Effect of ethoxyquin on the toxicity of the pyrrolizidine alkaloid monocrotaline and on hepatic drug metabolism in mice

Chem.-Biol. Interactions, 37 (1981)95--107

95

© Elsevier/North-Holland Scientific Publishers Ltd.

E F F E C T OF E T H O X Y Q U I N ON T H E TOXICITY O F T H E PYRROLI ZI DI NE A L K A L O I D M O N O C R O T A L I N E AND ON HEPATIC D R U G METABOLISM IN MICE

CRISTOBAL L. MIRANDA a, HILLARY M. CARPENTER a, PETER R. CHEEKEb and DONALD R. BUHLERa,c aDepartment of Agricultural Chemistry, bDepartment o f Animal Science and CEnvironmental Health Sciences Center, Oregon State University, Corvallis, OR 97331 (U.S.A.)

(Received June 20th, 1980) (Revision received March 16th, 1981 ) (Accepted March 20th, 1981)

SUMMARY Diets containing t he a nt i oxi da nt e t h o x y q u i n (6-ethoxy-2,2,4-trimethyl1,2
Abbreviations: ALT, alanine aminotransferase; AST, aspartate transferase; CDNB, 1-chloro-2,4-dinitrobenzene; PA, pyrrolizidine alkaloid.

96 INTRODUCTION Monocrotaline, a pyrrolizidine alkaloid (PA) f o u n d in Crotalaria spectabilis, produces a wide variety of lesions in experimental animals. It causes acute hemorrhagic centrilobular necrosis and upon chronic admini. stration, it produces megalocytosis of liver cells [1]. In monkeys, intra. gastric administration o f the alkaloid produces veno-occlusive hepatic disease [2]. Pulmonary arterial hypertension leading to right ventricula~ h y p e r t r o p h y and cor pulmonale has been observed in rats [3,4]. The mecha. nism by which monocrotaline produces these toxic manifestations is n o t well defined. Monocrotaline is converted to pyrrole metabolites b y liver microsomal enzymes [5] and a direct relationship between the amounts ot pyrrolic metabolites formed in the liver of rats and acute hepatotoxicity o] monocrotaline has been shown [6]. The PA pyrroles are strong alkylatin~ agents and cellular damage apparently results from the interaction ot pyrroles with nucleophilic components of cells [6]. Certain thiol c o m p o u n d s such as mercaptoethylamine and cysteine have been shown to protect rats against monocrotaline intoxication [3]. It it postulated that these c o m p o u n d s m a y bind the toxic metabolites of monocrotaline, reducing the alkylation of critical cellular sites. Although in vivc reaction products of monocrotaline with thiol c o m p o u n d s have n o t yet been isolated, in vitro interaction of a pyrrole metabolite of monocrotaline~ dehydroretronecine, with cysteine and glutathione has been found [7]. Reaction b e t w e e n retrorsine pyrrole and glutathione in vitro have also been reported [ 8]. Aside from its direct interaction with PA pyrroles, cysteine may be used as a precursor for the synthesis of glutathione. There is a doubling in hepatic glutathione levels in rats after the i.p. administration of cysteine [8] but nc increase in liver sulfhydryls following consumption of dietary cysteine [9]. The increased level of glutathione after i.p. administration of cysteine is suggested to be responsible for the decreased sensitivity of rats to the toxicity of retrorsine [8]. While non-enzymatic conjugation of pyrrolic metabolites of PAs with glutathione has been demonstrated [7], it is not known whether the reaction can be enhanced enzymatically. For many foreign compounds, formation of glutathione conjugates occurs both nonenzymatically and enzymatically, the latter being mediated by glutathione S-transferases [ 10]. The present work was carried out to determine the effects of a potent inducer of glutathione S-transferase activity, ethoxyquin or 6-ethoxy2,2,4-trimethyl-l,2
97 liver glutathione and microsomal drug-metabolizing enzyme activities in female mice. MATERIALS AND METHODS

Chemicals Ethoxyquin {marketed as Santoquin) was purchased from Monsanto Co., St. Louis, MO. Monocrotaline was a product of Trans World Chemicals, Washington, DC and was shown to be pure b y high pressure liquid chromatography. [8-14C]Styrene oxide (spec. act., 16.4 mCi/mmol) was purchased from Amersham-Searle Co., Arlington Heights, IL. Unlabelled styrene oxide was obtained from Aldrich Chemical Co., Milwaukee, WI. 1-Chloro-2,4dinitrobenzene {CDNB) was purchased from Eastman Kodak Co., Rochester, NY. NADP, glucose 6-phosphate, glucose-6-phosphate dehydrogenase and reduced glutathione were obtained from Sigma Chemical Co., St. Louis, MO. Sodium selenite (Na2SeO3), 1-ascorbic acid and DL-a-tocopherol acetate were purchased from ICN Pharmaceuticals, Inc., Cleveland, OH. Animals and diets Female CD-1 mice (Charles River Breeding Labs, Wilmington, MA), 6 weeks of age, were used in this study. Control animals were fed ground Purina Lab Chow (No. 5001) while the test groups were fed Lab Chow containing one o f the following compounds: 0.25% ethoxyquin, 0.1% ascorbic acid, 400 t~g/g vitamin E (as D L ~ - t o c o p h e r o l acetate), 1 ug/g selenium {as sodium selenite) or 1% cysteine. The Lab Chow contains trace amounts of vitamin C and has a vitamin E activity of approx. 29.8 IU/lb. It contains no ethoxyquin (Dr. D.C. Shelton of Ralston Purina, pers. comm.). The doses of the feed additives, although selected arbitrarily, were based on previous studies showing the stimulatory effects of ethoxyquin [11] and ascorbic acid [12] on glutathione S-transferase activity and the protective effects of vitamin E on methylmercury toxicity [13] and selenium on aflatoxin toxicity [14]. The level of 0.25% instead of 0.5% e t h o x y q u i n [11] was used in our study because preliminary results showed that 0.5% e t h o x y q u i n depressed food intake and weight gain of the female mice. After 10 days of feeding, the mice were killed and livers removed for biochemical and enzymatic assays. In the lethality experiments, mice were injected i.p. with monocrotaline after 10 days o f feeding the respective diets, using one or several dose levels and continued on the same diets for an additional 2--4 weeks. When plasma enzyme activities were determined, the animals were killed 2 and 24 h after monocrotaline. Preparation o f cytosol and microsomes Livers were homogenized in ice~cold 1.15% KC1/0.02 M Hepes (pH 7.4). The homogenate was centrifuged at 10 000 X g for 20 min and the resulting supernatant fraction then centrifuged at 1 0 5 0 0 0 X g for 1 h. The 105 000 X g supernatant (cytosol) was employed in the assay of glutathione

98 S - t r a n s f e r a s e a c t i v i t y while t h e m i c r o s o m a l pellet was s u s p e n d e d in 1.15% KC1/0.02 M H e p e s for use in t h e d e t e r m i n a t i o n o f m i c r o s o m a l m o n o . o x y g e n a s e activities.

Analytical methods Liver g l u t a t h i o n e levels w e r e d e t e r m i n e d using whole h o m o g e n a t e s a c c o r d i n g t o t h e p r o c e d u r e o f E l l m a n [ 1 5 ] . Liver m i c r o s o m a l c y t o c h r o m e P - 4 5 0 was e s t i m a t e d using t h e m e t h o d of O m u r a and S a t o [ 1 6 ] . A m i n o . p y r i n e N - d e m e t h y l a s e a c t i v i t y w a s d e t e r m i n e d b y m e a s u r i n g the a m o u n t ot f o r m a l d e h y d e f o r m e d [ 1 7 ] . T h e rate o f c o n v e r s i o n o f m o n o c r o t a l i n e tc p y r r o l i c m e t a b o l i t e s b y liver m i c r o s o m a l p r e p a r a t i o n s was e s t i m a t e d a~ d e s c r i b e d b y W h i t e [8]. G l u t a t h i o n e S - t r a n s f e r a s e activity was m e a s u r e d using t w o d i f f e r e n t s u b s t r a t e s , C D N B [ 1 8 ] a n d s t y r e n e o x i d e [ 1 9 ] . Protein was d e t e r m i n e d b y t h e m e t h o d o f L o w r y et al. [ 2 0 ] . T h e a m o u n t o f livel p y r r o l e s f o r m e d in vivo a f t e r m o n o c r o t a l i n e a d m i n i s t r a t i o n was e s t i m a t e d b y using t h e m e t h o d o f M a t t o c k s a n d W h i t e [ 2 1 ] . T h e activities o f alanine a m i n o t r a n s f e r a s e ( A L T ) a n d a s p a r t a t e a m i n o t r a n s f e r a s e ( A S T ) in p l a s m a w e r e d e t e r m i n e d a c c o r d i n g to t h e m e t h o d of R e i t m a n and F r a n k e l [ 2 2 ] , using r e a g e n t s p u r c h a s e d f r o m S i g m a C h e m i c a l Co., St. Louis, MO. T h e d a t a in T a b l e s I I I - - V I w e r e s u b j e c t e d t o an analysis of variance and t r e a t m e n t m e a n s w e r e c o m p a r e d t o c o n t r o l values using t h e D u n n e t t ' s procedure [ 23]. RESULTS

Acute toxicity o f monocrotaline T h e relative e f f e c t s o f various f e e d additives on t h e t o x i c i t y of m o n o c r o t a l i n e is s h o w n in T a b l e I. M o n o c r o t a l i n e was given at a d o s e o f 255 m g / TABLE I EFFECT OF FEED ADDITIVES ON MONOCROTALINE TOXICITY IN MICE Female mice were fed for 38 days. At 10 days, all mice were injected i.p. with monocro taline, except controls. Mortality was recorded until 4 weeks after injection. Feed additive a

None None Vitamin C (0.1%) Vitamin E (400 ug/g) Selenium (1/~g/g) Ethoxyquin (0.25%)

Mo nocrot aline (mg/kg) 0 255 255 255 255 255

Mortality (cumulative) 24 h

1 week

2 week

4 week

0/8 118 0/8 0/8 1]8 0/8

0/8 318 5/8 3/8 4/8 0/8

0/8 4/8 5/8 3/8 4/8 0/8

0/8 4/8 5/8 3/8 4/8 0/8

a Animals were fed Lab Chow containing the indicated additives throughout the entir~ test period.

99 kg i.p. t o s h o w e i t h e r e n h a n c e m e n t o r r e d u c t i o n o f m o r t a l i t y b y t h e v a r i o u s a g e n t s w i t h i n t h e s p e c i f i e d t i m e p e r i o d s . M o r t a l i t y at 2 o r 4 w e e k s f o l l o w i n g m o n o c r o t a l i n e a d m i n i s t r a t i o n i n t h e g r o u p s f e d d i e t s c o n t a i n i n g v i t a m i n C, v i t a m i n E or s e l e n i u m was essentially similar to t h a t of c o n t r o l s . However, t h e a n i m a l s f e d t h e e t h o x y q u i n d i e t w e r e all alive 4 w e e k s a f t e r m o n o crotaline injection. T o d e t e r m i n e LDs0-values [24], mice fed Lab C h o w with or w i t h o u t a d d i t i o n o f 0 . 2 5 % e t h o x y q u i n , 0 . 2 5 % e t h o x y q u i n p l u s 1% c y s t e i n e o r 1% c y s t e i n e a l o n e were i n j e c t e d w i t h m o n o c r o t a l i n e in increasing doses using a g e o m e t r i c f a c t o r o f 1.1. A t a d o s e o f 2 8 0 m g / k g , m o n o c r o t a l i n e k i l l e d all t h e c o n t r o l m i c e at 2 w e e k s ( T a b l e II). N o d e a t h s o c c u r r e d i n t h e e t h o x y q u i n - f e d m i c e a t t h i s d o s a g e level e v e n a f t e r 2 w e e k s . C y s t e i n e p r o v i d e d a TABLE II EFFECT OF ETHOXYQUIN AND CYSTEINE ON MONOCROTALINE TOXICITY IN MICE Female mice were fed for 24 days. At 10 days, monocrotaline was injected i.p. to test groups. Controls received saline alone. Mortality was recorded up to 2 weeks after injection. Feed additive

Monocrotaline (mg/kg)

Mortality (cumulative) 24h

72h

1week

2week

2week

----

None 211 232 255 280

0/4 0/4 1/4 1/4 0/4

0/4 0/4 1/4 1/4 0]4

0/4 0/4 1]4 2/4 2/4

0/4 0/4 1/4 3/4 4/4

243 (228--260)

+ + + + +

------

None 308 339 373 410

0/4 0/4 0/4 0/4 1/4

0/4 0/4 1/4 1/4 3/4

0/4 0/4 1/4 2/4 4/4

0/4 0/4 1/4 2/4 4/4

364 (338--391) b

--

4+

0/4 0/4 0/4 2/4

0/4 1/4 2/4 4/4

0/4 1/4 2/4 4/4

0/4 1/4 2/4 4/4

331 (308--356) b

0/4 0/4 2/4 0/4 0/4

0/4 0/4 2/4 2/4 4/4

0/4 0/4 2/4 3/4 4/4

0/4 0/4 2/4 3/4 4/4

382 (355--411) b

Ethoxyquin (0.25%) -

-

-

---

-

Cysteine (1%) -

-

-

-

--

+

--

+

280 308 339 373

+ + + + +

+ + + + +

None 339 373 410 451

-

-

LDso a

a LDs0 plus 95% confidence intervals shown in parenthesis [20 ]. b p < 0.05 for difference in LDs0-values between control and ethoxyquin, cysteine, or ethoxyquin plus cysteine treated animals.

100 TABLE III EFFECT OF ETHOXYQUIN AND CYSTEINE ON PLASMA ALANINE AMINOTRANSFERASE AND ASPARTATE AMINOTRANSFERASE ACTIVITIES IN FEMALE MICE Female mice were fed for 12 days. At 10 days, monocrotaline or saline was injected i.p. The animals were killed 2 days post-injection. Values represent means ± S.E. of 5 animals. Feed additive

Monocrotaline (mg/kg)

None (controls) Ethoxyquin (0.25%) Cysteine (1%)

0 0 0

None Ethoxyquin (0.25%) Cysteine (1%)

280 280 280

ALT (Sigma units/ml)

AST (Sigma units/ml)

27.8 ± 1.5 27.4 ± 1.2 24.6 ± 0.5

66.2 ± 3.9 68.4 ± 1.6 61.4 ± 2.9

280.0 ± 51.0 a 39.2 ± 15.3 244.0 ± 49.5 a

377.0 ± 70.0 a 70.0 ± 14.9 253.0 ± 63.5 a

a p < 0.05, from controls. slight b u t n o t statistically significant p r o t e c t i o n against m o n o c r o t a l i n e lethality w h e n it was a d d e d t o t h e e t h o x y q u i n diet. H o w e v e r , d i e t a r y c y s t e i n e alone d e c r e a s e d t h e m o n o c r o t a l i n e - i n d u c e d lethality.

Effect o f dietary ethoxyq uin and cysteine on acute hepatotoxicity of monocrotaline T h e effects o f d i e t a r y e t h o x y q u i n and c y s t e i n e on t h e h e p a t o t o x i c i t y of m o n o c r o t a l i n e as m e a s u r e d b y t h e activities o f A L T and A S T in plasma are given in T a b l e III. In t h e absence o f m o n o c r o t a l i n e t r e a t m e n t , t h e t w o feed additives had no significant e f f e c t o n the plasma e n z y m e activities. A f t e r ( 4 8 h) m o n o c r o t a l i n e a d m i n i s t r a t i o n , b o t h p l a s m a A L T and A S T were m a r k e d l y increased in mice fed n o feed additives. H o w e v e r , in mice fed e t h o x y q u i n diets, plasma A L T and A S T r e m a i n e d at c o n t r o l values f o l l o w i n g m o n o c r o t a l i n e a d m i n i s t r a t i o n . D i e t a r y c y s t e i n e h a d n o significant e f f e c t on t h e m o n o c r o t a l i n e - i n d u c e d increases in plasma A L T and AST.

Effect of feed additives on liver weight, glutathione levels and glutathione S-transf erase activities V i t a m i n C, selenium or c y s t e i n e had n o significant e f f e c t o n g l u t a t h i o n e levels (~g/g liver) w h e r e a s v i t a m i n E and e t h o x y q u i n significantly increased t h e l a t t e r b y a b o u t 11% a n d 40%, r e s p e c t i v e l y (Table IV). When expressed as mg/liver, g l u t a t h i o n e c o n t e n t o f e t h o x y q u i n - f e d rats were 71% higher t h a n t h e c o n t r o l s due to t h e increased weight of livers o f rats fed t h e e t h o x y q u i n diet. V i t a m i n C p r o d u c e d a small increase in h e p a t i c g l u t a t h i o n e S-transferase activity with s t y r e n e o x i d e a substrate. W h e n C D N B was used as a substrate, t h e activity of h e p a t i c g l u t a t h i o n e S-transferase was u n c h a n g e d in mice

6.19-+ 6.20 ± 5.80 ± 5.60± 8.72-+ 5.77-+

None Vitamin C (0.1%) Vitamin E (400 ug/g) S e l e n i u m ( l ~g/g) Ethoxyquin(0.25%) Cysteine(1%)

a CDNB and s t y r e n e o x i d e (SO) used as s u b s t r a t e s . b p < 0.05, f r o m controls. c p < 0.01, f r o m controls.

0.23 0.02 0.20 0.06 0.23 b 0.21

Liver wt. (g/100 b o d y w t . )

F e e d additive

2685-+ 21 2690 ± 121 2972 ± 31 c 2797-+ 53 3758-+ 98 c 2 6 1 8 ± 25

/~g/g

Liver g l u t a t h i o n e

4162± 4170 ± 4012 ± 3916± 7102± 3665±

ug/liver

215 155 219 137 288 c 171

Female mice were fed for 10 d a y s b e f o r e sacrifice. Values r e p r e s e n t m e a n s -+ S.E. of 4 animals.

a

1676± 1645 ± 1830 ± 1784 ± 8504± 2191±

CDNB

30 58 58 142 342 c 170 b

162 181 171 174 453 195

SO a

G l u t a t h i o n e S-transferase ( nmol conjugate/min/rag p r o t e i n activity)

-+ 6 ± 2b -+ 5 ±4 + 8c ± 6b

E F F E C T OF F E E D A D D I T I V E S ON L I V E R WEIGHT, G L U T A T H I O N E L E V E L S AND C Y T O S O L I C G L U T A T H I O N E S - T R A N S F E R A S E A C T I V I T I E S IN MICE

T A B L E IV

t==~

o

0.572 0.747 0.673 0,738 1.076 0.657

None Vitamin C (0.1%) Vitamin E (400 ug/g) Selenium (1 ug/g) E t h o x y q u i n (0.25%) Cysteine (1%)

a M o n o c r o t a l i n e was used as substrate. b p < 0,05, f r o m controls.

-+ 0 . 0 6 -+ 0.03 ± 0,05 ± 0,02 ± 0.10 b ± 0.01

C y t o c h r o m e P-450 (nmol/mg protein)

F e e d additive

12.38 13.36 14.28 12,70 12,83 12,27

-* 0.78 ± 0.14 ± 0.27 ± 0.18 ± 0,58 ± 0.43

Per mg p r o t e i n

21,6 17.9 21.3 17.2 11.9 18.7

± ± ± ± ± ±

0.08 0.27 0.57 0.12 0.13 b 0.78

Per n m o l c y t o c h r o m e P-450

A m i n o p y r i n e d e m e t h y l a s e activity (nmol HCHO/min)

Female mice were fed for 10 days b e f o r e sacrifice. Values r e p r e s e n t m e a n s ± S.E. o f 4 animals.

E F F E C T OF F E E D A D D I T I V E S ON L I V E R M I C R O S O M A L M O N O O X Y G E N A S E S IN MICE

TABLE V

1.81 2,07 2.17 1.75 2.27 2.05

± 0.04 ± 0,01 b ± 0.135 -+ 0.08 ± 0,11 b ± 0.04 b

Per mg p r o t e i n

3.20 2.77 3.22 2.38 2.12 3.11

-+ 0.19 ± 0.08 ± 0.07 -+ 0.155 ± 0.025 ± 0.08

Per n m o l c y t o c h r o m e P-450

Rate o f pyrrole f o r m a t i o n ( n m o l / 1 5 rain)

b-a

o tx~

103 pretreated with vitamin C, vitamin E or selenium (Table IV). In contrast, e t h o x y q u i n and cysteine significantly increased t he activity of this e n z y m e using either substrate.

Effect o f feed additives on hepatic microsomal cytochrome P-450 and related monooxygenases Since PA pyrroles are f o r m e d upon the microsomal metabolism of monocrotaline [5], t he effects of the various agents on hepatic m onooxygenases were examined. Vitamin C, vitamin E, selenium, or cysteine did not produce a significant change in the c y t o c h r o m e P-450 c o n t e n t or in am i nopyri ne d e m e t h y l a s e activity per milligram protein (Table V). However, e t h o x y q u i n increased hepatic c y t o c h r o m e P-450 c o n t e n t by 88% b u t did not alter a m i n o p y r i n e dem e t hyl a s e activity per milligram protein. On the o t h e r hand, the in vitro microsomal conversion o f m o n o c r o t a l i n e to pyrroles per milligram p r o tein was significantly increased by all the feed additives e x c e p t selenium. When e n z y m e activities were expressed per n a n o m o l e o f c y t o c h r o m e P-450, hepatic a m i nopyr i ne demethylase activity and the in vitro pyrrole p r o d u c t i o n were, however, f o u n d to be significantly reduced by dietary e t h o x y q u i n and selenium.

Effect of dietary ethoxyquin on the formation o f pyrrolic metabolites from monocro taline in vivo The effects o f dietary e t h o x y q u i n on the levels of liver pyrroles 2 h and 24 h after the i.p. administration of monocrotaline, 280 mg/kg, are presented in Table VI. T h e levels of liver pyrroles at either 2 h or 24 h after th e administration of t he alkaloid were n o t altered by dietary e t h o x y quin. Th er e was a marked decline in liver pyrroles of bot h cont rol and e t h o x y q u i n - f e d rats within 24 h after monocrotaline.

TABLE VI EFFECT OF ETHOXYQUIN ON LIVER PYRROLES OF MONOCROTALINETREATED MICE Female mice were fed control or 0.25% ethoxyquin diets for 11 days. At 10 days, all animals were injected i.p. with monocrotaline, 280 mg/kg. Five animals from each group were killed at 2 h and 24 h after monocrotaline. Values represent means -+ S.E. Treatment

Control 0.25% Ethoxyquin

Liver pyrroles (nmol pyrrole/g) 2h

24h

63.2 -+ 5.5 60.6 -+ 3.6

18.3 ± 3.2 22.1 -+ 3.2

104 DISCUSSION The results of these experiments demonstrate that ethoxyquin is an effective protective agent against the lethality and acute hepatotoxicity of monocrotaline in mice. The i.v. 7
105 treatment. However, on the basis of per nanomole of cytochrome P-450, both enzyme activities were significantly decreased b y dietary ethoxyquin. These results imply that the amount of a particular species of c y t o c h r o m e P-450 induced by ethoxyquin that is active in the biotransformation of monocrotaline and aminopyrine is rather small compared to the sum total of all species of c y t o c h r o m e P-450 induced. Whether ethoxyquin indeed causes a differential induction o f various forms of cytochrome P-450 in mouse liver is an interesting subject for future investigation. One possible explanation for the protective effect of ethoxyquin is the increased concentration o f liver glutathione when this antioxidant was fed to mice. Surprisingly, only ethoxyquin b u t not the other agents significantly increased the concentration (mg/liver) of liver glutathione. The presence of elevated levels of glutathione in liver may enhance detoxication of the PA pyrroles. A direct correlation has been shown between liver glutathione levels and the toxicity of the PA retrorsine [8]. The protective effect of dietary cysteine could not be due to glutathione synthesis since the amino acid failed to increase the glutathione content. Cysteine may have enhanced the conjugation of the pyrroles directly as demonstrated earlier [ 7]. Another possible mechanism b y which ethoxyquin can influence monocrotaline toxicity is its inductive effect on glutathione S-transferase activity. Using 4 different substrates including CDNB, Benson et al. [11] reported a 3--6.6-fold increase in glutathione S-transferase activity in mice fed diets containing 0.5% ethoxyquin. A 2.8--5-fold increase in glutathione S-transferase activity was seen after feeding the mice diets containing 0.25% ethoxyquin. The elevated glutathione S-transferase activity in ethoxyquinfed mice probably increases the rate of reaction of monocrotaline pyrroles with glutathione, forming relatively harmless conjugates [7]. However, we recently found that conjugation of dehydroretronecine, one of the pyrrole metabolites of monocrotaline was n o t enhanced by glutathione S-transferase (unpublished observation). It appears that glutathione S-transferase is not involved in the protective mechanisms. Attempts have been made to determine the protective effects of various antioxidants against PA toxicity. The antioxidant, N,N'-diphenyl-pphenylene-diamine is not protective against heliotrine [25]. a-Tocopherol or ubiquinone had no protective effect against the toxicity of lasiocarpine in rats [34]. However, mercaptoethylamine afforded protection against hepatic necrosis and death caused b y lasiocarpine [34]. Miranda et al. [35,36] reported that dietary butylated hydroxyanisole reduced mortalities in mice treated with monocrotaline. Liver glutathione levels were increased and microsomal PA pyrrole formation was decreased in the animals treated with butylated hydroxyanisole. Although the mechanism of action of ethoxyquin is not fully resolved it is evident that this c o m p o u n d has a potential value in protecting animals against monocrotaline toxicity.

106 ACKNOWLEDGEMENTS

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