each
run, bound pyrrole was quantified according to Mattocks and Jukes (1990b). The resin
(while still in the column) was rinsed with HiO followed by MeOH. The resin was
removed and suspended in 5 ml of MeOH. Aliquots (3) of suspension (1 ml) were
removed and I ml of Ehrlich reagent was added to each, followed by heating in a water
bath at 60 °C for I min. After the solution had cooled, 1 ml of 10% methanolic HgCK was
added and the solution was vortexed for 30 sec. The solution was brought to 5 ml with
MeOH and centrifuged to pellet the resin. The supernatant was read at 562 nm in a
spectrophotometer. Pyrrole content was determined by using absorbance 8.7 per |imol of
pyrrole released (as read in 5 ml) (Mattocks and Jukes, 1990b).
56
Studies of DHA Reactivity
Alkylaliun of DHAs by 4-(p-nilrobenzyl)pyridine
AJkylation reactions of DHAs by 4-(/7-nitrobenzyl)pyridine were performed as
described previously (Karchesy et ai, 1987). Briefly, 250 nmol of DHA, dissolved in 25
Hl dimethylformamide was added to 250 ^1 of 5% 4-{/7-nitrobenzyl)pyridine in acetone
acidified with 25 nl of formic acid. Reactions were carried out at room temperature (23°C).
Blanks contained no DHA. The reaction was quenched by the addition of ice cold
acetone:triethylamine, 1:1. The resultant blue-purple color was read spectrophotometrically
at 565 nm. Samples were diluted with ice-cold acetone as necessary. Results for each DHA
were plotted as total absorbance versus time. To determine rate constants, ^i, derived
curves were fit to a monoexponential equation (1), or a biexponential equation (2) in the
case of dehydroretrorsine, with the aid of a computer program (Karchesy et ai, 1987).
(1) A = A»-A«„e'*'
(2) A = A„-Be"*'-Ce"*'
Potentiometric measurement of hydrolysis of DHAs
Hydrolysis rates for a series of DHAs was determined potentiometrically by
recording the fall in pH on hydrolysis due to release of necic acid. DHAs (10 |imol
dissolved in 100 |al dimethylformamide) were rapidly added to a stirred HEPES solution
(10 ml, 0.5 mM, pH 8). The change in pH was continuously followed by a pH meter
connected to a chart recorder, previously calibrated from pH 3 to 10. A sigmoidal log-linear
plot was produced which was linear through at least two pH units. From this slope, the rate
constant, k, and the half-life of hydrolysis, txa could be estimated by assuming single
component exponential decay. Specifically, k was found by using the concentrations of the
base form of HEPES (pATa 7.55) at pH 6 and 7, calculated from the Henderson-
57
Hasselbalch equation. These values were then used to derive k from an equation describing
single component exponential decay, x = Ae ", which describes the linear portion of the
generated curves. Time r, measured directly from the chart recordings, is the time to go
from pH 7 to 6, A is the concentration of HEPES base at time 0 (pH 7), and x is the
concentration of HEPES base at time t (pH 6). Values derived represent the disappearance
of HEPES conjugate base, which was assumed to be directly proportional to appearance of
hydrogen ion liberated from ester hydrolysis. Half-lives were found by using the equation
0.691k. Data was analyzed for statistical differences by Newman-Keuls multiple analysis
with the aid of a computer program (Tallarida and Murray, 1986).
Inhibition of Hexokinase, in vitro
Yeast hexokinase assay
Yeast hexokinase (E.G. 2.7.1.1., from Baker's yeast) activity was measured in a
coupled assay with glucose-6-phosphate dehydrogenase by following the reduction of P-
NADP spectrophotometrically (Bennet er iz/., 1962; Puri e/a/., 1988). Assay volumes
were 1 ml and contained the following: triethanolamine (TEA) 39 mM; D-glucose, 0.01 -
216 mM; ATP, 0.74 mM; MgCU, 7.8 mM; P-NADP, 1.1 mM; hexokinase, 0.01 units:
glucose-6-phosphate dehydrogenase, 1.0 unit, pH 7.6. Mixtures were allowed to
equilibrate in quartz cuvettes (1 cm light padi) at 25 °C for 5 min prior to addition of
hexokinase. Reactions were read at 340 nm every 30 sec for 7 min. Final enzyme activity
was measured as change in absorbance per min over the 7 min assay. The enzyme rates
were linear over the course of measurement. For inhibition studies, hexokinase (0.75 units
in 960 III of 100 mM TEA buffer, pH 7.6, 25 °C) was incubated for 2 min with either
dehydrotrichodesmine, dehydroretrorsine, dehydro-7-acetyl-lycopsamine or
dehydrolycopsamine (dissolved in 40 ^1 dimethylformamide) at 0.01 - 20 mM. An aliquot
58
(15 nl, representing 0.01 units of hexokinase) of these incubations was then added to the
above described assay solution and residual phosphotransferase activity was measured.
Vehicle controls lacked DHA. DHP controls were performed by adding DHAs to the
incubation mixture 1 min prior to adding the hexokinase. Similar studies were performed
with mannose (10 mM) and ATP (10 mM) present in the incubation mixture to determine
whether they afforded protection to hexokinase against DHA inhibition. Data are plotted as
residual hexokinase activity versus concentration of DHA. Studies were also performed to
determine hexokinase activity in the presence of glucose concentrations from 0.01 to 5.0
mM. Prior to assaying, the enzyme was exposed to either vehicle, or dehydrotrichodesmine
at 2.5, 5.0 and 10 mM in the manner described above. Data were plotted as hexokinase rate
(nmol NADPH/min) versus concentration of glucose. and data were derived from
non-linear regression with the aid of a computer program (SigmaPlot, Jandel Scientific,
San Raphael, Ca) Data were also plotted as [S]/y versus [S] in order to graphically
illustrate non-competitive inhibition.
Rat brain hexokinase assay
Rat brain hexokinase was assayed according to Bennet et al. (1962). Male Sprague-
Dawley rats were used, weighing between 150-200 g. Animals were killed by decapitation,
the brain immediately removed and weighed. Brains were then homogenized in a Teflon-
glass homogenizer in 10 ml of ice cold potassium phosphate buffer (100 mM, pH 8.0) per
gram of tissue. Homogenates were then diluted with buffer to a concentration of 5 mg wet
tissue/ml and stored on ice. Assay solution (I ml) contained glycylglycine, 150 mM;
MgCU, 20 mM; ATP, 9 mM; P-NADP, 1 mM; and glucose, 25 mM. Assays were
conducted in quartz cuvettes at 37 °C. Solutions were prewarmed for 5 min prior to the
addition of 50 |al of brain homogenate (representing 0.25 mg wet tissue) and glucose-6-
59
phosphate dehydrogenase, 0.3 units. For inhibition studies, 960 |il of brain homogenate
was incubated wilii DHAs (2 min) or PAs (10 aiin), dissolved in 40 |i.l dinietliylforniaiuide
at 1.0 - 10 mM. Vehicle controls lacked DHA. Studies were also conducted in which
DHAs were added to the incubation buffer (910 ̂ ll) 5 min prior to adding a concentrated
aliquot of brain homogenate (50 [il undiluted homogenate). Assays were read every 30 sec
for 10 min at 340 nm using a spectrophotometer.
Glucose-6-phosphate dehydrogenase assay
Glucose-6-phosphate dehydrogenase (EC 1.1.1.49; from Torula yeast) was
assayed according to Noltman etal. (1961). In a 1 ml assay solution, concentrations were
glycylglycine, 50 mM, pH 7.4; D-glucose 6-phosphate, 2 mM; |3-NADP, 0.67 mM;
MgCli, 10 mM; glucose-6-phosphate dehydrogenase, 0.05 units. For inhibition studies,
glucose-6-phosphate dehydrogenase was prepared at 1 unit/ml in glycylglycine buffer, 50
mM, pH 7.4. Studies with DHAs were conducted in the same fashion as with yeast
hexokinase. Assays were conducted at 25 °C, read every 30 sec for 7 min at 340 nm.
60
Table 1
TLC Condilions for PAs aiid Pyrrolic Derivatives
PA or Pyrrole Plate Solvent Rf
Monocrotaline silica a 0.53
Trichodesmine C18 b 0.57
Incanine C18 b 0.42
Retrorsine silica c 0.35
Senecionine silica c 0.61
Seneciphylline silica c 0.63
Florosenine silica c 0.97
Lycopsamine* silica c 0.22
3-Acetyl-lycopsamine* silica c 0.46
7-Acetyl-lycopsamine* silica c 0.66
3,7-Diacetyl-lycopsamine* silica c 0.92
Symphytine* silica c 0.84
7-CYSDHP CIS d 0.74, 0.26
7-GSDHP silica e 0.54, 0.19', 0.86'
DHP CI8 f 0.83
7-Ethoxy DHP CI8 f 0.69
9-Ethoxy DHP C18 f 0.56
7,9-Diethoxy DHP CI8 f 0.31
^•BuOH : EtOH : H,0 : NH3OH (30%), 24 : 14 : II : 1 "EtOH : H.0 : NH;OH(30%), 60 : 39; I ^CHClj: MeOH : NH,OH (30%), 85 : 14 : I '' EtOH : H,0 : NH3OH, 89 : 10 : 1
MeOH : CHCI3 : H.O : EtjN. 80 : 8.5 : 11 : 0.5 f EtOH : H,0 : NH3OH, 50 : 49 : I. * One or more stereoisomers present, with identical Rf values ' trace
61
syringe pump
mixer
trapping column
delay loop
pump sepharose resin
37 "bath reservoir
Circulation Apparatus
Figure 7. Schematic of recirculating aqueous flow system for "trapping" of DHAs.
62
RESULTS
High-Speed Counter-Current Chromatography
High-speed counter-current chromatography was used for the separation of PA
mixtures isolated from Amsinckia tessellata, Symphytum spp. (comfrey root),
Trichodesma incamim, and Senecio douglasii var. longilobus (Figures I and 2). A two-
component solvent system was chosen, consisting of a chloroform mobile phase and
potassium phosphate buffer, 0.2 M, at various pH values (Table 2). as the stationary
phase. Up to 800 mg of sample could be separated in a single run, with excellent resolution
of alkaloids. However, the diastereomers lycopsamine and intermedine, or their
derivatives, could not be resolved.
A. tessellata collected near Tucson, Az., contained 0.02% PA per dry weight of
plant. These PA mixtures were isolated as a brown gum, as reported previously (Culvenor
and Smith; 1966 Roitman, 1983). Figure 8 shows the resolution of the PAs using an
aqueous phase pH of 7.4, from 710 mg of this gum. Three alkaloid peaks were obtained
and additional PA remained in the column. Peaks I-EH, plus the retained PA, yielded a
single spot on TLC. However, analysis by GC-MS indicated each peak consisted of an
isomeric pair, producing nearly identical mass spectra, but with slightly different retention
times. PAs were of both open mono- and diesters of retronecine. Peak I, 3,7-diacetyl-
lycopsamineZ-intermedine (a mixture of the two isomers 3,7-diacetyl-lycopsamine and 3,7-
diacetyl-intermedine) (5.0 mg) was retained sufficiently to elute after the colored impurities,
and was well resolved from peak H, 7-acetyl-lycopsamine/-intermedine (13.1 mg). Peak
EH, 3-acetyl-lycopsamine/-intermedine (55.1 mg), completely eluted by 2 hr, but the peak
exhibited some tailing. Lycopsamine/intermedine (70 mg) remained in the column and were
recovered by purging. Mass spectra were consistent with literature data (Kelley and Seiber,
63
1992). As reported by other researchers (Culvenor and Smith, 1966; Roitman, 1983), the
alkaloids, which were obtained as amber oils, could not be crystallized.
Several samples of commercial comfrey root from different sources were extracted,
with alkaloid content ranging from 0.01-0.09% dry weight. The PA fraction was isolated
as a brown gum, consisting of open mono- and diesters. Figure 9 shows the resolution of
586 mg of alkaloidal fraction from comfrey, using a stationary phase pH of 5.6. Two well
resolved peaks were obtained, with a third alkaloid remaining in the column. The colored
impurities, unretained by the stationary phase, eluted with the solvent front. TLC indicated
one spot for each eluted peak and the retained material, although GC-MS revealed the
presence of isomers. Peak I (19.0 mg) consisted of 3 isomeric PAs, all producing nearly
identical mass spectra, consistent with that reported for symphytine and its 3 known
diastereomers (Kelley and Seiber, 1992). Peak II, 7-acetyl-lycopsamine/-intermedine (13.6
mg), was retained until 113.1 min, due to the low pH of the stationary phase compared to
the Amsinckia separation. Lycopsamine and intermedine (210.6 mg) remained in the
column. Following chromatography, PAs were obtained as amber oils, but again could not
be crystallized.
S. douglasii var. longilobus collected from Sonoita, Az, contained ca. 0.03% PAs
per dry weight of plant material, isolated as a clear-brown gum. GC-MS analysis of the
alkaloidal fraction revealed the presence of the CI 2 macrocyclic type PAs senecionine,
seneciphylline, retrorsine, and the otonecine-based florosenine, previously unreported in S.
douglasii. The PAs had mass spectrum matching literature spectrum (Cava et al., 1968;
Quails and Segall, 1978). Riddelliine, reported to occur in S. douglasii var. longilobus,
was not detected (Adams and Govindachari, 1949). Figure 10 shows a chromatogram of
the separation of 600 mg of this alkaloidal fraction. Excellent resolution was achieved at a
stationary phase pH of 5.0. Peak I, florosenine (2.0 mg), was unretained at this or any
other pH however, and eluted together with the colored material at the solvent front.
64
Table 2
Results of Separation of Pyrrolizidine Alkaloid Fractions Using High-Speed Counter-Current Chromatography
Stationary t' * ' R
Species Alkaloid phase pH" (min) K"
Amsinckia 3,7-Diacetyl-lycopsamine/-intermedine 7.4 2.6 27.4
tessellata 7-Acetyl-lycopsamine/-intermedine 13.1 6.4
3-Acetyl-lycopsamine/-intermedine 74.0 1.3
Lycopsamine/Intermedine''
Symphytum Symphytine and/or isomers 5.6 14.5 6.4
spp. 7-Acetyl-lycopsamine/-intermedine
Lycopsamine/Intermedine''
113.1 0.9
Trichodesma Incanine 6.0 7.7 11.3
incaman Trichodesmine 36.6 2.6
Senecio Florosenine 5.0 0.9 52.0
douglasii
var. Senecionine 34.9 2.7
longilobus Seneciphylline
Retrorsine''
70.6 1.4
"Solvent system composed of CHCI3 mobile phase and phosphate buffer, 0.2 M, at indicated pH, as the stationary phase.
'detention time, defined as time of solute peak maximum minus time of solvent front. Tartition coefficients, K, estimated according to Zhang etal. (1988). ''Alkaloid(s) remained in colunui and were recovered as described in the Methods section.
65
m
s a
CN vO »o
(U CJ c rt jD
LA o zn
<
0
h
0 20 40 60 80 100 120 140
Time (min)
Figure 8. Separation of PAs from Amsinckia tessellata using high-speed counter-current chromatography. I = 3,7-Diacetyl-lycopsamine/-intermedine; II = 7-Acetyl-lycopsamine/-intermedine; EI = 3-Acetyl-lycopsamine/-intermedine. LycopsamineZ-intermedine was recovered from the column as described in Methods.
0.8 s c
CN vo irj © 0.6
0.2
0.0
80 100 120 140 160 180 0 20 40 60
Time (min)
Figure 9. Separation of PAs from Symphytum spp. using high-speed counter-current chromatography. I = Symphytine and isomers (symlandine, myoscorpine, echiumine); II 7-Acetyl-lycopsamine/-intermedine. LycopsamineZ-intermedine was recovered from the column as described in Methods.
67
0.4 r
0.3
0.2
0.1
0.0 w
J. J-
0 20 40 60 80
Time (min)
100 120 140
Figure 10. Separation of alkaloids from Senecio douglasii var. longilobus using high-speed counter-current chromatography. I = Florosenine; 11 = Senecionine; HI = Seneciphylline. Retrorsine remained in the column and was recovered as described in Methods.
Figure 11. Separation of PAs from Trichodesma incanum using high-speed counter-current chromatography. I = Incanine; II = Trichodesmine.
69
Peak n, senecionine (23.0 mg), and peak IE, seneciphylline (63.7 mg) were well resolved,
while relrorsine (216.6 mg) remained in the column, and was easily recovered after the
run. With the exception of florosenine, alkaloids were recrystallized from acetone. TLC
and GC-MS analysis of peaks revealed single alkaloids, although TLC failed to separate the
structurally similar senecionine and seneciphylline (Chizzola, 1994; Cooper et al., 1996).
Alkaloidal fraction from seeds and surrounding capsule of T. incanum yielded
1.1% PA by weight, isolated as white crystals. In agreement with an earlier account
(Yunusov and Plekhanova, 1957), the mixture of alkaloids consisted of two cyclic diesters
of retronecine, incanine and trichodesmine, as determined by GC-MS. Figure 11 shows the
chromatogram of a separation of 250 mg of the alkaloid fraction, using a stationary phase
pH of 6.0. Two well resolved peaks were obtained; peak I, incanine (7.3 mg) and peak II,
trichodesmine (238 mg). Separations were performed with up to 800 mg of alkaloid, but in
higher amounts the trichodesmine peak tailed considerably, although resolution was still
satisfactory. Although peak U exhibited slight shoulders, no other alkaloids were detected
from this peak by GC-MS. TLC and GC-MS analysis of each peak revealed the presence
of a single PA, confirming complete resolution. The mass spectrum of incanine, which has
not been previously reported, is shown in Figure 29.
Toxicity of Sulfur-Conjugated Pyrroles, in vivo
The role of sulfur-conjugated pyrroles as potential toxic metabolites of PAs was
investigated by examining the in vivo effects of the PA metabolite 7-GSDHP, and the
further putative metabolite, 7-CYSDHP. Animals were killed at 21 days because
pulmonary hypertension and cor pulmonale associated with a single dose of monocrotaline
is of delayed onset (Huxtable. 1990).
70
7-CYSDHP
7-CYSDHP was administered to male rats (60 - 80 g) via single tail vein injection at
3(X) and 600 ^mol/kg. Animals were necropsied, and organ and body weights (Tables 3-5)
was recorded as described in Methods. Over the course of the study, animals in the high
dose group gained significantly less weight than controls, although they appeared healthy.
Both dose groups had significantly reduced wet and dry liver to body weight ratios.
Pneumotoxicity was apparent by the presence of a dose-related increase in lung mass. Both
groups displayed significantly increased wet lung to dry lung weight ratios in comparison
to control animals. The high dose group also had significantly elevated wet lung/body
weight ratio. An increase in right heart size was observed in both treatment groups, but
values were not significantly different, possibly due to the large sample deviation.
7-GSDHP
7-GSDHP was administered to male rats (160 - 190 g) via single tail vein injection
at 600 p.mol/kg. Monocrotaline was given as a positive control at 185 |amol/kg
(approximately half the acute LDjg value). Rats treated with monocrotaline displayed
characteristic hepato- and pneumotoxicity (Tables 6-8). Significant increases in both wet
and dry organ/body weight ratios were observed in liver, lung, spleen, right ventricle and
RV/LV+S compared to control and 7-GSDHP treated animals. Contrary to some earlier
studies (Huxtable et al., 1990; Bowers et al., 1991), rats treated with 7-GSDHP showed
little signs of toxicity throughout the study. A mild increase in right heart size occurred, but
was not significant. The right heart/left heart plus septum ratios were significantly elevated
however, but less so than the monocrotaline treated group. An increase in this ratio
indicates right heart hypertrophy. However, this may be due to the correspondingly low
71
Table 3
Summary of Wet Organ Weight / Body Weight Ratios in Rats at 21 Days Following a Single Injection (i.v.) of 7-CYSDHF'
Control CYSDHP CYSDHP
Organ (Saline) (300 ^mol/kg) (600 M-mol/kg)
Liver 5.002 ± 0.285 4.614 ±0.095* 4.402 ± 0.242''
Lung 0.559 ± 0.057 0.648 ± 0.088 0.694 ± 0.084*
Spleen 0.312 ±0.027 0.336 ±0.017 0.324 ± 0.025
Thymus 0.277 ± 0.031 0.321 ±0.028 0.324 ± 0.049
Kidney 0.852 ± 0.052 0.876 ± 0.036 0.860 ± 0.020
Right Ventricle 0.069 ± 0.005 0.070 ±0.011 0.075 ± 0.008
Left Ventricle 0.272 ± 0.008 0.287 ±0.010 0.282 ±0.013
RV/LV+S^ 0.251 ±0.025 0.244 ±0.031 0.266 ± 0.029
Weight Gain (g) 146.1 ±4.596 139.7 ±11.25 132.3 ± 9.728*
"Organ weights expressed as means (x 10') ± SD (N = 6). ''Significantly different than controls at p < 0.05. "^V/LV+S = right ventricle weight/ left ventricle plus septum weight.
72
Table 4
Summary of Dry Organ Weight / Body Weight Ratios in Rats at 21 Days Following a Single Injection (i.v.) of 7-CYSDHP
Control CYSDHP CYSDHP
Organ (Saline) (300 ^lmol/kg) (600 nmol/kg)
Liver 1.559 ±0.058 1.377 ±0.038'' 1.322 ±0.102"
Lung 0.115 ±0.016 0.127 ±0.017* 0.136 ±0.018"
Spleen 0.068 ± 0.006 0.073 ± 0.005 0.070 ± 0.005
Thymus 0.123 ±0.015 0.132 ±0.008 0.133 ±0.015
Kidney 0.213 ±0.013 0.219 ±0.009 0.215 ±0.005
Right Ventricle 0.014 ±0.001 0.014 ±0.002 0.015 ±0.002
Left Ventricle 0.059 ±0.001 0.062 ± 0.002 0.060 ± 0.003
RV/LV+S' 0.231 ±0.018 0.222 ± 0.025 0.247 ± 0.028
"Organ weights expressed as means (x 10") ± SD (N = 6). ^Significantly different than controls zip < 0.05. '"RV/LV+S = right ventricle / left ventricle plus septum ratio.
73
Table 5
Summary of Wet Organ Weight / Dry Organ Weight Ratios in Rats at 21 Days Following a Single Injection (i.v.) of 7-CYSDHP
Control CYSDHP CYSDHP
Organ (saline) (300 |imol/kg) (6(X) |imol/kg)
Liver 3.205 ± 0.085 3.352 ±0.102 3.332 ± 0.093
Lung 4.873 ± 0.073 5.084 ±0.071'' 5.113 ±0.115"
Spleen 4.609 ± 0.099 4.591 ±0.077 4.604 ± 0.095
Thymus 4.944 ±0.091 4.963 ±0.103 4.896 ± 0.096
Kidney 4.395 ±0.147 4.472 ±0.186 4.119 ±0.067
Right Ventricle 4.961 ±0.125 5.055 ±0.313 5.026 ±0.215
Left Ventricle 4.634 ± 0.075 4.596 ± 0.054 4.673 ± 0.068
"Data expressed as means ± SD (N = 6). ''Significantly different than controls at p < 0.05.
74
Table 6
Summary of Wet Organ / Body Weight Ratios in Rats at 21 Days Following a Single Injection (i.v.) of 7-GSDHP or Monocrotaline"
Control Monocrotaline GSDHP
Organ (saline) (185 nmol/kg) (600 ^lmol/kg)
Liver 3.687 ± 0.292 4.319 ± 0.363" 3.996 ±0.197
Lung 0.494 ± 0.024 0.819 ±0.076" 0.497 ± 0.034
Spleen 0.279 ± 0.048 0.354 ± 0.029'' 0.260 ± 0.024
Thymus 0.250 ± 0.042 0.251 ±0.026 0.243 ± 0.020
Kidney 0.837 ± 0.032 0.858 ± 0.025 0.807 ± 0.052
Right Ventricle 0.067 ±0.014 0.089 ± 0.004" 0.075 ± 0.008
Left Ventricle 0.317 ±0.045 0.287 ±0.013 0.278 ±0.017
RV/LV+S'' 0.212 ±0.020 0.311 ±0.016" 0.268 ± 0.029'"
Weight Gain (g) 239.8 ±24.1 239.8 ± 7.4 253.0 ± 12.0
"Organ weight data expressed as means (x 10") ± SD (N = 5, except 7-GSDHP, N = 6). ''Significantly different from control and 7-GSDHP treated at p < 0.05. "Significantly different from control and monocrotaline treated at p < 0.05. ''RV/LV-t-S = right ventricle weight / left ventricle plus septum weight.
75
Table 7
Summary of Dry Organ / Body Weight Ratios at in Rats at 21 Days Following a Single Injection (i.v.) of 7-GSDHP or Monocrotaline"
Control Monocrotaline GSDHP
Organ (Saline) (185 limol/kg) (600 lamol/kg)
Liver 1.120 ±0.093 1.320 ± 0.064'' 1.29 ±0.080"
Lung O.I 11 ±0.011 0.162 ±0.011'' 0.105 ±0.006
Spleen 0.064 ± 0.009 0.081 ± 0.007'' 0.060 ± 0.005
Thymus 0.067 ± 0.002 0.055 ± 0.005" 0.053 ± 0.004
Kidney 0.201 ±0.012 0.207 ± 0.004 0.193 ±0.014
Right Ventricle 0.015 ±0.003 0.019 ±0.001" 0.017 ±0.002
Left Ventricle 0.067 ± 0.004 0.064 ± 0.003 0.064 ± 0.003
RV/LV-hS'' 0.220 ± 0.033 0.302 ± 0.020" 0.265 ± 0.025"
"Organ weight data expressed as means (x 10") ± SD (N = 5, except 7-GSDHP, N = 6). ''Significantly different from control and 7-GSDHP treated at p < 0.05. "Significantly different from control and monocrotaline treated at;? < 0.05. ''RV/LV-f-S = right ventricle weight/ left ventricle plus septum weight.
76
Table 8
Summary of Wet Organ / Dry Organ Weight Ratios in Rats at 21 Days Following a Single Injection (i.v.) of 7-GSDHP or Monocrotaline"
Control Monocrotaline GSDHP
Organ (Saline) (185 |imol/kg) (600 [xmol/kg)
Liver 3.122 ±0.088 3.263 ±0.212 3.082 ± 0.773
Lung 4.478 ± 0.253 5.045 ±0.174* 4.728 ±0.129
Spleen 4.363 ±0.140 4.395 ±0.189 4.321 ±0.078
Thymus 4.603 ± 0.050 4.600 ±0.051 4.622 ± 0.054
Kidney 4.175 ±0.121 4.159 ±0.189 4.321 ±0.052
Right Ventricle 4.467 ±0.179 4.654 ±0.177 4.399 ±0.131
Left Ventricle 4.748 ± 0.549 4.521 ±0.132 4.345 ±0.112
T>ata expressed as means (x 10") ± SD (N = 5, except 7-GSDI-IP, N = 6). ''Significantly different from controls and 7-GSDHP treated at /y < 0.05.
77
weights of the left heart from this group, which would artificially increase the ratio. A small
increase in iiver weight was observed, but was not significantly different than controls.
Chemistry of Sulfur-Bound Pyrroles
Identification ofThiopropyl-Sepaharose resin-bound pyrrole
Sulfur-bound pyrrole can be solvolized by silver or mercury ion (Mattocks and
Jukes, 1990a) with cleavage of the thioether bond. If this procedure is conducted in a
buffered ethanolic solvent, the former thioether will be replaced with an ethoxy
substitution. The pyrrole can be identified on TLC or GC-MS, establishing the position of
the ethoxy group (C7 or C9), and thus the position of sulfur-binding. We have used this
procedure with Thiopropyl-Sepharose resin exposed to different DHAs. Each DHA (10
Hmol in 1 ml dimethylformamide) was continuously injected into the recirculating system
over a period of one hour. Delay times were 1.5 and 30 sec (5 cm and 400 cm tubing
length, respectively). Resin exposed to dehydrotrichodesmine, dehydromonocrotaline,
dehydroseneciphylline and dehydroretrorosine, and dehydro-7-acetyl-lycopsamine yielded
7-ethoxy DHP, regardless of the delay time. Thus, time of exposure to the buffer (1.5 or
30 sec) did not affect the pattern of alkylation. Evidence of bialkylation was not observed
as 7,9-ethoxy DHP was not detected. Pyrrole released from resin exposed to
dehydrolycopsamine after a 1.5 sec hydrolysis time was identified as 9-ethoxy DHP. This
indicates that all DFIAs except dehydrolycopsamine bound the resin at C7 of the pyrrole
moiety. Dehydrolycopsamine bound the resin at C9. When dehydrolycopsamine was
allowed a 30 sec exposure to the buffer prior to trapping, no pyrrole could be detected from
the resin.
78
Chemistry of liver sulfur-bound pyrroles
Bile was collected from isolated rat livers perfused with monocrotaline,
seneciphylline, retrorsine, trichodesmine and a mixture of lycopsamine and intermedine
(Yan et at., 1995). Rats were given single i.p. doses of the same alkaloids at 65-90% acute
LDjo (3-5 day) levels (Mattocks, 1986; Wild, 1994) and killed at 24 hr. Liver homogenates
from rats injected with PAs, and bile from PA perfused livers were treated with Tris-
buffered ethanolic silver nitrate to determine the position of sulfur-binding of pyrrole
metabolites. When bound pyrroles were cleaved from either liver or bile sulfhydryls, 7-
ethoxy DHP was produced, as determined by TLC and GC-MS (Method I). This indicated
the presence of C7 pyrrole conjugates from liver and bile. As with the resin, no evidence of
sulfhydryl crosslinking was detected, as a pyrrolic diether was not produced in the silver
nitrate reactions. Pyrrolic ethers were not detected in bile or tissue from rat liver perfused
with the C9 monoesters lycopsamine and intermedine. Thus a C9 sulfur adduct did not
form, or was present in very small amounts. Lung and brain from the PA treated animals
were treated in the same manner as the livers, but pyrrolic ethers were not detected. This
was attributed to the fact that tissues were assayed under buffered conditions, which
decreases the sensitivity of this procedure (Mattocks and Jukes, 1990a).
Quantitation ofDHA binding to Thiopropyl-Sepharose resin
The relative reactivity of a series of DHAs toward a large molecular weight
sulfhydryl compound was studied by using Thiopropyl-Sepharose as a model nucleophile.
Equimolar amounts of DHAs (6 nmol) were injected into the recirculating aqueous flow
system, where they were instantaneously mixed with the buffer and passed through the
activated resin after a 1.5 sec delay (5 cm tubing). The resin-bound pyrrole was cleaved by
ethanolic silver nitrate and quantified with Ehrlich Reagent. Table 9 shows the amount of
79
Table 9
Relative Quantity of Pyrrole Bound to Thiopropyl-Sepharose Resin After Exposure to DHAs"
Bound pyrrole
DHA (^imol) {% of total injected)
Dehydromonocrotaline 0.80 ±0.01 13.31 ±0.26
Dehydroseneciphylline 0.76 ± 0.05 12.71 ± 0.81
Dehydro-7-acetyl-Iycopsamine 0.58 ±0.08 9.59 ± 1.39
Dehydroretrorsine 0.51 ± 0.05 8.50 ± 0.76
Dehydrotrichodesmine 0.42 ±0.04 7.07 ± 0.62
Dehydrolycopsamine* 0.05 ± 0.02 0.85 ± 0.24
"Data from recirculating system as described in Methods. DHAs (6 ^mol) were exposed to the aqueous environment for approximately 1.5 sec prior to passing through the resin. Values are means ± S.D. (N = 4-9 experiments). different from other groups at p < 0.01.
80
pyrrole subsequently bound to the resin after exposure to different DHAs. The highest
quantity of pyrrole was trapped on passing dehydromonocrotaline and
dehydroseneciphylline through the resin, with 0.80 ^mol and 0.76 nmol trapped,
representing 13.3 % and 12.7%, respectively, of total DHA injected. Pyrrole trapped from
dehydro-7-acetyl-lycopsamine, dehydroretrorsine and dehydrotrichodesmine was 0.57
M-mol, 0.51 ^imol, and 0.42 ^lmol, representing 9.6% , 8.5%, and 7.1% respectively, of
total injected DHA. A mixture of the 9-monoesters, dehydrolycopsamine and
dehydrointerraedine, was found to react minimally with the resin, with 0.09 [imol trapped
pyrrole, or 0.85% of the total injected. DHP, 7-GSDHP, and parent PAs did not react with
the resin in this system.
Hydrolysis of DHAs
When a DHA is introduced into an aqueous environment, the ester rapidly
hydrolyzes forming DHP and the corresponding necic acid. As a result, the pH of the
solution falls. DHAs, prepared according to established methods (Mattocks et al., 1989),
were rapidly added to a stirred HEPES solution (0.5 mM, pH 8.0). Change in pH was
followed potentiometrically, producing a sigmoidal log-linear curve which was near linear
through at least 2 pH units (Figure 12). From the slope of the near linear portion, the rate
constant, k, and the half-life, of hydrolysis under the studied conditions could be
estimated. Calculations were based on the disappearance of HEPES conjugate base, which
was assumed to be directly proportional to liberation of protons by ester hydrolysis.
Relative rates of hydrolysis for the DHAs are shown in Table lO.Under the conditions
studied, the two open esters, dehydrolycopsamine and 7-acetyl-lycopsamine, had the
shortest half-lives; 1.02 and 0.39 sec, respectively. Dehydroretrorsine and
81
9
8
7
6
4
3 20 35 40 5 10 15 25 30 0
Time (sec)
Figure 12. Representative potentionietric recording of the hydrolysis of dehydroretrorsine and dehydrotrichodesmine. The fall in pH was due to the release of protons on hydrolysis.
82
Table 10
Rate Constants and Half-Lives of Hydrolysis of DHAs"
DHA )t(sec ')±S.D.' S.D/
Dehydro-7-acetyl-lycopsamine 1.77 ±0.09 0.39 ± 0.02
Dehydrolycopsamine 0.68 ±0.12 1.02 ±0.22
Dehydroretrorsine 0.65 ±0.10 1.06 ±0.20
Dehydroseneciphylline 0.43 ± 0.06 1.60 ±0.28
Dehydromonocrotaline 0.20 ± 0.06 3.39 ± 0.99
Dehydrotrichodesmine 0.13 ±0.02 5.36 ± 0.92
"Values were determined potentiometrically as described in Methods. Data represent means ± SD from a minimum 6 experiments.
''All values were statistically different at p < 0.01 except for dehydrolycopsamine and dehydroretrorsine, whose values did not differ significantly.
Tlalf-lives calculated by Q.69/k
83
dehydroseneciphylline were slightly more stable, with half-lives of 1.06 and 1.60 sec,
respectively. Dehydromonocrotaline and dehydrotrichodesmine were the slowest to
hydrolyze, with half-lives of 3.39 and 5.36 sec, respectively. All half-life values were
significantly different at /? < 0.01, except for dehydroretrorsine and dehydrolycopsamine,
which did not differ.
Alkylation of 4-(p-nitrobenzyl)pyridine by DHAs
The DHAs reacted with 4-(p-nitrobenzyl)pyridine under pseudo-first-order
conditions (Karchesy and Deinzer, 1987), (Table 11) forming an adduct which could be
detected colorimetrically after quenching with base at 565 nm (Figures 13, 14). As time
increased, the amount of detected color increased, indicating buildup of product. The two
C12 DHAs reacted at the highest rates with 4-(p-nitrobenzyl)pyridine. Dehydroretrorsine
was the most reactive, the data fitting a biexponential rate expression (/:,, = 0.226 min ',
= 0.021 min"'). Dehydroseneciphylline, another C12 macrocycle, reacted about 4.5 times
slower (fc, = 0.041 min ') with the nucleophile. The faster reaction, of dehydroretrorsine
and 4-(p-nitrobenzyl)pyridine appeared to be mostly over by 20 min, at which time the
slower, second color-forming reaction, could be observed. Dehydrotrichodesmine (A:, =
0.013 min"'), a CI 1 DHA, was the slowest reacting compound tested.
Dehydromonocrotaline (fc, = 0.026 min"') reacted at twice the rate of
dehydrotrichodesmine. The alkylation of dehydroseneciphylline, dehydromonocrotaline
and dehydrotrichodesmine fit monoexponential rate expressions. The open ester DHAs
were also reactive toward 4-(/?-nitrobenzyl)pyridine. The diester dehydro-7-acetyl-
lycopsamine reacted twice as fast as its 9-monoester derivative, dehydrolycopsamine, with
values of 0.152 and 0.076 min"', respectively.
Table 11
Rate Constants for the Alkylation of DHAs by 4-(/?-nitrobenzyl)pyridine''
DHA A:, (min ') ± S.E. r''
Dehydroretrorsine 0.226 ± 0.063 0.996
Dehydro-7-acetyl-lycopsamine 0.152 ±0.011 0.980
Dehydrolycopsamine 0.076 ± 0.003 0.994
Dehydroseneciphylline 0.041 ±0.002 0.999
Dehydromonocrotaline 0.026 ± 0.002 0.993
Dehydrotrichodesmine 0.013 ±0.001 0.994
"Reactions were carried out under pseudo-first order conditions at 22 °C. Rate constants calculated from mono- or biexponential rate equation. 'Tlegression coefficient.
85
• Dehydroretrorsine
A DehydroseneciphylHne
• Dehydromonocrotaline
• Dehydrotrichodesmine
0 -
10 20 30 40 50 60
Time (min)
Figure 13. Rates of alkylation of macrocyclic DHAs by 4-05-nitrobenzyl)pyridine. Data are means ± standard deviations from 3 observations. Data for dehydroretrosine was fit to a biexponential rate expression. Data for the remaining DHAs were fit to a monoexponential equation.
86
• Dehydro-7-acetyl-lycopsamine
• Dehydrolycopsamine
c c ca vo
<u (J c
X3 U« O C/3
X) <
0 0 10 20 30 40 50 60
Time (min)
Figure 14. Rates of alkylation of open-ester DHAs by 4-(/7-nitrobenzyl)pyridine. Data are means ± standard deviations from 3 observations. Data were fit to a monoexponential rate equation.
87
Inhibition of Hexokinase by DHAs, in vitro
Yeasl hexokinase
The phosphotransferase activity of yeast hexokinase was irreversibly inhibited by
dehydrotrichodesmine in a dose-dependent fashion (Figure 15). Inhibition of the enzyme
occurred at concentrations of DHA above 1 mM and complete, or near complete
inactivation of hexokinase occurred at 20 mM dehydrotrichodesmine. Concentrations of
DHAs above 20 mM were not tested because of excessive polymerization in the incubation
mixture by the pyrrole. Dehydroretrosine and dehydro-7-acetyl-lycopsamine, DHAs widi a
7-ester, both inhibited the enzyme in a similar fashion to dehydrotrichodesmine. The 9-
monoester DHA, dehydrolycopsamine, was a weaker inhibitor of hexokinase than the
diester DHAs (Figure 16). Under the smdied conditions, the IC50S for
dehydrotrichodesmine and dehydrolycopsamine were 5.3 and 52.4 mM, respectively. IC50
values for other 7-ester DHAs did not differ from dehydrotrichodesmine (Data not shown).
The inhibitory action of the DHAs was lost if they were added to the incubation mixture 1
min prior to adding hexokinase, indicating that DHP and necic acid did not affect enzyme
activity under the studied conditions. Hexokinase samples incubated 30 or 60 min with
DHAs showed no recovery of activity. At 20 mM, the parent alkaloids trichodesmine and
retrorsine had no effect on hexokinase activity.
In order to assess the nature of enzyme inhibition by DHAs in terms of competitive
or non-competitive, hexokinase inhibition assays were conducted in the presence of
increasing concentrations of substrate. Figure 17 indicates that dehydrotrichodesmine
produced a dose-dependent decrease in the of hexokinase. Additionally, because a
rightward shift in the concentration-effect curves was not observed, the data suggest a non
competitive interaction between enzyme and inhibitor. Non-linear regression analysis of the
data indicate a dose-related decrease in while values are similar (Table 12). When
88
the data were plotted as [substrate]/!^ versus [substrate], the regressions were also
suggestive of a non-competitve interaction (Figure 18).
Experiments were also conducted in which hexokinase was preincubated with
mannose (10 mM) or ATP (10 niM) prior to dehydrotrichodesmine exposure. Mannose, a
high affinity substrate for hexokinase, provided the enzyme minimal protection against
inactivation by dehydrotrichodesmine (Figure 19). However, ATP, a co-substrate,
provided even greater protection to hexokinase against inactivation by
dehydrotrichodesmine (Table 13). From this experiment, die IC50 value for
dehydrotrichodesmine was 5.84 mM. When hexokinase was pretreated with mannose or
ATP, the IC50 values for dehydrotrichodesmine were 7.14 and 23.99 mM, respectively.
Rat brain hexokinase
Preliminary investigations were performed using rat brain hexokinase to determine
if inhibition occurred toward the mammalian enzyme. Rat brain hexokinase, assayed in
vitro, was significantly inhibited by 1 min incubation with 5 mM macrocyclic DHAs
(Figure 20) compared to controls. The enzyme was only slighdy inhibited by the DMF
vehicle (Figure 20). No effect on hexokinase was observed with dehydrolycopsamine at 5
mM or 10 mM (Figures 20, 21). Preincubation of brain homogenates with 1 mM glucose
afforded slight, but significant protection to hexokinase against 10 mM
dehydrotrichodesmine (Figure 21). Dehydrotrichodesmine was significantly more active
against hexokinase when the brain homogenates were preincubated with
P-mercaptoethanol (0.1%) for 24 hr prior to assay (Figure 21). p-Mercaptoethanol has a
reducing effect on several of the hexokinase disulfides (Lenzen et ai, 1989). As with yeast
hexokinase, inhibitory action of DHAs was lost if the compounds were added to the
89
incubation buffer I min prior to brain homogenate. Additionally, parent PAs had no
inhibitory effect on enzyme activity.
Glucose-6-phosphate dehydrogenase
In comparative assays, dehydrotrichodesmine, at 1.0, 5.0 or 20 mM, did not
significantly inhibit glucose-6-phosphate dehydrogenase in relation to controls (data not
shown).
90
Table 12
Effect of Dehydrotrichodesmine on Yeast Hexokinase Phosphotransferase Activity in the Presence of Increasing Substrate Concentrations"
[Dehydrotrichodesmine]
(mM)
V max
(nmol NADPH/min) (mM glucose) r'
0.0 9.76 ± 0.64 0.122 ±0.034 0.988
2.5 9.12 ± 1.28 0.165 ±0.089 0.969
5.0 4.80 ± 0.05 0.148 ±0.007 0.999
10.0 1.76 ±0.06 0.168 ±0.001 0.992
"Values were derived from non-linear regression of data from Figure 17 with the aid of a computer program (SigmaPlot®, Jandel Scientific, San Raphael, CA). Data represent means ± standard errors from 5 observations. '' Coefficient of regression.
Table 13
Effect of Mannose (10 mM) and ATP (10 mM) Pretreatment on the Inhibition of Yeast Hexokinase by Dehydrotrichodesmine"
Treatment ICjg
Dehydrotrichodesmine 5.84 ±1.06
Dehydrotrichodesmine + Mannose 7.14 ± 1.03
Dehydrotrichodesmine + ATP 23.99 ±1.05
"Data derived from regression analysis from linear portion of log concentration-effect curves (Figure 19) with the aid of a computer program (Tallarida and Murray, 1986). Data represent means ± standard errors from 5 observations. All data are statistically different at p < 0.05.
[Dehydrotrichodesmine] (mM)
Figure 15. Concentration-effect curve of the Inhibition of yeast hexokinase by dehydrotrichodesmine. Data represent means ± standard deviations from 5 observations.
100
80
60
Dehydrotrichodesmine Dehydrolycopsamine
40
20
t I t U 5 10
[DHA] (mM)
20
Figure 16. Concentration-effect curve for the inhibition of yeast hexokinase by dehydrotrichodesmine and dehydrolycopsamine. Data represent means ± standard deviations from 5 observations.
93
10.0
8.0
6.0
4.0
2.0
• 0.0 mM Dehydrotrichodesmine
T 2.5 mM Dehydrotrichodesmine
• 5.0 mM Dehydrotrichodesmine
• lO.O mM Dehydrotrichodesmine
0.0
-U- • • • '
0.01 O.l 10
[Glucose]
Figure 17. Saturation curves for the inhibition of yeast hexokinase by dehydrotrichodesmine. Curves represent logistic non-linear regression fit with the aid of a computer program. Data represent means ± standard deviations from 5 observations.
94
500
400
300
200
100
0 9 3 5 4
[Substrate] mM
Figure 18. [Substrate]A^ versus [Substrate] plot of the inhibition of yeast hexokinase by dehydrotrichodesmine. Data, transformed from Figure 17, represent means from 5 observations.
95
120 • Dehydrotrichodesmine
• Dehydrotrichodesmine + Mannose (10 mM) • Dehydrotrichodesmine + ATP (10 mM)
100
80
60
£
'•w U < (U c« ca c • o X (U
± 40 C3 3
u
^ 20
0 .J L
10 20
[Dehydrotrichodesmine] (mM)
Figure 19. Effect of ATP and mannose on the inhibition of yeast hexokinase by dehydrotrichodesmine. Data represent means ± standard deviations from 5 observations. Hexokinase was preincubated with ATP or mannose for 5 min prior to exposure to dehydrotrichodesmine.
96
• Control
OH Vehicle Control
Dehydrotrichodesmine (5 mM)
Dehydromonocrotaline (5 mM)
Hi Dehydroseneciphylline (5 mM)
Dehydroretrorsine (5 mM)
V/Wi Dehydrolycopsamine (5 mM)
Figure 20. Effect of DHAs on rat brain hexoidnase, in vitro. Data represent means ± standard deviations from 5 observations. *Indicates group mean is different than control at p < 0.05.
97
160
140
120
100
80
- 60
40
20
0
• Vehicle Control
mnH Dehydrolycopsamine (10 mM)
Dehydrotrichodesmine (10 mM)
Dehydrotrichodesmine (10 mM) + Glucose Pretreatment (1 mM)
EZ3 P-MEtOH Pretreatment (0.1 %) Control
P-MEtOH Pretreatment (0.1 %) + Dehydrotrichodesmine (10 mM)
Figure 21. Effect of dehydrotrichodesmine and dehydrolycopsamine on rat brain hexokinase, in vitro. Hexokinase was preincubated 24 hr with P-mercaptoethanol and 5 min with glucose prior to dehydrotrichodesmine exposure. Data represent means ± standard deviations from 5 observations. *Indicates group means are different from control at p < 0.05. **Indicates group means are different from control and dehydrotrichodesmine at p < 0.05.
98
DISCUSSION
PAs are found in a wide variety of flowering plant families distributed throughout
the world. They are toxic to animals, including man. Humans ingest PAs through the use
of herbal products or by the consumption of contaminated grains (Huxtable, 1989).
Hepatotoxicity is the trademark of PA poisoning, although other organs may be affected in
certain instances. In humans, ingestion of PAs results in veno-occlusive disease of the liver
and neurotoxicity in some cases (Ismailov etal., 1970). In addition to hepatotoxicity, PAs
such as monocrotaline produce pulmonary toxicity and associated cor pulmonale in the rat.
Hepatic metabolism of PAs by cytochrome P450s to pyrroles is a requirement for toxicity.
Metabolism by other organs has not been demonstrated, suggesting that the liver is the sole
source of toxic metabolites. Toxicity is believed to result from the formation of pyrrolic
ester intermediates (DHA), which are highly reactive electrophiles. Covalent binding of
DHAs to important tissue nucleophiles presumably initiates the events leading to toxicity.
In recent years, much information has emerged regarding the metabolism of PAs. It is now
widely accepted that DHAs are the proximate toxin, responsible for both hepatic and
extrahepatic toxicity. However, many important questions remain unanswered. Perhaps
most interesting is the observation that structurally similar PAs often produce markedly
different toxicities, in terms of both lethality and organs affected. The Senecio alkaloids,
such as seneciphylline and retrorsine are hepatotoxic in rats, except under unusual
circumstances (White et al., 1972; Mattocks and White, 1973; Ohtsubo et ai, 1977), in
which pneumotoxicity has been reported. The acute LD50S (3-5 day) of retrorsine and
seneciphylline in male rats are 97 and 230 [imol/kg, respectively (Mattocks, 1972; Bull et
ai, 1968). Crotalaria alkaloids such as monocrotaline are pneumotoxic in rats. The LDjg of
monocrotaline is 335 p.mol/kg (Mattocks, 1972). Trichodesmine is neurotoxic in humans,
rats and mice (Ismailov, 1970), yet differs from monocrotaline only by an isopropyl
99
substitution at C14 rather than a methyl. Trichodesmine is also approximately six-fold more
lethal than monocrotaline, having an LD50 of 57 |imol/kg in rats. The open ester PAs are
only known to produce hepatotoxicity. The 9-monoesters are weak hepatotoxins in
comparison to macrocyclic diester PAs. Indicine, a stereoisomer of lycopsamine, has a
LD50 of greater dian 3.3 mmol/kg in rats (Schoental, 1968). The toxicity of open diester
PAs appears to vary greatly, although they are often as toxic as macrocyclic diesters.
Symphytine, a diester alkaloid of comfrey root, has an of 340 |imol/kg in rats (Hirono
et al., 1979). The reasons for these differences in toxicity are poorly understood.
We believe that the differences in PA toxicity are a reflection of differences in rates
of formation of DHAs and their further metabolism. We posmiate that the metabolic
disposition of DHAs is partially determined by differences in their stability, especially at the
7-ester position. The aim of this dissertation was to investigate the relationship between
physicochemical properties of structurally varied DHAs and observed toxicity of the
corresponding parent PAs. The goals of this research were to: 1) to develop an efficient
chromatographic system for the separation and purification of large quantities of PAs, 2)
investigate the role of sulfur-conjugated pyrroles as potential toxic metabolites of PAs, 3)
to use in vitro and in vivo methods to investigate differences in reactivity and stability of
structurally similar DHAs, 4) to examine the relationship between the nucleophilic
reactivity of these DHAs and patterns of their further metabolism in the isolated perfused rat
liver, and to the toxicity produced by the parent PA.
Counter-Current Chromatography
As plants typically contain several alkaloids, there has been a need for convenient,
rapid separation and purification methods for these compounds. The separation of PAs on
newer high speed counter-current chromatography instruments has not been reported until
recently (Cooper et al., 1996), and had been little used for alkaloids in general compared to
100
other natural products of plant origin (Marston and Hostettman, 1994). We demonstrate the
preparative separation and purification of PAs isolated ixom Amsinckia tesseiiata,
Symphytum spp. (comfrey root), Trichodesma incanum, and Senecio douglasii var.
longilobus, using high speed CCC.
A chloroform (mobile) and phosphate buffer (stationary) solvent system was
chosen for separations of PAs. The large density difference between the two solvents
promotes rapid phase separation and excellent stationary phase retention even at high
mobile phase flow rates (Marston and Hostettman, 1994). PAs are easily recoverable from
a chloroform mobile phase. With an aqueous buffer stationary phase, good solute
resolution can be obtained since structurally similar PAs differ in their values
(Mattocks, 1986). Additionally, the colored impurities often associated with PA isolations
are largely unretained in an aqueous stationary phase, and elute at the solvent front.
Resolution of PAs is determined by partitioning between the two liquid phases. Partition
coefficients, K, defined as concentration of solute in the mobile phase divided by solute
concentration in the stationary phase, ideally fall between 2 and 0.5 for the most efficient
separations (Conway, 1990), but a wide range of K values may be acceptable depending
on the amount and retention times of impurities (Zhang et al., 1988). Partitioning of PAs
between solvent phases depends on the proportion of alkaloid in the ionized form, as well
as its lipid solubility in the free base form. Partition coefficients and retention time, of
PAs will therefore be profoundly affected by the stationary phase pH. Because the
stationary phase is a liquid, retained PAs are easily recovered, and their adsorptive loss to
various solid chromatographic media as reported by Zalkow etal. (1988), is prevented.
Amsinckia tessellata (Boraginaceae) is a member of a small genus of around 14
species native to North and South America and introduced to Australia (Culvenor and
Smith, 1966; Kelley and Seiber, 1992). Alkaloid content of Amsinckia is variable, but all
contain hepatotoxic PAs, dominated by the diastereomers lycopsamine and intermedine,
l O I
with smaller quantities of their acetylated derivatives and other uncommon PAs. These are
all open mono- and diesters of retxonecine and heliotridine, 28 of which have been
identified (Kelley and Seiber, 1992). The abundance of A. tessellata in the vicinity of
Tucson, Arizona, makes this plant an ideal source for the open ester PAs used in this
dissertation. Roitman (1983) used silica gel column chromatography for preparative
separation of PAs from A. menziesii, while Culvenor and Smith (1966) employed counter-
current distribution to resolve PAs from several Amsinckia species. Both these techniques
proved time-consuming since successive runs were necessary for complete resolution. In
the genus Amsinckia, the relative quantities of stereoisomers is variable between both
species and different populations of the same species (Kelley and Seiber, 1992). Because
of the difficulty in resolving isomeric PAs on a preparative scale, comparative toxicological
studies for such stereoisomers have not been performed. Human poisonings by Amsinckia
have not been reported, but they are implicated in livestock poisonings in California
(Roitman, 1983).
Comfrey, consisting of dried root or leaves of Symphytum officinale (common
comfrey) or 5. x uplandicum Nyman (Russian comfrey, Boraginaceae), is widely used by
humans as a herb, vegetable and food supplement (Huxtable, 1990). Symphytum spp.
usually contain a complex mixmre of PAs, consisting of open mono- and diesters of
retronecine. They are mildly hepatotoxic and are carcinogenic in rats (Hirono et al., 1978),
and have been implicated in human poisonings (Huxtable, 1990; Ridker et al., 1985).
Culvenor et al. (1980) used counter-current distribution with a solvent system of
chloroform and phosphate buffer, pH 7.5, to resolve 1.8 g of an alkaloid fraction from S.
X uplandicum, yielding eight PAs. Due to the expense of comfrey root in the local
supermarkets, the variable PA content and difficulty in botanical identification, Amsinckia
was the preferred source for the open-ester PAs.
102
The large genus Senecio (Asteraceae) has over 100 PA-containing species
(Huxtable, 1989). Senecio doiigiasii var. iongiiobus (Asteraceae), contains the hepatotoxic
PAs riddelliine, senecionine, retrorsine and seneciphylline (Adams and Govindachari,
1949), all structurally similar cyclic diesters of retronecine. In the southwestern U.S., S.
douglasii has been used extensively for ethno-medicinal purposes (Huxtable, 1990). Infant
poisonings and deaths have occurred in Arizona due to consumption of herbal preparations
containing S. douglasii var. Iongiiobus (Stillman etal., 1977; Fox etal., 1978). Zalkow et
al. (1988) using DCCC with a CHClj-CgHg-MeOH-HjO solvent system, separated a 3 g
mixture of 10 alkaloids from S. anonymus, including partial resolution of the diastereomers
senecionine and integerrimine. This separation demonstrates the resolving power of
counter-current techniques, although DCCC separations take longer, and choice of solvent
systems are limited (Conway, 1990). Resolution of the PAs senecionine and seneciphylline
in preparative systems has been difficult. Separation was achieved on silica gel
chromatography only by the use of sequential runs (Adams and Govindachari, 1949). The
two PAs were unresolvable by TLC in these and other studies (Chizzola, 1994). Despite
these earlier difficulties, they were well resolved in the present CCC system. Rorosenine
has been reported from Cacalia floridana, Senecio aureus, S. floridanus, S. fluviatilis
(Mattocks, 1986), but not from S. douglasii var. Iongiiobus (Adams and Govindachari,
1949). The former Senecio species are not known to occur in Arizona (Kearney and
Peebles, 1960), therefore contamination by another florosenine containing plant was
unlikely.
The small genus Trichodesma (Boraginaceae), native to Africa and Asia, contains
about 35 species, 3 of which are known to contain toxic PAs (Huxtable, 1989). T.
incanum has been responsible for outbreaks of human and livestock poisonings, reviewed
in the Russian literature (Smimov etal., 1959a, 1959b; Ismailov, 1970; Ismailov etal.,
1970). T. incanum has been shown to contain two neurotoxic PAs, incanine and
103
trichodesmine (Yunusov and Plekhanova, 1957), both cyclic diesters of retronecine.
Previously, Irichoclesinine isolated from various Crotalaria was separated from other PAs
by column chromatography using Celite and Florosil, preparative TLC and DCCC (Adams
and Gianturco, 1956; Rothschild et al., 1979; Roeder et al., 1992). In the present study,
incanine and trichodesmine were co-isolated as white crystals. The lack of non-alkaloid
contaminants allowed for rapid separation times (< 2 hr), since the PA did not have to be
retained longer than impurities which elute near the solvent front. Thus, the high K value
for incanine (Table 2) was acceptable.
The use of high speed CCC in the preparative separation and purification of natural
products has become well established (Marston and Hostettman, 1994). Our results show
the value of this method for PAs. Plant compounds, including PAs, which are often
isolated as a gum or tar, as in the case of Senecio, Amsinckia and Symphytum alkaloid
fractions in this study, can be safely injected onto the column without fear of contaminating
or fouling a solid matrix. Thus pre-separation clean-up steps are unnecessary. Further
advantages demonstrated in this investigation include rapid separation times and low
solvent consumption (< 2 1 per run). The simple chloroform-phosphate buffer solvent
system used in these experiments was sufficient for the separations performed, although
the resolving power was insufficient to separate diastereomers. Preliminary experiments
indicate that ternary solvent systems of CHClj-MeOH-HjO may also be useful in
separating PAs. More elaborate solvents and techniques will become necessary with the
separation of increasingly complex PA mixtures. New techniques such as pH-zone-refining
CCC described by Ma etal. (1994), which utilizes a pH gradient, should prove especially
useful in cases where PAs possess large differences in K values.
104
Toxicity of 7-CYSDHP and 7-GSDHP
Glutatliione is aii iniporlaiil cellular component in the defense against damage
produced by electrophilic compounds. Glutathione is found in high concentrations in many
mammalian cell types, participating in a variety of reactions. The nucleophilic sulfur atom
of the cysteine residue serves to neutralize many electrophilic metabolites. Conjugation is
generally mediated by the enzyme glutathione 5-transferase, although some highly
electrophilic compounds can react non-enzymatically (Koob and Dekant, 1991). Depending
on its disposition, a glutathione 5-conjugate may be further metabolized to a mercapturic
acid (A'-acetyl-cysteine conjugate) prior to excretion. This is accomplished by renal
y-glutamyltranspeptidase and dipeptidases which cleave the glutathione 5-conjugate to its
corresponding cysteine S'-conjugate. In the kidney, cysteine 5-conjugates are //-acetylated
by A^-acetyl-transferase, forming a mercapturic acid which can be excreted in the urine.
While this type of phase n metabolism serves to detoxify most compounds, several groups
of chemicals may be bioactivated to toxic metabolites by initially forming a glutathione
conjugate (Anders etai, 1988; Vamvakas and Anders, 1990). Several such mechanisms
have been elucidated. Further metabolism of a cysteine S-conjugate by p-lyase, located in
the renal proximal tubules, yields ammonia, pyruvate and reactive thiol compounds which
can form nephrotoxic agents. Certain halogenated alkenes produce renal toxicity by this
mechanism (Koob and Dekant, 1991). Some compounds are also believed to react with
glutathione in a reversible manner. This could allow a conjugate to circulate in a relatively
stable form where it could be reactivated to cause toxicity in other tissues.
Conjugation with glutathione in the liver is a major route of metabolism for certain
PAs (LaFranconi etal., 1985; Lame etal., 1990; Mattocks etai, 1991; Yan and Huxtable,
1994; Lame et ai, 1995). The presence of an AZ-acetyl-cysteine conjugated pyrrole in the
urine of rats treated with monocrotaline or senecionine is evidence of formation of a
cysteine 5-conjugate (Estep et al.. 1990). We have shown that 7-GSDHP formation occurs
105
in an isolated rat liver preparation perfused with the macrocyclic PAs monocrotaline,
Irichodesmine, seneciphylline and retrorsine (Yan etai., 1995). No evidence of GSH
conjugation was observed when a mixture of the 9-monoesters lycopsamine and
intermedine were perfused in this preparation, as detected by TLC and treatment of bile
with ethanolic silver nitrate. Mattocks et al. {1991) reported a similar occurrence when the
9-monoester heliotrine was tested in the same preparation. This may be due to the low rate
of metabolism of these alkaloids, as well as a lesser reactivity of C9 on the pyrrole nucleus
towards sulfhydryl groups. Formation of 7-GSDHP is likely a result of the non-enzymatic
reaction between the electrophilic DHA and the nucleophilic glutathione. In vitro studies
with hepatic microsomes incubated with senecionine have shown that the addition of GSH
5-transferase (from rat liver cytosol) to the preparation did not increase the rate of 7-
GSDHP formation (Reed et al., 1992). Dehydromonocrotaline reacts rapidly at its 7
position with both cysteine and glutathione (Mattocks et al., 1991). DHP, the hydrolytic
product of DHAs, does not react significantly with glutathione or cysteine at physiological
pH, nor is its ^-conjugation catalyzed by glutathione 5-transferases (Robertson et al.,
1977; Reed et al., 1992). Recent studies have suggested that 7-GSDHP and 7-CYSDHP
may be involved in the extrahepatic toxicity of monocrotaline (LaFranconi et al., 1985;
Huxtable etal., 1990). Although pneumotoxicity is dependent on metabolism, the
metabolite(s) responsible have yet to be identified. Dfff is not responsible for the
pneumotoxicity associated with this PA, and whether the highly reactive
dehydromoncrotaline can survive transport to the lungs is uncertain. This makes die sulfur-
conjugated pyrroles attractive candidates as pneumotoxic metabolites of PAs. They may act
as a transport mechanism, delivering a pyrrolic nucleus to the lungs where it could be
released in a reactive form. In light of recent evidence of glutathione-dependent
bioactivation of xenobiotics, we have investigated the role of 7-GSDHP and the putative
further metabolite, 7-CYSDHP in the toxicity of PAs.
106
In contrast to earlier studies (Huxtable et al., 1990; Bowers et al., 1991), but in
agreement with anodier (.Pan et al., 1993), 7-GSDHP did not produce toxicities associated
with the PA monocrotaline. Aldiough a small but significant increase in right heart / left
heart ratio was observed, this appears due to the small size of the left heart. Additionally,
because a corresponding increase in lung mass was not observed in these animals, the
increase in right heart size was probably artifactual. The lack of observed toxicity may be
related to the age of the rats studied, as younger rats are clearly more sensitive to PA
intoxication than older ones (Schoental, 1959, 1970; Jago, 1970, 1971; Mattocks and
White, 1973). Huxtable et al. (1990) and Bowers et al. (1991) gave 7-GSDHP to rats
weighing 50-70 g, while in the present study and that by Pan et al. (1993), animals
weighed from 160 to 220 g. The larger rats in this study were used to facilitate the i.v. tail
vein injections.
The putative further metabolite, 7-CYSDHP produced some of the toxicities
associated with monocrotaline. most notably an increase in lung mass. Only a slight
accompanying increase in right heart size was observed, attributable to the relatively small
increase in lung mass. Rats treated with 7-CYSDHP had a small, but significant decrease in
liver weight compared to controls. Because treated groups were smaller than controls,
livers may have been smaller because livers are proportionally smaller in smaller rats
(Mattocks, 1986). Expression of toxicity by 7-CYSDHP required high doses (300 and 600
^imol/kg). Similar changes to the lungs, liver and heart were produced by 7-GSDHP at
these same doses by Bowers etal. (1991) in comparable sized rats. By contrast, greater
pneumotoxicity (as measured by increased lung mass) and right heart hypertrophy is
produced by dehydromonocrotaline at 9-15 ^imol/kg or the parent PA at 185 |j.mol/kg. Yan
and Huxtable (1994) have demonstrated that less than 2% of perfused monocrotaline is
released into the perfusate as 7-GSDHP from isolated rat liver preparation.
107
Studies indicate that 7-GSDHP is excreted primarily into the bile (LaFranconi et al..
1985; Yan and Huxtable. 1994; Yan ei ai., 1995; Lame ecaL, 1995). Thus the doses of
sulfur-conjugated pyrroles used in this study do not likely reflect their in vivo disposition.
As well, they would be released slowly from the liver rather than as a bolus dose. The
sulfur-conjugated pyrroles are highly water soluble, and do not show alkylating activity.
They are unreactive towards Thiopropyl-Sepharose resin or 4-(/?-nitrobenzyl)pyridine
unless silver nitrate is present to cleave the thioether bond, reforming the pyrrole-stabilized
carbonium ion (Mattocks and Jukes, 1990a). Renal toxicity in rats has been reported with
the PA monocrotaline (Ratnoff and Mirick, 1949; Roth et al., 1981; Kurozumi et al.,
1983). Despite the localization in the kidney of enzymes involved in further metabolism or
generation of toxic metabolites of GSH S'-conjugates, any role of 7-GSDPIP in renal
toxicity is not supported by our findings or those by other researchers. It also does not
appear that 7-GSDHP produces pneumotoxicity at relevant doses. Retrorsine-perfused rat
livers released into the bile four times more, and seneciphylline-perfused livers two times
more 7-GSDHP than monocrotaline-perfiised liver (Yan etal., 1995). Yet the former two
alkaloids are not normally pneumotoxic. Due to the low toxicity and disposition of 7-
GSDHP and 7-CYSDHP, we conclude that hepatic conjugation with glutathione by
metabolites of PAs likely represents a detoxication pathway.
Chemistry of Sulfur-Bound Pyrroles
Thiopropyl-Sepharose 6B resin, a solid phase reagent containing immobilized thiol
groups (anonymous, 1977), is useful in the chromatography of many compounds. The
resin must be "activated" prior to use with a reducing agent such as P-mercaptoethanol.
This uncovers the thiol groups, as they are substituted with a 2-thiopyridyl moiety which
serves as a protective agent. The thiopropyl group is immobilized to the Sepharose matrix
by an ether linkage, and is stable under normal conditions.
108
It has been established that DHAs react covalently with activated Thiopropyl-
Sepharose resin, forming a stable thioether pyrroie (Mattocks and Juices. 1990b; Mattocks
et ai, 1990). This resin was used to the demonstrate the release of DHAs from isolated
liver perfused with PAs (Mattocks et al., 1990). Glowaz et al. (1992) used the resin to
show production of DHAs by in vitro hepatic microsomal systems incubated with PAs.
Thiopropyl-Sepharose is useful in these studies because of its selectivity for the DHA.
PAs, and metabolites including DHP and 7-GSDHP will not bind to the resin at
physiological pH (Mattocks and Jukes, 1990a, 1990b; Glowaz et al., 1992). As
summarized in Figures 22 and 23, thioether bonds are cleaved in the presence of silver
nitrate or mercuric chloride, solvolizing the pyrrole moiety. Mattocks and Jukes (1990a)
proposed that the metal cation cleaves the sulfide bond, producing a pyrrole stabilized
carbonium ion which reacts with an available nucleophile. This reaction is analogous to the
cleavage of allylic thioethers by silver ion (Saville, 1962). When the pyrrole-bound resin is
treated with silver nitrate in the presence of excess ethanol, a pyrrolic ethyl ether is formed.
This reaction is termed a solvolysis reaction because the solvent, in this case ethanol,
serves as the nucleophile. The position of the ethoxy substitution on the pyrrole represents
the location of the former thioether bond. However, the ethanol must be buffered to prevent
acid-catalyzed alkoxide exchange of hydroxyl substitutions for an ethoxy group (Mattocks
and Jukes, 1990a). Protons released during solvolysis would catalyze diether formation,
preventing true determination of the position of the thioether linkage. A DHA can alkylate
sulfhydryls at either its 7 or 9 position. The subsequent 7- or 9-ethoxy pyrrole is easily
extracted and can be identified by TLC and GC-MS (Table 1 and Figures 23, 39 and 40).
109
OCOR' ROCO
AgNO
Figure 22 . Proposed reaction of DHAs and Thiopropyl-Sepharose resin. The pyrrole is subsequent solvolized in ethanolic silver nitrate or mercuric chloride, producing an ethoxy pyrrole.
1 1 0
A) CH
CH
SR
HgCl Nu
OH HO
7,9-Diethoxy DHP
9-Ethoxy DHP 7-Ethoxy DHP
Figure 23. A) Proposed mechanism of cleavage of sulfur-bound pyrroles by mercuric chloride or silver nitrate. From Mattocks (1986). B) Structures of pyrrolic ethyl ethers formed by solvolysis of sulfur-bound pyrroles by ethanolic silver nitrate.
I l l
This cleavage procedure can be applied to tissue homogenates from animals exposed to
PAs lo establish the position of 5-aikylation in vivo (Mattocks and White, 1970; Mattocks
and Jukes, 1992). Alternatively, by the same procedure, pyrrole bound to resin or tissues
may be released and quantified with Ehrlich Reagent (Mattocks and Jukes, 1990b; Glowaz
et ai, 1992).
Identification of Sulfur-Bound Pyrrole
When resin exposed to macrocyclic DHAs was treated with buffered ethanolic
silver nitrate, 7-ethoxy DHP was produced. 9-Ethoxy or 7,9-diethoxy DHP was not
detected, indicating that the bound pyrrole was in the form of a 7-thioether. In earlier
experiments. Mattocks and Jukes (1990b) presented evidence that when
dehydromonocrotaline was allowed to hydrolyze for 12 sec prior to passing over the resin,
binding occurred at the C9 position, as indicated by the formation of 9-ethoxy DHP from
the resin. Our results do not support the theory that the 9-ester can bind the resin once the
7-ester has hydrolyzed. Further, this is not supported by the results of our in vivo studies,
and studies by others (Mattocks and Jukes, 1990a), as only 7-sulfur adducts were detected
in tissues of animals dosed with macrocyclic PAs. Alkylation at C7 is favored as it forms a
secondary carbonium ion as opposed to the primary carbonium ion formed at C9. The
secondary ion is more stable (the positive charge is delocalized and stabilized by
hyperconjugation with the adjacent alkyl substitutions), and hence forms more easily. The
concept that DHAs can cross-link or bialkylate macromolecules is not supported by these
results, as 7,9-diethoxy DHP has not been detected. Possibly steric factors are involved, as
two large molecular weight nucleophiles would have to be favorably situated to be bound
by the same pyrrole moiety. Only a minimal amount of dehydrolycopsamine bound the
resin, which when treated in the manner above, yielded a trace quantity of 9-ethoxy DHP.
1 1 2
Wild (1994) observed that the 9-monoester dehydroheliotrine was unreactive towards the
resin.
Relative Reactivity ofDHAs Toward Thiopropyl-Sepharose
We attempted to use the recirculating system coupled with Thiopropyl-Sepharose
resin developed by Mattocks and Jukes (1990b) and described in Methods, for the
determination of aqueous half-lives of DHAs. As mentioned earlier, the system proved
cumbersome and results were difficult to reproduce. Thus the system was superseded by
the use of the potentiometric method. However, some interesting observations were found
with the former system. We examined the relative reactivity of a series of DHAs towards
Thiopropyl-Sepharose resin under aqueous conditions at physiological pH. The reactivity
of DHAs toward the resin is a function of the ability of the DHA to form a carbonium ion
when introduced to the aqueous environment. The quantity of pyrrole binding is dependent
on the competition between water (hydrolysis) and die immobilized sulfhydryls. In half-life
studies, the log quantity of resin bound verse time of hydrolysis (length of delay tubing) is
plotted. The slope represents the rate of hydrolysis. This slope is unaffected by inherent
differences in reactivity towards the resin by DHAs. We found that the system was not
sufficiently sensitive to indicate Oxie differences in 7-ester DHA reactivity, however.
Therefore the studies were terminated in favor of the potentiometric method.
The most important finding was that the 9-monoester dehydrolycopsamine was
only minimally reactive toward the resin. However, the results are consistent with binding
of macrocyclic DHAs to the resin, in which 9-adducts were not detected. Additionally,
sulfur-conjugated pyrroles could not be detected in bile from rat livers perfused with the 9-
monoester PAs heliotrine or lycopsamine (Mattocks etal., 1991; Yan and Huxtable,
unpublished observations). When dehydro-7-acetyl-lycopsamine was tested, it was found
1 1 3
to be quite reactive towards the resin in comparison to the latter. This demonstrates the
importance of the 7-esler in terms of reactivity towards large molecular weight
nucleophiles.
Hydrolysis of DHAs
The quantity and physicochemical properties of a DHA released into the circulation
from the liver probably determine the nature of extra-hepatic toxicity. Because DHAs are
short-lived, differences in their half-lives may be a factor in the type and extent of observed
toxicity. The aim of these experiments was to establish the aqueous half-lives of a series of
DHAs. The instability of the DHAs makes it impossible to determine their half-lives in
vivo. In vitro determination is also difficult. Mattocks and Jukes (1990b) used the
recirculating system described above to estimate DHA half-lives. The system is
cumbersome, slow, and inaccurate at longer time points. The results are difficult to
reproduce because only a small proportion of the DHAs binds the resin (Table 9). The
potentiometric method we have developed for estimating aqueous half-lives of DHAs is
simple, rapid and reproducible. Ester hydrolysis can be either acid or base catalyzed.
However, the near log-linearity of the results (Figure 12) indicates that the hydrolysis rates
of the DHAs were not significantly affected by the pH of the aqueous medium over the
range used. Thus the use of a pH stat device was unnecessary.
When a DHA enters an aqueous environment, the ester rapidly hydrolyzes by an
1 mechanism to the corresponding pyrrolic alcohol and necic acid. The rate of hydrolysis
is related to the structure of the esterifying acid. Because the departing carboxylate may
remain momentarily attached at the 9 position, there is a possibility the ester may reform,
slowing the rate of hydrolysis. For example, the larger isopropyl group at C14 of
dehydrotrichodesmine 5.36 sec) compared to the methyl of dehydromonocrotaline
(r,/2 = 3.39 sec) could produce greater hindrance to an incoming nucleophile, or could
1 1 4
cause the departing acid to remain closer to the carbonium ion. This would increase the
probability liial Liie esler would reform, hence slowing hydrolysis. Such a hypothesis
remains consistent with an I mechanism. In support of this observation is the high rate
of hydrolysis of dehydro-7-acetyllycopsamine (^,/2= 0.39 sec), an acyclic diester. Once the
7-acetate dissociates, it is unlikely to reform because it is not attached at the 9 position. The
acyclic 9-monoester lycopsamine 1.02 sec) also hydrolyzes faster than the
macrocyclic DHAs.
The stability of the departing carboxylate may also play a role in DHA reactivity.
The esters of DHAs hydrolyze rapidly because carboxylates are good leaving groups. This
is because in the carboxylate the negative charge of the oxygen anion can be spread over
both oxygen atoms, stabilizing the molecule. In contrast, hydroxyls are poor leaving
groups because they are strongly basic. Their negative charge is localized to one oxygen
and is therefore much less stable. Thus, the weaker the base (or stronger the acid), the
better the leaving group. Synthetic acetyl ester pyrroles are more reactive than
corresponding pivalyl esters (Mattocks, 1986). Acetic acid has a pAT^ of 4.72 compared to
5.01 for pivalic acid. A substituent attached to the carboxyl group which has an electron
withdrawing effect will further stabilize the carboxylate anion (lowering the pATJ, making it
a better leaving group. Dehydroretrorsine (?,/2= 1-06 sec) is significandy more reactive than
dehydroseneciphylline (?,/2= 1-60 sec). Because their structural differences all well
removed from the C7 ester, retronecic acid and seneciphyllic acid may have similar pAT^s.
The reason for their differences in reactivity is unclear. The results of these experiments
clearly establishes the fact the DHAs differ significantly in their rates of hydrolysis. The
relationship of these results and other physicochemical factors to metabolism and toxicity
will be discussed below.
1 1 5
Alkylation of 4-(/7-nitrobenzy[)pyridine by DHAs
Pyrrolic metabolites of PAs, as well as synthetic pyrroles, have ijeen shown to
alkylate the nucleophile 4-(p-nitrobenzyl)pyridine, forming a colored adduct which can be
detected spectrophotometrically (Figure 24) (Mattocks, 1969; Karchesy and Deinzer, 1981;
Karchesy et al., 1987).
NO-. NO-.
ROCO OCOR
CH- CHi "OH
N^ .OH
NO.
^max 565 nm
Figure 24. Proposed reaction between DHAs and 4-(p-nitrobenzyl)pyridine.
This reaction is useful in the assessment of relative alkylating reactivity of pyrroles
(Mattocks, 1969). Alkylation takes place in aqueous acidified acetone in the presence of
excess nucleophile and is therefore pseudo-first order. DHAs (pyrrolic esters) are more
reactive toward 4-(/7-nitrobenzyl)pyridine than the corresponding alcohol pyrrole, DHP.
Presumably, the pyrrole forms an adduct at its most reactive center by covalently binding
the cyclic nitrogen of 4-(p-nitrobenzyl)pyridine, via an S^l mechanism (Mattocks, 1986).
A second, slower color-forming reaction has been shown to occur as well. Karchesy et al.
(1987) have used mass spectral techniques to show that this reaction (represented by A:,) is
a result of oligomerization reactions by the pyrrole ring, and is not related to the initial
1 1 6
reactivity of the DHA. Although DHAs are considered bifunctional alkylating agents,
alkylation of the pyrrole at both C7 and C9 is unlikeiy due to the proximity of the positive
charges formed by the 4-(/7-nitrobenzyl)pyridinium ions (Karchesy etal., 1987).
As all die compounds tested were retronecine based (as opposed to heliotridine
based), as with hydrolysis, differences in reactivity of the DHAs with A-{p-
nitrobenzyI)pyridine are a reflection of the different esterifying moieties on die pyrrole
nucleus. Because the same chemical factors which determine hydrolysis rates probably
influence alkylation by 4-(p-nitrobenzyl)pyridine, the results of the two expetiments are
similar. The same rank order of reactivity is observed for the macrocyclic diester PAs
toward 4-(/7-nitrobenzyI)pyridine as toward water (hydrolysis) (Tables 10 and 11).
Karchesy et al. (1987) observed a similar rank order of reactivity for
dehydromonocrotaline, dehydroretrorsine and dehydroseneciphylline as found in diis
study. Dehydro-7-acetyI-lycopsamine reacted at approximately twice the rate of
dehydrolycopsamine in both systems, demonstrating the greater reactivity of the 7-ester
position of the pyrrole compared to the 9-ester. The increased reactivity of C7 compared to
C9 is also demonstrated by 2,3-dihydro-l//-pyrrolizine-l-ol and dehydrosupinidine
(Figure 25). The hydroxyl of the former reacts at 2.5 times the rate with
4-(/7-nitrobenzyl)pyridine as the hydroxyl of the latter (Karchesy et al., 1987).
Figure 25. Structure of 2,3-dihydro-l//-pyrrolizine-l-ol and dehydrosupinidine.
2,3-dihydro-iff-pyiTolizine-l-ol dehydrosupinidine
1 1 7
This ratio is comparable to dehydro-7-acetyl-lycopsamine and dehydrolycopsamine. While
the reaction between DHAs and 4-(/7-nitrobenzyi)pyridine is not conducted under
physiological conditions, the results provide further support for the view that DHAs differ
significantly in reactivity.
Inhibition of Yeast and Rat Brain Hexokinase by DHAs, in vitro
Hexokinase is a globular protein of approximately 100-kDa, found in microbes,
plants and animals (Wilson. 1995). The enzyme is divided roughly in half by a deep cleft,
with each "subunit" having a mass of approximately 50-kDa. The C-terminus half contains
the binding sites for both ATP and glucose, and is thus considered the catalytic subunit.
Hexokinase catalyzes the first step of the glycolysis pathway, by phosphorylating glucose
to produce glucose-6-phosphate at the expense of ATP. The hexokinases are sulfhydryl-
rich, although the cysteine residues are not well conserved across the family, indicating
they are probably not essential for catalysis (Colowick, 1973; Chou and Wilson, 1974).
Bovine type I hexokinase contains 21 cysteine residues, at least 12 of which exist as free
sulfhydryls. Yeast hexokinases contains at least 8 cysteine residues, but no disulfide
bridges (Wilson, 1995). The yeast enzyme and rat brain enzymes are closely related
(Middleton, 1990). Many studies have shown that reagents reacting with cysteine residues
inactivate hexokinase (Colowick, 1973; Puri etal., 1988; Lenzen etal., 1990; Munday et
al., 1993). The effects of non-specific alkylating agents on yeast hexokinase has also
recently been studied (Puri and Roskoski, 1993; 1994). Because hexokinase is relatively
uncomplicated to assay, and appears sensitive to a variety of alkylating agents, we have
chosen this enzyme as a model to study the interactions between DHAs and an important
cellular macromolecule.
1 1 8
Yeast and rat brain hexokinase phosphotransferase activity was measured by
following the reduction of NADP to NADPH in a coupied assay with giucose-6-phosphate
dehydrogenase (Figure 26).
Flcxokitiflsc Glucose + ATP :r GIucose-6-Phosphate + ADP
Mg-^
and Glucose-6-Phosphate
Glucose-6-Phosphate + NADP + H.,0 Dehydrogenase ^
6-Phosphogiuconate + NADPH + 2H"^
Figure 26. Schematic of the hexokinase assay. The buildup of NADPH is followed spectrophotometrically at 340 nm.
Yeast hexokinase was rapidly inactivated by 7-ester DHAs, as determined by loss of
phosphotransferase activity of the enzyme. Complete inactivation occurred after a one
minute incubation with 20 mM dehydrotrichodesmine. The rapid time course was expected,
as virtually all alkylating activity of the DHAs would be lost after a one minute exposure to
the incubation solution. That dehydrotrichodesmine, dehydroretrorsine and dehydro-7-
acetyl-lycopsamine had similar IC50 values indicates that alkaloid structure did not effect the
ability of the DHA to inhibit hexokinase. While the actual rate of alkylation may be affected
by DHA structure, the process is too rapid to easily evaluate. The 9-monoester
dehydrolycopsamine was a much weaker inhibitor of hexokinase than
dehydrotrichodesmine. This may be related to the comparatively low alkylating activity of
9-monoesters toward sulfur nucleophiles. as reported in Table 9 and by Wild (1994).
DHP, the hydrolysis product of DHAs, was not active towards hexokinase under the
1 1 9
studied conditions. DHP is a weak alkylating agent, except under acidic conditions
(Mallocks and Bird, 1983b). The 7.6 pH of the incubation solution would not favor
alkylation by DHP in a short time period. Consistent with their relative unreactivity, parent
PAs had no effect on hexokinase. In parallel assays, the activity of glucose-6-phosphate
dehydrogenase was not inhibited by dehydrotrichodesmine. The Candida utilis (Torula
yeast) form of the enzyme used in these assays is inactivated by various nucleosides, but
not by compounds which modify cysteine residues (Domshke etal., 1970; Levy, 1979).
Why hexokinase, and not glucose-6-phosphate dehydrogenase was inhibited by DHAs is
unknown, but sulfhydryl modification is a possible explanation. At high concentrations,
DHAs are susceptible to rapid polymerization in an aqueous environment, especially under
acidic conditions (Mattocks, 1986). In the 10 and 20 mM DHA incubations with enzyme,
some pyrrole polymerization was visible as a slight pinkish tint to the solution. Because
glucose-6-phosphate dehydrogenase was not inhibited by DHAs, enzyme was not
precipitating with, or "coated" by a pyrrolic polymer (Mattocks, 1986). This suggests that
hexokinase inhibition was not due to such a process, providing support for an alkylation
reaction with DHAs.
Experiments were conducted to generate saturation curves for hexokinase in the
presence and absence of dehydrotrichodesmine (Figure 17). This type of analysis provides
the and for hexokinase under assay conditions. Because dehydrotrichodesmine
caused a decrease in without significantly affecting K^, the inhibition appears to result
from a non-competitive interaction. The effect on the assay system is the same as removing
enzyme; is lowered because it is concentration dependent, while is not affected.
When the data were plotted in double-reciprocal fashion, the regressions were also
suggestive of non-compedtive inhibition (Figure 18). Because the DHAs are powerful
alkylating agents, they likely bind to the enzyme in a irreversible, non-selective manner.
That mannose was only mildly protective against inhibition by dehydrotrichodesmine
120
suggests that little DHA is reaching the glucose binding site, which is located at the bottom
of a hydrophobic cleft between the two halves (Wilson, 1995). Possibly DHAs are too
large to reach diis site, or do not have time to reach it because of their short half-lives. ATP
provided considerable protection against inactivation by DHAs. This suggests that
alkylation by DHAs at or near the ATP binding site is partially responsible for inhibition.
The ATP binding site has not been conclusively identified, because a crystal structure of
hexokinase-ATP has not been produced. It has been suggested however, that the site is
located on the surface of the C-terminus subunit, near the glucose cleft (Shoham and Stietz,
1980). Such a location could be more accessible to a highly reactive DHA. Alternatively,
ATP may be protecting another important site on the enzyme by inducing a conformational
change, which then does not allow DHAs access to the site.
In fact, these studies do not prove that DHAs are covalently binding to hexokinase.
However, that the parent alkaloid and DHP do not inhibit the enzyme suggests alkylation
by the DHA as a mechanism of inhibition. The many reactive sulfhydryl groups of
hexokinase are potential sites of alkylation (Colowick, 1973; Wilson, 1995). Compounds
that covalently react with these sulfhydryls, such as alloxan (Munday etai, 1993),
inactivate the enzyme. Sulfhydryl modification likely results in an alteration of the
enzyme's conformation or steric obstruction of a catalytic site (Wilson, 1995), as the
cysteine residues are considered non-essential for catalysis (Colowick, 1973). In our
studies, glucose-6-phosphate dehydrogenase was from Candida utilis . This form of the
enzyme contains 8 thiol groups per mole of enzyme, 2 of which are reactive to Ellman's
Reagent without partial denamration (Domshke et al., 1970). The sensitivity of this enzyme
family to sulfhydryl alkylating agents is variable (Levy, 1979). Glucose-6-phosphate
dehydrogenase from C. utilis was shown to be resistant to inhibition by sulfhydryl
alkylating agents such as 5,5'-dithiobis(2-nitrobenzoic acid). The lack of enzyme inhibition
by dehydrotrichodesmine may be related to the low quantity of reactive thiols. Although we
1 2 1
have not demonstrated that DHAs inactivate hexokinase by sulfhydryl binding, it remains
an attractive hypothesis.
Preliminary experiments show that rat brain hexokinase was inhibited by DHAs in a
similar fashion to the yeast enzyme. All 7-ester DHAs significandy inhibited the enzyme.
The 9-monoester, dehydrolycopsamine, DHP, or parent PA did not inhibit the enzyme. An
interesting finding was that inhibition by dehydrotrichodesmine was increased when the
enzyme was pretreated for 24 hr with P-mercaptoethanol (Figure 21). Sulfhydryl reagents
such as P-mercaptoethanol and cysteine increase the activity of rat brain hexokinase (Figure
21) (Bennet et al., 1962), possibly by reducing disulfides. Whether the increase in
inhibition by dehydrotrichodesmine was associated with additional sulfhydryl binding is
unknown, however. Mild protection was afforded to brain hexokinase by glucose against
inhibition by dehydrotrichodesmine, providing support for the formation of an enzyme-
pyrrole adduct. These experiments demonstrate that DHAs, probably through alkylation,
can adversely affect the activity of a critical cellular component. This does not imply that
inhibition of hexokinase occurs in vivo, yet similar types of interactions might be expected
between DHAs and macromolecules.
The Relationship Between the Physicochemical Properties of DHAs and
Their Further Metabolism and Toxicity
Macrocyclic diester PAs
Results from this dissertation, as well as odier studies in our laboratory, have
identified physicochemical properties associated with the quantitative and qualitative
differences in toxicity of PAs (Yan and Huxtable, 1995; Yan etai, 1995; Huxtable etal.,
1996). The toxicity produced by PAs is a consequence of their hepatic metabolism to
DHAs, which alkylate cellular nucleophiles. Lethality and specific organs affected are
122
related to both the rate of formation of DHAs, and the pattern of their further metabolism.
As discussed earlier, rate of pyrrole foniiation is primaiily a function of PA lipophilicily
and an acid structure which favors dehydrogenation rather than hydrolysis and A/-oxidation
(Mattocks, 1986). From studies in this laboratory (Yan et al., 1995), Table 14 shows
differences in rates of pyrrole formation in the isolated rat liver perfused with PAs.
Consistent with other studies, retrorsine was the most extensively metabolized macrocyclic
PA (Mattocks, 1972, 1986). Of the two primarily hepatotoxic PAs, significantly more
bound pyrrole was detected in the liver following perfusion with retrorsine than
seneciphylline. The higher rate of pyrrole formation may account for the lower LDjg and
greater hepatotoxicity (Culvenor et al., 1975) of retrorsine compared to seneciphylline.
Mattocks (1972) has demonstrated a strong correlation between hepatotoxicity and the
quantity of liver-bound pyrrole following PA exposure. Wild (1994) has shown that when
PAs are administered to rats by i.p. injection, significantly more liver-bound pyrrole is
found following retrorsine injection than either monocrotaline or trichodesmine. This is in
keeping with the greater hepatotoxicity and higher rate of metabolism of retrorsine than
monocrotaline (Culvenor et al., 1975). While the hepatotoxicity of trichodesmine has not
been assessed, its metabolism is associated with significantly less liver-bound pyrrole than
monocrotaline (Table 15), despite the faster metabolism of the former (Yan et al., 1995;
Huxtable er a/., 1996).
In addition to rate of pyrrole formation, toxicity of PAs is related to the pattern of
further metabolism of the DHA. This pattern is in turn related to DHA stability, particularly
at the 7-ester position. As shown in studies with the isolated perfused liver (Table 14), the
more resistant a DEIA is to nucleophilic attack (the longer the half-life), the greater the
proportion that is released from the liver, increasing the availability of DHA for extrahepatic
reactions (Yan et al., 1995). The more reactive DHAs of retrorsine and seneciphylline favor
hydrolysis or nucleophilic attack closer to their sites of formation. Thus, a greater
123
proportion of the total pyrrole produced is released from the isolated liver as non-toxic 7-
GSDHP, and a smaller proportion of DHA is released into the perfusate compared to
monocrotaline and trichodesmine. Quantitatively, the amounts of DHA released into the
perfusate by retrorsine, seneciphylline and monocrotaline perfused livers were all similar
(but represent a variable proportion of the total pyrrole produced). Monocrotaline is the
only one of these PAs associated with extrahepatic toxicity, suggesting that of the
corresponding DHAs, only dehydromonocrotaline is sufficiently stable to reach the lungs
in quantities necessary to produce toxicity. Wild (1994) demonstrated that monocrotaline-
treated rats had more lung pyrrole than retrorsine-treated animals. Following exposure to
the PAs, four times as much DHA is released from the liver perfused with trichodesmine
than monocrotaline. This is related to the higher rate of metabolism of trichodesmine and
the longer half-life of its DHA (Table 14). Consequently, in trichodesmine-treated animals,
more DHA will be available to produce toxicity away from the liver. Such a conclusion is
supported by the observed pattern of tissue alkylation in vivo 18-24 hr after i.p. injection of
the PAs. Following a dose of PA approximately 60% of the LDjq, trichodesmine-treated
rats had significantly higher brain pyrrole than monocrotaline-treated animals (Table 15)
(Huxtable et al., 1996). When tested on an equimolar basis, brain pyrrole was ten-fold
higher in the trichodesmine-treated animals (Wild, 1994). The differences in brain pyrrole
levels are probably a reflection of greater delivery and lipophilic character of
dehydrotrichodesmine. Due to the isopropyl substimtion at CI4, trichodesmine has a
greater lipid:aqueous partition coefficient than monocrotaline (Huxtable etaL, 1996).
Pyrrole was not detected in brains of retrorsine-treated rats (Wild, 1994), in keeping with
the very short half-life of its DHA. Consistent with its pneumotoxicity, monocrotaline-
treated animals had higher lung pyrrole levels than those administered trichodesmine (Table
15). The slower reactivity of dehydrotrichodesmine and dehydromonocrotaline with water
or GSH ensures a greater proportion of DHA will be released from the liver into the
124
circulation (Table 14). This stability also allows DHA to reach sites downstream from the
liver to produce extraliepatic toxicity. Tlie higher rate of tnelaboHsm of Irichodesmine
compared to monocrotaline is attributable to the isopropyl grouping of the former. The
increased lipophilicity may make it a better substrate for P450, or the bulkier acid may be
more resistant to hydrolysis by hepatic esterases.
Open ester DHAs
The low toxicity of PA monoesters such as lycopsamine (LD50 > 3 mmol/kg
(Fowler et al., 1967)) can also be related to its structural properties. Monoesters are not
metabolized to pyrroles by hepatic P450s (Glowaz et al. 1992, Yan et al., unpublished
observations) as extensively as diester PAs, which may be related to their hydrophilicity.
thus promoting excretion. Retronecine based monoesters, including lycopsamine, are also
susceptible to hydrolysis by esterases. Once metabolized to a pyrrole, open ester DHAs
appear to have very short half-lives (Cooper and Huxtable, 1996), consistent with the
observation that they only produce hepatotoxicity (Mattocks, 1986). Additionally, our
studies indicate that 9-monoester DHAs are poor alkylating agents of sulfur
macromolecules under physiological conditions, although the reason for this selectivity is
unknown.
The hepatotoxicity and lethality of open diester PAs are highly variable (Mattocks,
1986). Because they all contain a 7-ester, their corresponding DHAs are strong alkylating
agents. The relative toxicity of the diesters is primarily dependent on the structure of the
esterifying acid at CI. Larger, branched acids such as tiglic or angelic are more resistant to
hydrolysis by esterases than smaller acids such as acetic (Mattocks, 1982). Culvenor et al.
(1975) found 7-acetyl-heliotrine to be equally hepatotoxic as heliotrine, which he attributed
to loss of the 7-acetyl group to hydrolysis. The more resistance to hydrolysis, the greater
125
Table 14
Relationship Between Pattern of Metabolism of PAs in the Isolated Perfused Rat Liver and DHA Half-Life"
PA % Metabolized 7-GSDHP^ DHA''
(with LDso) to Pyrrole* (% of total pyrrole) (% of total pyrrole) r,^of DHA
Retrorsine (97 nmol/kg)
31.3 54.4 ± 2.7 10.2 ±0.43 1.06 ±0.20
Seneciphylline (230 |imol/kg)
17.0 45.3 ±3.7 18.5 ±2.1 1.60 ±0.28
Monocrotaline (335 |imol/kg)
9.7 36.4 ±3.2 23.4 ± 4.0 3.39 ± 0.99
Trichodesmine (57 |imol/kg)
15.8 9.6 ± 0.8 56.2 ± 7.7 5.36 ± 0.92
Terfusion data taken from Yan etal. (1995). Isolated rat livers were perfused with PAs at 0.5 mM for I hr. Pyrrolic metabolites that were released into the perftisate and bile, or bound to the liver, were quantified with Ehrlich Reagent.
^Percentage of total perfused PA metabolized to pyrrole over the one hour period. '7-GSDHP released into the bile, expressed as a percentage of total pyrrolic metabolites detected.
"IDHA released into the perfusate, expressed as a percentage of the total pyrrolic metabolites detected.
126
Table 15
Tissue-Bound Pyrrolic Metabolites 24 hr After i.p. Injection of PAs in Rats
Parent Dose Tissue bound pyrrole (nmol/g tissue)
Alkaloid" (jimol/kg) Liver Lung Brain
Monocrotaline 65 17.1 ±0.9 10.4 ± 0.9 1.74 ±0.6
Trichodesmine 15 6.7 ± 1.4 7.7 ± 0.4 3.74 ± 0.3
"Doses represent approximately 60% of the acute LDj^ (Mattocks, 1986; Wild, 1994). Values for each tissue are significantly different at p < 0.05 (From Huxtable nt al., 1996).
127
the quantity of PA available for metabolism to pyrrole. PAs such as lasiocarpine and
symphytine, have larger, branched esterifying acids at C7 and are quite lipophilic (Mattocks
and Bird, 1983a; Cooper et al., 1996). Because of extensive metabolism to a DHA with a
reactive 7-ester, these PAs are as lethal as some macrocyclics, with LD50S of 340 and 185
|j.mol/kg for symphytine and lasiocarpine, respectively (Bull et ciL, 1958; Hirono et ai.
1979).
Conclusion
This research has investigated how certain physicochemical properties of PA
metabolites relate to the toxicity produced by the parent alkaloids. The relationship between
structure and toxicity is complex and only partially understood. The results of this
dissertation suggest that differences in stability of macrocyclic DHAs are associated with
differences in the pattern of metabolite release observed in the isolated, perfused liver. In
turn, the pattern of metabolite release is related to the quantitative (lethality) and qualitative
(organs affected) toxicity.
Using a potentiometric method, we have shown that the Senecio type DHAs,
dehydroseneciphylline and dehydroretrorsine, are more reactive in an aqueous environment
than the Crotalaria alkaloids, dehydromonocrotaline and dehydrotrichodesmine. Reactivity
of these DHAs involves nucleophilic substitution at the 7 position of the ring system by an
S^I mechanism. As demonstrated in the isolated, perfused liver, the more reactive the
DHA, the greater the proportion reacting with GSH, the greater the proportion alkylating
liver macromolecules, and the lower the proportion released as DHA into the circulation.
Lower quantities of DHAs (with short half-lives) escaping the liver decreases the
probability that other organs will be affected. On the other hand, the more stable DHAs of
monocrotaline and trichodesmine are associated with lower proportions of 7-GSDHP
release and liver alkylation, and higher proportions of DHA released into the circulation.
128
The higher proportion of the latter increases the risk of extrahepatic toxicity, as greater
quantities of DHA will be delivered to extrahepatic sites. The high iiposoiubiiity, longer
half-life, and greater rate of formation of dehydrotrichodesmine compared to
dehydromonocrotaline favor increased access of DHA to the brain for
dehydrotrichodesmine.
Other contributions of this research include the development of an efficient method
of separating and purifying PAs using high-speed counter-current chromatography. This
technique is useful in supplying the large quantities of PAs necessary for chemical and
toxicological studies. We have also shown that glutathione and cysteine-conjugated
pyrroles have low toxicity, and probably represent a major detoxication pathway for PAs.
This provides further support for DHAs as the primary toxic metabolite of PAs. Overall,
the results of this research gives more insight into some of the toxic properties of certain
types of PAs. In general, this work provides further information on the relationship
between hepatic metabolism of xenobiotics and extrahepatic toxicity.
129
APPENDIX A
Electron Impact-Mass Spectra of PAs
43
53
120
93
136
157 164
\ / 194
236 I
256 281 325 (M-")
40 80 120 160 200
m/z
240 280 320 360
Figure 27. Electron impact-mass spectrum of monocrotaline.
130
100
90
80 -3
70
60
50
40
30
20
10
0 J
222 206 /
14.
264
J. 338
\
353 (M^-)
—I—1 r-—f—I—r-—I 1—r— 1
40 80 120 160 200 240 280 320 360
m/z
Figure 28. Electron impact-mass spectrum of trichodesmine.
1 3 1
100
90 H
80 -
70 A g ^ 6 0 - 3 •w 'cfl C ''J sr, tS 50 -
•s 40 A
13 0^ 30
20
10
0 J
206 250
-Lull
292
\ 337 (M"'
I
T X
160 200 m/z
240 280 320 360
Figure 29. Electron impact-mass spectrum of incanine.
132
220
246
—r"—'—I— 240 280
320 351 (M-")
—I 320 160 200
m/z
360
Figure 30. Electron impact mass-spectrum of retrorsine.
133
100 q 136
90
80
^ 70
§ 60 c (U
•S 50 I
"S Oj 40 ^
30
20
10
0
\20
1151
246
jiL
218
L
288 289 333 (M^)
\ , / /
I ' ' ' '"I "" ' I I I
40 80 120 160 200 240 280 320 360
m/z
Figure 31. Electron impact mass-spectrum of seneciphylline.
100 120
93
80 -
.-s 60 c u
ta 40 -
20
0 J
53
I—^
40
80
80
106
136
151 220 289 291 335 (M^)
1' \ I \ i i
120 160 200 240 280 320 360
m/z
Figure 32. Electron impact-mass spectrum of senecionine.
135
100 n
80
^ 60
c D
^ 40 ga (U
20 •
69
55 110
122
94
150
168
184
250 304 336
238
264
281
40 80 120
r" ̂ 1>—^-"1——
160 200 240 280 320
423 (m-^) 364 \ /
Jlj—, I I .
360 400 440
m/z
Figure 33. Electron impact mass-spectrum of florosenine.
138
90 -i
80 H
43
93
80
67
lU
120
LjkJlL
156
40 80 1 1—
120 160 200
254 299(m
240
—I ̂
280
m/z
Figure 34. Electron impact-mass spectrum of lycopsamine/intermedine.
137
100 1 138
43
93
67 120
i ii In. j.ii jij 1 iwtL 156 181
Jj •I 198 J H-
160 200
m/z
255 298 —l-
341 (m"")
T-
40 80 120 240 280 320 360
Figure 35. Electron impact-mass spectrum of 3-acetyl-lycopsamine/-intermedine-
100 T
90
80 -
g 70 -
'33 60 -c <u •w c
•Z 5 0 ^
i 40 (U " 0^
30 .
20 r
10 r
0 -
180
43
i| li jflllj
120
93
80 136
L
198
+ 281 296 34 j
\ I \
40 80 120 160 200 240 280 I
320 360
m/z
Figure 36. Electron impact-mass spectmm of 7-acetyl-lycopsamine/-intermedine.
139
100 T 180
90
80 -
70
60 H
C/D S 50
u 40
-4-* — <u 0^ 30
20
10
0
93
'T-80
120
136
198 381 (m-^)
291 341 J L-
120 160 200 240 280 320 360 400
m/z
Figure 37. Electron impact-mass spectrum of 3,7-diacetyl-lycopsamine/-intermedine.
100 T 220
90 -
80
70
60 -
C/3
I 50
•>: 40 -
30 -
20 -
10 H
0 J
43 55
93
I
83
67
120
40 80
136 I
141
/
120 160
m/z
238
200 240
281
280
Figure 38. Electron impact-mass spectrum of symphytine and isomers (symlandine, myoscorpine and echiumine). Because a molecular ion was not observed, mass was confirmed by flow-injection APCI.
100 T 136
90 T
80 -
70 H
35 60 ••
50 -HJ >
13 40 -
30
20 -
10 -
0
29 41
80
51 68
III., Jill
40 80
106
1 1 8
120
152 I
181 (M^-)
160 200
m/z
Figure 39. Electron impact-mass spectrum of 7-ethoxy DHP.
142
100
90
80 -
70 -
'ES 60 -i
^ 50 -
S 40 -
30 •
20 -
10
0
29
53
39
94
79
65
118
106
136
163
152
181 (M-")
20 40 60 80 100 120 140 160 180 200
m/z
Figure 40. Electron impact-mass spectrum of 9-ethoxy DHP.
100 n
90
80 ^
70
60
50
40
30
20
10
0 J
120
29
53 93 106
79
1 ii. • .111 (1 i
134
40 80 120
m/z
164
160
209 (M-^)
JU L
200
Figure 41. Electron impact-mass spectrum of 7,9-diethoxy DHP.
100
90 -
80
70 •
£ ^ 60 • c/a C o a
<u
•s 40 -— 13
30 -
20 -
10
0 J r
154 matrix
136 matrix
107
120
120
x l O — ^
219
207
171 189
n—
160
•T——r-'
200
239
257 (M+H)-"
307 273 matrix
289 matrix
326
240 280
-I—' 1—
320
m/z
Figure 42. Positive ion FAB mass spectrum of 7-CYSDHP. Matrix was 3-NBA.
145
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