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Effect of vitamin E dietary intake on in vitro activation of aflatoxin B1
Mutation Research, 319 (1993) 309-316 © 1993 Elsevier Science Publishers B.V. All rights reserved 0165-1218/93/$06.00
309
MUTGEN 01941
Effect of vitamin E dietary intake on in vitro activation of aflatoxin B1 Pierrette Cassand *, Sophie Decoudu, Franqois L6v~que, Michble Daubbze and J. Francois Narbonne Laboratoire de Toxicologie Alimentaire, Universit~ Bordeaux I, R~seau de Toxicologie Nutritionnelle MRES, Avenue des Facult~s, 33405 Talence Cedex, France (Received 2 February 1993) (Revision received 15 June 1993) (Accepted 16 June 1993) Keywords: Vitamin E; Mutagenesis; Aflatoxin B1; Metabolic activation
Summary The molecular mechanism of action of vitamin E on mammalian cells remains to be elucidated. In this study, vitamin E dietary intake was assessed for its effects on the initiation phase of carcinogenesis. We have conducted a dose-effect relationship between vitamin E dietary intake and aflatoxin B1 (AFB1) genotoxicity measured in vitro. Thus AFB1 induced mutagenesis in Salmonella typhimurium TA98 was investigated and compared to effect of vitamin E dietary intake on hepatic microsomal P-450 content and specific activities involved in AFB1 metabolism. Rats were fed ad libitum a diet containing 0, 0.05, 0.5 or 5 IU of a-tocopherol for 8 weeks. Modulation of vitamin E level in postmitochondrial and microsomal fractions resulted in nutritional effects. Cytochrome P-450 content was not modified by the level of vitamin E in the diet. The microsomal P-450 activities, P-450 IIB1 and IliA, were decreased in the deficient group to - 3 5 % and - 1 6 % , respectively, as compared with control diet (0.05 IU). Diet supplemented with 0.5 IU of vitamin E increased P-450 liB and IliA activities (+28% and +37%, respectively) whereas a diet highly supplemented in vitamin E (5 IU) reduced these specific P-450 activities. Lipid peroxidation, estimated by the formation of thiobarbituric acid reactive products, increased in the dietary vitamin E free diet (+ 20%) and strongly decreased in the supplemented group (-99%). This study establishes that in vivo, dietary vitamin E protects directly membrane against damage induced by lipid peroxidation and indirectly hepatic microsomal monooxygenase activities. However, vitamin E accumulation seems to alter membrane structure and function. The nutritional effect of vitamin E on hepatic microsomal cytochrome P-450 activities modified the AFB1 genotoxicity measured in vitro.
* Corresponding author. Abbreviations: AFB1, aflatoxin B1; AFM1, aflatoxin M1; AFQ1, aflatoxin Q1; AFP1, aflatoxin P1; TBARS, thiobarbituric acid reactive products; GSH, glutathione; GST, glutathione S-transferase; EROD, 7-ethoxyresorufin Odealkylase; BzND, benzphetamin N-demethylase; ERMD, erythromycin N-demethylase; PTROD, pentoxyresorufin Odealkylase.
Vitamin E is well known as a potent biological antioxidant inhibiting lipid peroxidation and protecting cell membranes and possibly nucleic acids from oxidative damage (Machlin and Brin, 1980). Thus a preventive role of a-tocopherol against development of cancer has been considered (Birt, 1989; Wang et al., 1989). The literature with regard to possible effects of vitamin E on chemi-
310
cal carcinogenesis is conflicting (Chen, 1988). Vitamin E may exert multiple functions. It could play a role as an antioxidant preventing lipid formation and it is also involved in maintenance of the integrity of the cellular and subcellular membrane. In the complicated processes of carcinogenesis initiation, promotion and growth, there are many potential sites at which vitamins can serve as either negative or positive influence factors, and the mechanism of action of this vitamin against other carcinogens is not clearly understood. As suggested by Chen et al. (1988), a-tocopherol can modify the mutagenicity of carcinogens through one or more of the following mechanisms: (i) shifting the equilibrium between metabolic activation and inactivation of carcinogenic compounds, (ii) scavenging of the reactive intermediate a n d / o r (iii) reducing the DNA binding of mutagenic metabolites. Aflatoxin B1 (AFB1) is a potent hepatocarcinogen for several animal species widely used for in vivo studies (Wogan, 1973; Garner and Martin, 1979). This xenobiotic requires metabolic activation to be converted to chemically reactive forms (Essigman et al., 1982). Metabolic conversion is catalyzed predominantly by the hepatic microsomal cytochrome P-450 dependent monooxygenase system (Miller and Miller, 1976). The predominant AFB1 metabolite that binds to DNA in animal and human tissues is the AFB1-2,3-oxide (Garner et al., 1972; Swenson et al., 1974). In human tissues, many isozymes of cytochrome P450 are involved in AFB1 metabolism (Forrester et al., 1990; Oyama et al., 1990). In rat tissues, Ueno et al. (1985) revealed that isozyme IA possesses a high potentiality for activation of AFB1. Other isozymes, essentially IA1 and IliA, catalyze the formation of small quantities of AFMI, AFQ1 and AFP1, metabolites biologically much less active than AFB1. Long term studies in rats indicated that diets deficient in vitamin E resulted in a lower incidence and severity of aflatoxin B1 induced hepatomas (Alfin-Slater et al., 1976). In short term in vitro assays, vitamin E was able to exert inhibitory effects on AFB1 induced mutagenesis in Salmonella typhimurium (Raina and Gurtoo, 1985; Decoudu et al., 1990). Conversely addition of atocopherol in Ames plates does not modify AFB1
genotoxicity. Vitamin E deficiency is known to decrease the cytochrome P-450 content and P450-related activities (Mounier, 1988; Murray, 1991). Vitamin E has been reported to modify the activities involved in metabolism related to peroxidation activity (Narbonne et al., 1990; Murray, 1991). Raina and Gurtoo (1985) have reported that this fat soluble vitamin affects metabolic as well as postmetabolic levels of mutagenesis in the Salmonella typhimurium assay. The aim of the present study is to evaluate the relationship between activities of drug metabolizing enzymes which especially activate or deactivate AFB1, and AFB1 genotoxicity measured in vitro. This investigation is assessed in a dose-response study with rats fed increasing levels of vitamin E. Material and methods
Bacterial strain. Salmonella typhimurium TA98 strain was obtained from Dr. B.N. Ames (University of California, Berkeley, CA, USA). Chemicals. Chemicals were obtained as follows: DNA, ethoxyresorufin, erythromycin and benzphetamin from Sigma Chemicals, St. Louis, MO (USA); AFB1 and thiobarbituric acid from Aldrich Chimie, Strasbourg (France); glutathione from Merck, Darmstadt (Germany); (lac)styrene oxide from Fluka AG, Buchs (Switzerland), radioactive labeled compound from Amersham, Les Ulis (France). Animals and tissue preparation. Twenty-four male Sprague-Dawley rats were obtained from Charles River (St Aubin les Elboeuf, France) at a weight of 40-50 g. The rats were fed ad libitum for 8 weeks with diets containing 0, 0.05, 0.5 or 5 IU of vitamin E (o-a-tocopherol) per gram (INRA, Jouy en Josas). In this study, 0 IU of vitamin E was referred to as D (deficient), 0.05 IU as C (control), 0.5 IU as $1 (supplement 1) and 5 IU as $2 (supplement 2). 0.05 IU of vitamin E per gram is considered in rats to be the daily acceptable intake. Each vitamin E dietary group comprised six animals. Rats were killed by decapitation. The livers were removed aseptically, minced and homoge-
311
nized in 3 volumes of ice-cold 0.15 M KCI solution. The postmitochondrial fraction was obtained by centrifugation of the homogenate at 9 0 0 0 - g for 15 min, and was stored at -80°C until used for mutagenesis and lipid peroxidation studies. The microsomal and cytosolic fractions were prepared from the remaining $9 by centrifugation for 1 h at 105,000- g. Final microsomal preparations were in buffer pH 7.4 (Tris 10 mM, KCI 150 mM, EDTA 0.1 mM). The subcellular fractions were stored at -80°C for 3 months maximum.
scored. The experiment was carried out in triplicate with five concentrations of AFB1 (2.5, 10, 25, 50 and 100 ng/plate). Lipid peroxidation was estimated by the formation of thiobarbituric acid-reactive products (TBARS) as described by Buege and Aust (1978).
Microsomal and cytosolic parameters. Cytosolic glutathione (GSH) was determined according to the method of Akerboom and Sies (1981). Cytosolic glutathione S-transferase (GST) activity was measured with (14C)styrene oxide as substrate (Marniemi and Parkki, 1975). Cytochrome P-450 microsomal content was assayed by the Omura and Sato procedure (Omura and Sato, 1964). The measured microsomal monooxygenase activities were 7-ethoxyresorufin O-dealkylase (EROD), benzphetamin N-demethylase (BzND) and erythromycin N-demethylase (ERMD), respectively specific for subfamilies IA, liB and IliA of cytochrome P-450. The fluorescence of resorufin was measured according to the method of Rifking and Muschick (1983). Formaldehyde was quantified by the method of Nash (1953).
Liver parameters. The protein contents were determined by the method of Lowry et al. (1951). Retinyl palmitate and a-tocopherol were extracted and quantified by HPLC method (Periquet et al., 1985). The HPLC analysis was performed on a Philips PU 4021 UV detector at 325nm, with a Pherisorb $ 5 0 D S reverse phase column. The mobile phase was methanol at 0.5 ml/min. Postmitochondrialparameters. We studied the ability of $9 liver fractions to activate AFB1 in the Ames test conditions. The procedure described by Maron and Ames (1983) was followed. To 2.5 ml of molten top agar were added in this sequence: 0.1 ml of AFB1 solution in DMSO, 0.1 ml of the bacterial suspension and 0.5 ml of the $9 mix which contained 20% $9. The mixed was poured on minimal glucose agar plates and incubated at 37°C for 48 h after which the number of histidine-independent (His +) revertant colonies in Salmonella typhimurium TA98 was
Statistical analysis. The interactions between the treatments and the comparison of the means obtained for each group were calculated as described by Duncan (1955). Results
Liver parameters. As shown in Table 1, the body and liver weights of animals on vitamin E deficient diet did not present any significant dif-
TABLE 1 E F F E C T O F V I T A M I N E D I E T A R Y INTAKE ON G R O W T H A N D L I V E R P A R A M E T E R S IN RATS Vitamin E diet ( I U / g ) 0 Body weight (g) Liver weight (g/100 g bodyweight) Liver tocopherol ( g g / g ) Liverretinylpalmitate(/.~g/g) Liver ascorbate ( ~ g / g ) Liver G S H ( m g / g )
416
0.05 +14
2.9 + 6.7 + 178.2 + 242.3 + 2.79+
0.03 0.05 ** 2.5 * 5.5 * * 0.06 *
435
0.5 +13
3.1+ 70 + 181.1+ 269.6 + 3.1+
0.03 8.6 2 4 0.06
433
5 + 5.6
2.7+ 340 + 162.7+ 259.2 + 2.8+
0.02 12 ** 1.1"* 13 0.12
The results are expressed as the mean of six determinations + SD. Statistically significant difference as compared to the 0.05 IU vitamin E group: ** P < 0.01, * P < 0.05.
411
+5.8
2.8 +0.05 710 + 7 ** 146 + 7 ** 274.5 + 7.5 2.86+0.03 *
312 TABLE 2 EFFECT OF VITAMIN E DIETARY INTAKE ON MICROSOMAL, CYTOSOLIC AND POSTMITOCHONDRIAL PARAMETERS IN RAT LIVER Vitamin E diet (IU/g) Microsomes microsomal proteins (mg/g) cytrochrome P450 (pmole/mg MP) EROD activity (pmole/mg MP) BzND activity (nmole/min/mg MP) ERMD activity (pmole/min/mg MP) Cytosol cytosolic proteins (rag/g) GST activity (nmole/min/mg CP)
0
0.05
0.5
5
18.5+ 0.9 1034 +51 209 + 3.8 ** 4.7_+ 0.83 * 678 _+80"*
19.9 + 1.7 932 +50 190 + 3.8 7.29 + 1 802 +70
19.3 _+ 0.8 995 -+70 176.3 +14 9.32 + 0.52 * 1096 + 5 2 " *
18.9 + 1 1055 +35 ** 162.7 -+18.5 8.08_+ 0.27 * * * 987 _+71"**
73.9+ 3.1 99.8+ 7
79.7 + 6.8 99.1 + 5
77.1 + 3.41 97.1 + 9.8
75.5 + 3.8 117.9 _+11"*
Results are expressed as the mean + SD of three determinations. Significantly different from rats as compared to the 0.05 IU vitamin E group: * * P < 0.01, * P < 0.05. Significantly different from rats as compared to the 0.5 IU vitamin E group: * * * P < 0.05.
f e r e n c e f r o m controls. T h e h e p a t i c level of a t o c o p h e r o l ( l o g / z g / g ) is positively c o r r e l a t e d with t he level o f v i t a m i n E d ie ta r y intake ( I U / g ) ( r = 0.723, P < 0.05). H e p a t i c levels o f retinyl palmit a t e a nd g l u t a t h i o n e d e c r e a s e in v i t a m i n E deficient or s u p p l e m e n t e d diet. H e p a t i c a s c o r b a t e c o n t e n t also d e c r e a s e s in v i t a m i n E f r e e diet c o n d i t i o n s ( - 1 0 % , P < 0.01) b u t is n o t significantly c h a n g e d with s u p p l e m e n t e d diets.
Microsomes. M i c r o s o m a l p r o t e i n c o n t e n t is not significantly c h a n g e d by v i t a m i n E d ie ta r y intake ( T a b l e 2). T h e c o n t e n t o f c y t o c h r o m e P-450 is not significantly m o d i f i e d by t h e level o f vitamin E in t h e diet. C o m p a r e d to t h e c o n t r o l
group, t h e m i c r o s o m a l m o n o o x y g e n a s e activities B z N D an d E R M D w e r e slightly d e c r e a s e d in t h e d e f i c i e n t group, an d weakly i n c r e a s e d in t h e S1 an d $2 groups. H o w e v e r , t h e m a x i m u m differe n c e was o b t a i n e d b e t w e e n t h e d e f i c i e n t an d S1 groups ( + 98% an d + 61%, P < 0.001 for B z N D and E R M D , respectively).
Cytosolic fraction. T a b l e 2 also s u m m a r i z e s t h e results o b t a i n e d in t h e cytosolic fraction. Only t h e G S T activity was i n c r e a s e d ( + 26%, P < 0.05) in t h e $2 g r o u p as c o m p a r e d to t h e control. Postmitochondrial fraction. C o m p a r e d to control value, T B A R S p r o d u c t s w e r e i n c r e a s e d in
TABLE 3 EFFECT OF VITAMIN E DIETARY INTAKE ON LIPID PEROXIDATION AND AFB1 MUTAGENIC ACTIVATION IN POSTMITOCHONDRIAL ($9) FRACTIONS IN RATS Vitamin E diet (IU/g) Proteins(mg/g) TBARS (nmole/3 h/mg S9P) Mutagenesis (TA98 rev/ng AFB1)
0
0.05
0.5
5
92.5 +5.1 14.9 +0.8 * 1.37 + 0.2 * *
99.5 +3.5 12.4 +0.6 2.88 -l-0.5
96.4 +4.8 0.15+0.02 ** 2.6 + 0.3
94.3 +4 0.11+0.01 ** 2.13 + 0.6 *
Results are expressed as the mean + SD of three determinations. Significantly different from rats as compared to the 0.05 IU vitamin E group: * * P < 0.01, * P < 0.05.
313 HIS+ revertants TA98
80O
jl
j~
jJJ
i
600
°
jJJ
// / //
//
,
//
400
/
/
/,
//
/~///
2ooI I
//
A-"
J" ! I
0
20
40
60
100
80
120
ng AFB1 per plate
+0
UI
+005
UI
+05
UI
+5
uI
Fig. 1. Dose-response effect of vitamin E dietary intake on $9 dependent mutagenic activity of AFB1 in TA98. Each point represents the average value of three plates +SD. The spontaneous mutation rate (32) was subtracted from each point.
phimurium
Salmonella ty-
the vitamin E deficient group (+ 20%, P < 0.05) and strongly decreased in the supplemented groups ( - 9 9 % , P < 0.01) (Table 3).
Mutagenicity assay. We report in Fig. 1 the ability of $9 fractions from vitamin E treated rats to activate AFB1 in Ames test conditions. In order to assess the influence of vitamin E dietary intake, we have expressed the AFB1 mutagenic activity in revertants per ng of AFB1 calculated from the slope of the linear section of the doseresponse curve (Table 3). Vitamin E free diet and high dietary level of vitamin E significantly decreased the mutagenic activity as compared to control values ( - 18%, P < 0.01 and -47.3%, P < 0.05, respectively). Mutagenic activity is unaffected by diet supplemented with 0.5 IU of vitamin E. Discussion
The present data demonstrate that vitamin E dietary intake influences AFB1 mutagenicity esti-
mated by the Ames test. This effect seems to be related to the activities of hepatic enzymes involved in AFB1 metabolism. Vitamin E content in liver fraction is related to the a-tocopherol dietary intake in accordance with Sevanian et al. (1982) and Machlin (1983). Thus an a-tocopherol dose effect may be analyzed on microsomal and nuclear fractions. Nutritional factors have the capacity to alter the activity of hepatic microsomal drug oxidases (Murray, 1991). The modulation of P-450 enzyme expression implicates several mechanisms of action. Thus some nutrients which are substrates of P-450 enzymes in microsomal fraction are able to exert a competitive inhibition with EROD, PTROD (pentoxyresorufin O-dealkylase) and ERMD activities. Such an inhibitory effect was observed with retinol (Koch, 1991) and flavonoids (Siess et al., 1990) and can be partially explained by a direct interaction with the active site of cytochrome P-450. On the basis of literature reports, a-tocopherol may not be considered a P-450 enzyme inhibitor. This vitamin has never been reported as a substrate of cytochrome P-450 dependent enzymes (Csallany et al., 1962; Nelly et al., 1988). Murray (1991) has reported that vitamin E does not directly interact with the cytochrome P-450 at its active site. Nutrients may also directly or indirectly alter the microsomal membrane and thus modify microsomal enzyme functions. Moreover small changes in the microsomal concentration of P-450 specific isozymes may result in an increase in mRNA level. Such a nuclear induction has been reported with retinol. Vitamin E is accumulated in rat hepatic nuclei (Patnaik and Nair, 1977; Machlin, 1980). However this component does not seem to induce the synthesis of P-450 isozymes implicated in AFB1 metabolism. Only the content of P-450 isozyme I I C l l has been reported to increase with vitamin E dietary supplementation (Murray, 1991). Vitamin E has been shown to influence the microsomal environment by modifying the susceptibility to lipid peroxidation (Wefers and Sies, 1989; Tirmenteins and Reed, 1989; Graham et al., 1989). The concentration of TBARS has been found to be indicative of the ultimate potential for peroxidation of tissue lipids in vitro, as well as an estimate of in vivo malondialdehyde produc-
314
tion (Buckingham, 1985). Thus the inverse relationship between vitamin E liver concentration and TBARS production indicates that rats fed a vitamin E deficient diet strongly increase the level of hepatic lipid peroxides as compared to rats fed a vitamin E supplemented diet. These results are in conformity with others (Buckingham, 1985; De and Darad, 1988; Graham et al., 1989). Several authors have reported that lipid derived radicals may be responsible for protein fragmentation (Dean and Cheeseman, 1987; Thomas et al., 1989). Cytochrome P-450 appears to be one of the most sensitive enzymes to lipid peroxide damage (Hrycay and O'Brien, 1971; Mimnaugh et al., 1981). The results reported by Ando and Tappel (1985) and Mounier (1988) are in accordance with this observation. However, in this study, the vitamin E free diet group shows a change in P-450 content in vitamin E deficiency; to counteract the increased tissue peroxidizability, the enzymatic antioxidant defense system has been shown to be more active (Chen et al., 1980). Thus a decrease in the hepatic level of the antioxidant substrates vitamin A, GSH and vitamin C was observed. This phenomenom may protect the hemoprotein P-450 against lipid derived radical attack. However, peroxidation has been shown to alter the integrity and permeability of the microsomal membrane, which can modify enzyme function (H6gherg et al., 1973; Hruszkewycz et al., 1978; Ando and Tappel, 1985). Thus in absence of vitamin E, P-450 IIB and IIIA expression was decreased. In microsomes from rats supplemented with 0.5 IU of vitamin E (S1 group), the increased P-450 IIB and IIIA activities confirm that a-tocopherol is indispensable for a maximum activity of oxidative drug metabolism. However the weak induction of the subfamilies liB and IliA of cytochrome P-450 does not seem to modify the AFB1 mutagenic activation in Ames test. High doses of o~tocopherol in microsomal membrane have been shown to decrease activities of cytochrome P-450 dependent enzymes (Mounier, 1988). Thus the membrane structure and function seem to be altered by vitamin E accumulation. The $9 fraction used in the Ames test involves the activity of GST located in the cytosolic fraction and catalyzing the AFB1-GSH formation,
considered to be the dominant epoxide detoxication mechanism (O'Brien et al., 1983; Lotlikar et al., 1989). In agreement with Ando and Tappel (1985), the activity of epoxide conjugation to GSH is not modified under vitamin E free diet conditions. Thus the inhibitory effect of vitamin E deficiency on AFB1 genotoxic activity in the Ames test can be related to the modulation of the activities of P-450 enzymes. In the $2 group, the induction of AFB1 detoxication and metabolic activation reduces the ability of AFB1 to revert TA98. The mutagenic activity in the 5 IU diet decreases as compared to the control group. However the activities of cytochrome P-450 dependent enzymes between these two groups are not signicantly different. Thus a nuclear mechanism may be envisaged. The antimutagenic effect of vitamin E can also be related to its ability to influence AFB1-DNA adduct formation, a-Tocopherol can be transformed by reaction with singlet quenching oxygen into tocopheryl quinone, tocopherol hydroquinone and other polar metabolites which are able to react directly with DNA (Csallanay et al., 1962; Nelly et al., 1988; Wang et al., 1989). Thus chromatin binding of D-a-tocopherol metabolites may change DNA accessibility to xenobiotics and thus may inhibit intercalation of AFB1 epoxide on DNA. In conclusion, the results presented here suggest that the inhibitory effect of vitamin E deficiency on AFB1 genotoxicity is indirectly related to an antioxidant mechanism. However high dietary vitamin E intake suggests an important role of a-tocopherol on microsomal membrane structure.
Acknowledgement This investigation was supported by a grant from the Minist~re de la Recherche et de la Technologie.
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Reduced glutathione effects on alpha-tocopherol concentration of rat liver microsomes undergoing NADPH-dependent lipid peroxidation, Lipids, 24, 909-914. Harris, T.M., M.P. Stone and S. Gopalakrishnan (1989) Aflatoxin bl epoxide, the ultimate carcinogenic form of aflatoxin bl: Synthesis and reaction with DNA, J. Toxicol. Toxin Rev., 8, 111-120. H6gherg, J., A. Bergstrang and S.V. Jakobsson (1973) Lipid peroxidation of rat liver microsomes: Its effect on the microsomal membrane and some membrane-bound microsomal enzymes, Eur. J. Biochem., 37, 51-59. Hruszkewycz, A.M., E.A. Glende and R.O. Recknagel (1978) Destruction of microsomal cytochrome P450 and glucose6-phosphatase by lipid extracts from peroxidized microsomes, Toxicol. Appl. Pharmacol., 46, 695-702. Hrycay, E.G., and P.J. O'Brien (1971) Cytochrome P450 as a microsomal peroxidase utilizing a lipid peroxidase substrate, Arch. Biochem. Biophys., 147, 14. Koch, B. (1991) Effets des x6nobiotiques sur le transport, la distribution et le m&abolism oxydatif de la vitamin A chez le rat, Ph.D. Thesis, Bordeaux. Lotlikar, P.D., H.G. Raj, L.S. Bohm, L.L. Ho, E.-C. Jhee, K. Tsuji and P. Gopalan (1989) A mechanism of inhibition of aflatoxin B1-DNA binding in the liver by phenobarbital pretreatment of rats, Cancer Res., 49, 951-957. Lowry, O.M., J.N. Rosebrough, A.I. Farr and R.J. Randall (1951) Protein measurement with Folin phenol reagent, J. Biol. Chem., 192, 265-275. Machlin, L.J. (1983) in: L.J. Machlin (Ed.), Handbook of Vitamins, Dekker, New York, pp. 100-145. Machlin, L.Y., and M. Brin (1980) in: R.B. Aflatin-Slater and D. Kritchevrki (Eds.), Human Nutrition - A Comprehensive Treatise, Plenum, New York, pp. 245-266. Marniemi, J., and M.G. Parkki (1975) Radiometrical assay of glutathione-S epoxide transferase and its enhancement by phenobarbital in rat liver in vivo, Biochem. Pharmacol., 24, 1569-1572. Maron, D.M., and B.N. Ames (1983) Revised methods for the Salmonella mutagenicity test, Mutation Res., 113, 173-215. Miller, E.C., and J.A. Miller (1976) The metabolism of chemical carcinogens to reactive electrophiles and their possible mechanism of action in carcinogenesis, in: C.S. Searle (Ed.), Chemical Carcinogens, ACS Monograph 173, Am. Chem. Soc., Washinton, DC, pp. 737-762. Mimnaugh, E.G., M.A. Trush, E. Ginsburg, Y. Hirokata and T.E. Gram (1981) The effects of adriamycin in vitro and in vivo on hepatic microsomal drug-metabolizing enzymes: role of microsomal lipid peroxidation, Toxicol. Appl. Pharmacol., 61, 313-325. Mounier, J. (1988) Influence de facteurs nutritionnels lipidiques sur la r6gulation des enzymes h6patiques du m&abolisme des x6niobiotiques. Th~se d'Etat, Universit6 de Bourgogne. Murray, M. (1991) In vitro and in vivo studies of the effect of vitamin E on microsomal cytochrome P450 in rat liver, Biochem. Pharmacol., 42, 2107-2114.
316 Narbonne, J.F., P. Cassand, M. Daubeze, C. Colin and F. Leveque (1990) Chemical mutagenicity and cellular status in vitamins A, E and C, Food Addit. Contam., 7, $48-$52. Nash, T. (1953) The colorimetric estimation of formaldehyde by means of the Hantzsch reaction, Biochem. J., 55, 416421. Nelly, W.C., J.M. Martin and S.A. Barker (1988) Products and relative reaction rates of the oxidation of tocopherols with singlet molecular oxygen, Photochem. Photobiol., 48, 423428. O'Brien, K., E. Moss, D. Judah and G. Neal (1983) Metabolic basis of the species difference to aflatoxin bl induced hepatotoxicity, Biochem. Biophys. Res. Commun., 114, 813-821. Omura, T., and R. Sato (1964) The carbon monooxide-binding pigment of liver microsomes. II: Solubilization, purification and properties, J. Biol. Chem., 239, 2379-2385. Patnaik, R.N., and P.P. Nair (1977) Studies on the binding of d-a-tocopherol to rat liver nuclei, Arch. Biochem. Biophys., 178, 333-341. Periquet, B., A. Bailly, J. Ghisolfi and J.P. Thouvenot (1985) Determination of retinyl palmitate in homogenates and subcellular fractions of rat by liquid chromatography, Clin. Chim. Acta, 147, 41-49. Raina, V., and H.L. Gurtoo (1985) Effects of vitamins A, C and E on aflatoxin Bl-induced mutagenesis in Salmonella typhimurium TA98 and TA100, Teratogen. Carcinogen. Mutagen., 5, 29-40. Rifking, A.B., and H. Muschick (1983) Benoxaprofen suppression of polychlorinated biphenyl toxicity without alteration of mixed function oxidase function, Nature, 303, 524-526. Sevanian, A., A.D. Hacker and N. Elsayed (1982) Influence of
vitamin E and nitrogen dioxide on lipid peroxidation in rat lung and liver microsomes, Lipids, 17, 269-277. Siess, M.H., A.M. Le Bon, M. Suschetet and P. Rat (1990) Inhibition of ethoxyresorufin deethylase activity by natural flavonoids in human and rat liver microsomes, Food Addit. Contam., 7, $178-$181. Swenson, D.H., E.C. Miller and J.A. Miller (1974) Aflatoxin b l 2,3 oxide: evidence for its formation in rat liver in vivo by human liver microsome in vitro, Biochem. Biophys. Res. Commun., 60, 1036-1043. Thomas, S.M., J.M. Gebicki and R.T. Dean (1989) Radical initiated alpha-tocopherol depletion and lipid peroxidation in mitochondrial membranes, Biochim. Biophys. Acta, 1002, 189-197. Tirmenteins, M., and D.J. Reed (1989) Effect of glutathione on the alpha-tocopherol-dependent inhibition of nuclear lipid peroxidation, J. Lipid Res., 30, 959-965. Ueno, Y., F. Tashiro and H. Nakaki (1985) Mechanism of metabolic activation of aflatoxin B1, GANN Monogr. Cancer Res., 30, 111-124. Vaca C.E., J. Wilhelm and M. Harms-Ringdahl (1988) Interaction of lipid peroxidation products with DNA, Mutation Res., 195, 137-149. Wang, Y., M. Purewal, B. Nixon, D. Li and D. SolysiakPawluczuk (1989) Vitamin E and cancer prevention in an animal model, Ann. NY Acad. Sci., 570, 383-390. Wefers, H., and H. Sies (1989) Antioxidant effects of ascorbate and glutathione in microsomal lipid peroxidation are dependent on vitamin E, Adv. Biosci., 76, 309-315. Wogan, G.N. (1973) Aflatoxin carcinogenesis, Methods Cancer Res., 8, 309-344.
309
MUTGEN 01941
Effect of vitamin E dietary intake on in vitro activation of aflatoxin B1 Pierrette Cassand *, Sophie Decoudu, Franqois L6v~que, Michble Daubbze and J. Francois Narbonne Laboratoire de Toxicologie Alimentaire, Universit~ Bordeaux I, R~seau de Toxicologie Nutritionnelle MRES, Avenue des Facult~s, 33405 Talence Cedex, France (Received 2 February 1993) (Revision received 15 June 1993) (Accepted 16 June 1993) Keywords: Vitamin E; Mutagenesis; Aflatoxin B1; Metabolic activation
Summary The molecular mechanism of action of vitamin E on mammalian cells remains to be elucidated. In this study, vitamin E dietary intake was assessed for its effects on the initiation phase of carcinogenesis. We have conducted a dose-effect relationship between vitamin E dietary intake and aflatoxin B1 (AFB1) genotoxicity measured in vitro. Thus AFB1 induced mutagenesis in Salmonella typhimurium TA98 was investigated and compared to effect of vitamin E dietary intake on hepatic microsomal P-450 content and specific activities involved in AFB1 metabolism. Rats were fed ad libitum a diet containing 0, 0.05, 0.5 or 5 IU of a-tocopherol for 8 weeks. Modulation of vitamin E level in postmitochondrial and microsomal fractions resulted in nutritional effects. Cytochrome P-450 content was not modified by the level of vitamin E in the diet. The microsomal P-450 activities, P-450 IIB1 and IliA, were decreased in the deficient group to - 3 5 % and - 1 6 % , respectively, as compared with control diet (0.05 IU). Diet supplemented with 0.5 IU of vitamin E increased P-450 liB and IliA activities (+28% and +37%, respectively) whereas a diet highly supplemented in vitamin E (5 IU) reduced these specific P-450 activities. Lipid peroxidation, estimated by the formation of thiobarbituric acid reactive products, increased in the dietary vitamin E free diet (+ 20%) and strongly decreased in the supplemented group (-99%). This study establishes that in vivo, dietary vitamin E protects directly membrane against damage induced by lipid peroxidation and indirectly hepatic microsomal monooxygenase activities. However, vitamin E accumulation seems to alter membrane structure and function. The nutritional effect of vitamin E on hepatic microsomal cytochrome P-450 activities modified the AFB1 genotoxicity measured in vitro.
* Corresponding author. Abbreviations: AFB1, aflatoxin B1; AFM1, aflatoxin M1; AFQ1, aflatoxin Q1; AFP1, aflatoxin P1; TBARS, thiobarbituric acid reactive products; GSH, glutathione; GST, glutathione S-transferase; EROD, 7-ethoxyresorufin Odealkylase; BzND, benzphetamin N-demethylase; ERMD, erythromycin N-demethylase; PTROD, pentoxyresorufin Odealkylase.
Vitamin E is well known as a potent biological antioxidant inhibiting lipid peroxidation and protecting cell membranes and possibly nucleic acids from oxidative damage (Machlin and Brin, 1980). Thus a preventive role of a-tocopherol against development of cancer has been considered (Birt, 1989; Wang et al., 1989). The literature with regard to possible effects of vitamin E on chemi-
310
cal carcinogenesis is conflicting (Chen, 1988). Vitamin E may exert multiple functions. It could play a role as an antioxidant preventing lipid formation and it is also involved in maintenance of the integrity of the cellular and subcellular membrane. In the complicated processes of carcinogenesis initiation, promotion and growth, there are many potential sites at which vitamins can serve as either negative or positive influence factors, and the mechanism of action of this vitamin against other carcinogens is not clearly understood. As suggested by Chen et al. (1988), a-tocopherol can modify the mutagenicity of carcinogens through one or more of the following mechanisms: (i) shifting the equilibrium between metabolic activation and inactivation of carcinogenic compounds, (ii) scavenging of the reactive intermediate a n d / o r (iii) reducing the DNA binding of mutagenic metabolites. Aflatoxin B1 (AFB1) is a potent hepatocarcinogen for several animal species widely used for in vivo studies (Wogan, 1973; Garner and Martin, 1979). This xenobiotic requires metabolic activation to be converted to chemically reactive forms (Essigman et al., 1982). Metabolic conversion is catalyzed predominantly by the hepatic microsomal cytochrome P-450 dependent monooxygenase system (Miller and Miller, 1976). The predominant AFB1 metabolite that binds to DNA in animal and human tissues is the AFB1-2,3-oxide (Garner et al., 1972; Swenson et al., 1974). In human tissues, many isozymes of cytochrome P450 are involved in AFB1 metabolism (Forrester et al., 1990; Oyama et al., 1990). In rat tissues, Ueno et al. (1985) revealed that isozyme IA possesses a high potentiality for activation of AFB1. Other isozymes, essentially IA1 and IliA, catalyze the formation of small quantities of AFMI, AFQ1 and AFP1, metabolites biologically much less active than AFB1. Long term studies in rats indicated that diets deficient in vitamin E resulted in a lower incidence and severity of aflatoxin B1 induced hepatomas (Alfin-Slater et al., 1976). In short term in vitro assays, vitamin E was able to exert inhibitory effects on AFB1 induced mutagenesis in Salmonella typhimurium (Raina and Gurtoo, 1985; Decoudu et al., 1990). Conversely addition of atocopherol in Ames plates does not modify AFB1
genotoxicity. Vitamin E deficiency is known to decrease the cytochrome P-450 content and P450-related activities (Mounier, 1988; Murray, 1991). Vitamin E has been reported to modify the activities involved in metabolism related to peroxidation activity (Narbonne et al., 1990; Murray, 1991). Raina and Gurtoo (1985) have reported that this fat soluble vitamin affects metabolic as well as postmetabolic levels of mutagenesis in the Salmonella typhimurium assay. The aim of the present study is to evaluate the relationship between activities of drug metabolizing enzymes which especially activate or deactivate AFB1, and AFB1 genotoxicity measured in vitro. This investigation is assessed in a dose-response study with rats fed increasing levels of vitamin E. Material and methods
Bacterial strain. Salmonella typhimurium TA98 strain was obtained from Dr. B.N. Ames (University of California, Berkeley, CA, USA). Chemicals. Chemicals were obtained as follows: DNA, ethoxyresorufin, erythromycin and benzphetamin from Sigma Chemicals, St. Louis, MO (USA); AFB1 and thiobarbituric acid from Aldrich Chimie, Strasbourg (France); glutathione from Merck, Darmstadt (Germany); (lac)styrene oxide from Fluka AG, Buchs (Switzerland), radioactive labeled compound from Amersham, Les Ulis (France). Animals and tissue preparation. Twenty-four male Sprague-Dawley rats were obtained from Charles River (St Aubin les Elboeuf, France) at a weight of 40-50 g. The rats were fed ad libitum for 8 weeks with diets containing 0, 0.05, 0.5 or 5 IU of vitamin E (o-a-tocopherol) per gram (INRA, Jouy en Josas). In this study, 0 IU of vitamin E was referred to as D (deficient), 0.05 IU as C (control), 0.5 IU as $1 (supplement 1) and 5 IU as $2 (supplement 2). 0.05 IU of vitamin E per gram is considered in rats to be the daily acceptable intake. Each vitamin E dietary group comprised six animals. Rats were killed by decapitation. The livers were removed aseptically, minced and homoge-
311
nized in 3 volumes of ice-cold 0.15 M KCI solution. The postmitochondrial fraction was obtained by centrifugation of the homogenate at 9 0 0 0 - g for 15 min, and was stored at -80°C until used for mutagenesis and lipid peroxidation studies. The microsomal and cytosolic fractions were prepared from the remaining $9 by centrifugation for 1 h at 105,000- g. Final microsomal preparations were in buffer pH 7.4 (Tris 10 mM, KCI 150 mM, EDTA 0.1 mM). The subcellular fractions were stored at -80°C for 3 months maximum.
scored. The experiment was carried out in triplicate with five concentrations of AFB1 (2.5, 10, 25, 50 and 100 ng/plate). Lipid peroxidation was estimated by the formation of thiobarbituric acid-reactive products (TBARS) as described by Buege and Aust (1978).
Microsomal and cytosolic parameters. Cytosolic glutathione (GSH) was determined according to the method of Akerboom and Sies (1981). Cytosolic glutathione S-transferase (GST) activity was measured with (14C)styrene oxide as substrate (Marniemi and Parkki, 1975). Cytochrome P-450 microsomal content was assayed by the Omura and Sato procedure (Omura and Sato, 1964). The measured microsomal monooxygenase activities were 7-ethoxyresorufin O-dealkylase (EROD), benzphetamin N-demethylase (BzND) and erythromycin N-demethylase (ERMD), respectively specific for subfamilies IA, liB and IliA of cytochrome P-450. The fluorescence of resorufin was measured according to the method of Rifking and Muschick (1983). Formaldehyde was quantified by the method of Nash (1953).
Liver parameters. The protein contents were determined by the method of Lowry et al. (1951). Retinyl palmitate and a-tocopherol were extracted and quantified by HPLC method (Periquet et al., 1985). The HPLC analysis was performed on a Philips PU 4021 UV detector at 325nm, with a Pherisorb $ 5 0 D S reverse phase column. The mobile phase was methanol at 0.5 ml/min. Postmitochondrialparameters. We studied the ability of $9 liver fractions to activate AFB1 in the Ames test conditions. The procedure described by Maron and Ames (1983) was followed. To 2.5 ml of molten top agar were added in this sequence: 0.1 ml of AFB1 solution in DMSO, 0.1 ml of the bacterial suspension and 0.5 ml of the $9 mix which contained 20% $9. The mixed was poured on minimal glucose agar plates and incubated at 37°C for 48 h after which the number of histidine-independent (His +) revertant colonies in Salmonella typhimurium TA98 was
Statistical analysis. The interactions between the treatments and the comparison of the means obtained for each group were calculated as described by Duncan (1955). Results
Liver parameters. As shown in Table 1, the body and liver weights of animals on vitamin E deficient diet did not present any significant dif-
TABLE 1 E F F E C T O F V I T A M I N E D I E T A R Y INTAKE ON G R O W T H A N D L I V E R P A R A M E T E R S IN RATS Vitamin E diet ( I U / g ) 0 Body weight (g) Liver weight (g/100 g bodyweight) Liver tocopherol ( g g / g ) Liverretinylpalmitate(/.~g/g) Liver ascorbate ( ~ g / g ) Liver G S H ( m g / g )
416
0.05 +14
2.9 + 6.7 + 178.2 + 242.3 + 2.79+
0.03 0.05 ** 2.5 * 5.5 * * 0.06 *
435
0.5 +13
3.1+ 70 + 181.1+ 269.6 + 3.1+
0.03 8.6 2 4 0.06
433
5 + 5.6
2.7+ 340 + 162.7+ 259.2 + 2.8+
0.02 12 ** 1.1"* 13 0.12
The results are expressed as the mean of six determinations + SD. Statistically significant difference as compared to the 0.05 IU vitamin E group: ** P < 0.01, * P < 0.05.
411
+5.8
2.8 +0.05 710 + 7 ** 146 + 7 ** 274.5 + 7.5 2.86+0.03 *
312 TABLE 2 EFFECT OF VITAMIN E DIETARY INTAKE ON MICROSOMAL, CYTOSOLIC AND POSTMITOCHONDRIAL PARAMETERS IN RAT LIVER Vitamin E diet (IU/g) Microsomes microsomal proteins (mg/g) cytrochrome P450 (pmole/mg MP) EROD activity (pmole/mg MP) BzND activity (nmole/min/mg MP) ERMD activity (pmole/min/mg MP) Cytosol cytosolic proteins (rag/g) GST activity (nmole/min/mg CP)
0
0.05
0.5
5
18.5+ 0.9 1034 +51 209 + 3.8 ** 4.7_+ 0.83 * 678 _+80"*
19.9 + 1.7 932 +50 190 + 3.8 7.29 + 1 802 +70
19.3 _+ 0.8 995 -+70 176.3 +14 9.32 + 0.52 * 1096 + 5 2 " *
18.9 + 1 1055 +35 ** 162.7 -+18.5 8.08_+ 0.27 * * * 987 _+71"**
73.9+ 3.1 99.8+ 7
79.7 + 6.8 99.1 + 5
77.1 + 3.41 97.1 + 9.8
75.5 + 3.8 117.9 _+11"*
Results are expressed as the mean + SD of three determinations. Significantly different from rats as compared to the 0.05 IU vitamin E group: * * P < 0.01, * P < 0.05. Significantly different from rats as compared to the 0.5 IU vitamin E group: * * * P < 0.05.
f e r e n c e f r o m controls. T h e h e p a t i c level of a t o c o p h e r o l ( l o g / z g / g ) is positively c o r r e l a t e d with t he level o f v i t a m i n E d ie ta r y intake ( I U / g ) ( r = 0.723, P < 0.05). H e p a t i c levels o f retinyl palmit a t e a nd g l u t a t h i o n e d e c r e a s e in v i t a m i n E deficient or s u p p l e m e n t e d diet. H e p a t i c a s c o r b a t e c o n t e n t also d e c r e a s e s in v i t a m i n E f r e e diet c o n d i t i o n s ( - 1 0 % , P < 0.01) b u t is n o t significantly c h a n g e d with s u p p l e m e n t e d diets.
Microsomes. M i c r o s o m a l p r o t e i n c o n t e n t is not significantly c h a n g e d by v i t a m i n E d ie ta r y intake ( T a b l e 2). T h e c o n t e n t o f c y t o c h r o m e P-450 is not significantly m o d i f i e d by t h e level o f vitamin E in t h e diet. C o m p a r e d to t h e c o n t r o l
group, t h e m i c r o s o m a l m o n o o x y g e n a s e activities B z N D an d E R M D w e r e slightly d e c r e a s e d in t h e d e f i c i e n t group, an d weakly i n c r e a s e d in t h e S1 an d $2 groups. H o w e v e r , t h e m a x i m u m differe n c e was o b t a i n e d b e t w e e n t h e d e f i c i e n t an d S1 groups ( + 98% an d + 61%, P < 0.001 for B z N D and E R M D , respectively).
Cytosolic fraction. T a b l e 2 also s u m m a r i z e s t h e results o b t a i n e d in t h e cytosolic fraction. Only t h e G S T activity was i n c r e a s e d ( + 26%, P < 0.05) in t h e $2 g r o u p as c o m p a r e d to t h e control. Postmitochondrial fraction. C o m p a r e d to control value, T B A R S p r o d u c t s w e r e i n c r e a s e d in
TABLE 3 EFFECT OF VITAMIN E DIETARY INTAKE ON LIPID PEROXIDATION AND AFB1 MUTAGENIC ACTIVATION IN POSTMITOCHONDRIAL ($9) FRACTIONS IN RATS Vitamin E diet (IU/g) Proteins(mg/g) TBARS (nmole/3 h/mg S9P) Mutagenesis (TA98 rev/ng AFB1)
0
0.05
0.5
5
92.5 +5.1 14.9 +0.8 * 1.37 + 0.2 * *
99.5 +3.5 12.4 +0.6 2.88 -l-0.5
96.4 +4.8 0.15+0.02 ** 2.6 + 0.3
94.3 +4 0.11+0.01 ** 2.13 + 0.6 *
Results are expressed as the mean + SD of three determinations. Significantly different from rats as compared to the 0.05 IU vitamin E group: * * P < 0.01, * P < 0.05.
313 HIS+ revertants TA98
80O
jl
j~
jJJ
i
600
°
jJJ
// / //
//
,
//
400
/
/
/,
//
/~///
2ooI I
//
A-"
J" ! I
0
20
40
60
100
80
120
ng AFB1 per plate
+0
UI
+005
UI
+05
UI
+5
uI
Fig. 1. Dose-response effect of vitamin E dietary intake on $9 dependent mutagenic activity of AFB1 in TA98. Each point represents the average value of three plates +SD. The spontaneous mutation rate (32) was subtracted from each point.
phimurium
Salmonella ty-
the vitamin E deficient group (+ 20%, P < 0.05) and strongly decreased in the supplemented groups ( - 9 9 % , P < 0.01) (Table 3).
Mutagenicity assay. We report in Fig. 1 the ability of $9 fractions from vitamin E treated rats to activate AFB1 in Ames test conditions. In order to assess the influence of vitamin E dietary intake, we have expressed the AFB1 mutagenic activity in revertants per ng of AFB1 calculated from the slope of the linear section of the doseresponse curve (Table 3). Vitamin E free diet and high dietary level of vitamin E significantly decreased the mutagenic activity as compared to control values ( - 18%, P < 0.01 and -47.3%, P < 0.05, respectively). Mutagenic activity is unaffected by diet supplemented with 0.5 IU of vitamin E. Discussion
The present data demonstrate that vitamin E dietary intake influences AFB1 mutagenicity esti-
mated by the Ames test. This effect seems to be related to the activities of hepatic enzymes involved in AFB1 metabolism. Vitamin E content in liver fraction is related to the a-tocopherol dietary intake in accordance with Sevanian et al. (1982) and Machlin (1983). Thus an a-tocopherol dose effect may be analyzed on microsomal and nuclear fractions. Nutritional factors have the capacity to alter the activity of hepatic microsomal drug oxidases (Murray, 1991). The modulation of P-450 enzyme expression implicates several mechanisms of action. Thus some nutrients which are substrates of P-450 enzymes in microsomal fraction are able to exert a competitive inhibition with EROD, PTROD (pentoxyresorufin O-dealkylase) and ERMD activities. Such an inhibitory effect was observed with retinol (Koch, 1991) and flavonoids (Siess et al., 1990) and can be partially explained by a direct interaction with the active site of cytochrome P-450. On the basis of literature reports, a-tocopherol may not be considered a P-450 enzyme inhibitor. This vitamin has never been reported as a substrate of cytochrome P-450 dependent enzymes (Csallany et al., 1962; Nelly et al., 1988). Murray (1991) has reported that vitamin E does not directly interact with the cytochrome P-450 at its active site. Nutrients may also directly or indirectly alter the microsomal membrane and thus modify microsomal enzyme functions. Moreover small changes in the microsomal concentration of P-450 specific isozymes may result in an increase in mRNA level. Such a nuclear induction has been reported with retinol. Vitamin E is accumulated in rat hepatic nuclei (Patnaik and Nair, 1977; Machlin, 1980). However this component does not seem to induce the synthesis of P-450 isozymes implicated in AFB1 metabolism. Only the content of P-450 isozyme I I C l l has been reported to increase with vitamin E dietary supplementation (Murray, 1991). Vitamin E has been shown to influence the microsomal environment by modifying the susceptibility to lipid peroxidation (Wefers and Sies, 1989; Tirmenteins and Reed, 1989; Graham et al., 1989). The concentration of TBARS has been found to be indicative of the ultimate potential for peroxidation of tissue lipids in vitro, as well as an estimate of in vivo malondialdehyde produc-
314
tion (Buckingham, 1985). Thus the inverse relationship between vitamin E liver concentration and TBARS production indicates that rats fed a vitamin E deficient diet strongly increase the level of hepatic lipid peroxides as compared to rats fed a vitamin E supplemented diet. These results are in conformity with others (Buckingham, 1985; De and Darad, 1988; Graham et al., 1989). Several authors have reported that lipid derived radicals may be responsible for protein fragmentation (Dean and Cheeseman, 1987; Thomas et al., 1989). Cytochrome P-450 appears to be one of the most sensitive enzymes to lipid peroxide damage (Hrycay and O'Brien, 1971; Mimnaugh et al., 1981). The results reported by Ando and Tappel (1985) and Mounier (1988) are in accordance with this observation. However, in this study, the vitamin E free diet group shows a change in P-450 content in vitamin E deficiency; to counteract the increased tissue peroxidizability, the enzymatic antioxidant defense system has been shown to be more active (Chen et al., 1980). Thus a decrease in the hepatic level of the antioxidant substrates vitamin A, GSH and vitamin C was observed. This phenomenom may protect the hemoprotein P-450 against lipid derived radical attack. However, peroxidation has been shown to alter the integrity and permeability of the microsomal membrane, which can modify enzyme function (H6gherg et al., 1973; Hruszkewycz et al., 1978; Ando and Tappel, 1985). Thus in absence of vitamin E, P-450 IIB and IIIA expression was decreased. In microsomes from rats supplemented with 0.5 IU of vitamin E (S1 group), the increased P-450 IIB and IIIA activities confirm that a-tocopherol is indispensable for a maximum activity of oxidative drug metabolism. However the weak induction of the subfamilies liB and IliA of cytochrome P-450 does not seem to modify the AFB1 mutagenic activation in Ames test. High doses of o~tocopherol in microsomal membrane have been shown to decrease activities of cytochrome P-450 dependent enzymes (Mounier, 1988). Thus the membrane structure and function seem to be altered by vitamin E accumulation. The $9 fraction used in the Ames test involves the activity of GST located in the cytosolic fraction and catalyzing the AFB1-GSH formation,
considered to be the dominant epoxide detoxication mechanism (O'Brien et al., 1983; Lotlikar et al., 1989). In agreement with Ando and Tappel (1985), the activity of epoxide conjugation to GSH is not modified under vitamin E free diet conditions. Thus the inhibitory effect of vitamin E deficiency on AFB1 genotoxic activity in the Ames test can be related to the modulation of the activities of P-450 enzymes. In the $2 group, the induction of AFB1 detoxication and metabolic activation reduces the ability of AFB1 to revert TA98. The mutagenic activity in the 5 IU diet decreases as compared to the control group. However the activities of cytochrome P-450 dependent enzymes between these two groups are not signicantly different. Thus a nuclear mechanism may be envisaged. The antimutagenic effect of vitamin E can also be related to its ability to influence AFB1-DNA adduct formation, a-Tocopherol can be transformed by reaction with singlet quenching oxygen into tocopheryl quinone, tocopherol hydroquinone and other polar metabolites which are able to react directly with DNA (Csallanay et al., 1962; Nelly et al., 1988; Wang et al., 1989). Thus chromatin binding of D-a-tocopherol metabolites may change DNA accessibility to xenobiotics and thus may inhibit intercalation of AFB1 epoxide on DNA. In conclusion, the results presented here suggest that the inhibitory effect of vitamin E deficiency on AFB1 genotoxicity is indirectly related to an antioxidant mechanism. However high dietary vitamin E intake suggests an important role of a-tocopherol on microsomal membrane structure.
Acknowledgement This investigation was supported by a grant from the Minist~re de la Recherche et de la Technologie.
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