- Email: [email protected]
Comparison of the effect of low- and high-dose dietary butylated hydroxytoluene on microsome-mediated aflatoxin B1-DNA binding
CANCER LETTERS
ELSEVIER
Cancer
Letters
114 (1997)
217-220
Comparison of the effect of low- and high-dose dietary butylated hydroxytoluene on microsome-mediated aflatoxin B 1-DNA binding A. Allameh* Department
of Biochemistry,
Faculty
of Medical
Sciences,
Tarbiat
Moafaress
University,
PO Box 14155-4838,
Tehran,
I.R. Iran
Abstract Butylated hydroxytoluene (BHT) is known to inhibit tumor formation due to several chemical carcinogens including aflatoxin Bl (AFBl). Pre-treatment of laboratory animals with high doses of BHT (0.75% for 15 days in diet) is associated with pathological effects. The mechanism of action of BHT against AFB 1 carcinogenesis is by induction of liver glutathione (GSH) S-transferases. As a result, the formation of AFB 1-DNA binding is effectively inhibited. In the present study, effects of low and high doses of dietary BHT on in vitro activation and inactivation of AFB 1 was examined. BHT feeding to rats at a dose of 0.75% for 15 days caused a significant increase in the GSH S-transferase activity. Addition of liver cytosolic fractions prepared from the rats pre-treated with high dose BHT to the cell free system caused a 48% inhibition in AFBl-DNA binding. In contrast, low dose BHT feeding (0.06% for 6 months) had little influence on GSH S-transferase activity. This was corroborated when addition of liver cytosol from low dose BHT-treated rats failed to inhibit microsome-mediated AFBlDNA binding as compared to that of control. It is concluded that the permitted dose of BHT, added to processed food as preservative, plays no role in the biotransformation of AFBI. 0 1997 Elsevier Science Ireland Ltd. Keywords:
Butylated hydroxytoluene; Aflatoxin B 1; Glutathione transferases; DNA adduct
1. Introduction A variety of naturally occurring compounds which are present in the human diet have been shown to inhibit experimental carcinogenesis [l]. Examples of such compounds are retinoids, which appear to act at the initiation stage, and selenium, phenolic antioxidants and ethoxyquin, which may act either at the initiation or promotion stages [2]. The exact mechanism of action of these compounds as anti-carcinogens is not well understood; however, it has been shown that they produce changes in mixed function oxidase and phase II drug metabolising enzymes [3]. * Tel.: +98 21 8007830;
fax: +98 21 8006544.
0304-3835/97/$17.00 0 1997 Elsevier PII SO304-3835(97)04667-3
Science
Ireland
Butylated hydroxytoluene (BHT) is one of the several antioxidants commonly used as a preservative in processed food. The effective dose of BHT is known to be between 0.5 and 0.75% for a period of 1-2 weeks. BHT inhibits the carcinogenicity and mutagenicity of many chemicals including aflatoxin Bl (AFBl) in various laboratory animals [4,5]. Dietary BHT inhibits the binding of carcinogens to cellular macromolecules, particularly DNA [6]. AFB 1 conjugate formation to liver GSH, catalysed by various hepatic cytosolic GSH S-transferases, is one of the detoxification pathways of the ultimate carcinogen, AFB 1-epoxide. Cytosolic GSH S-transferases play an important role in the inhibition of AFB 1-DNA binding, in vitro and in vivo [7]. High-dose BHT treatment, although effective in
Ltd. All rights reserved
218 Table
1
Effect of dietary BHT dose (c/c)
BHT on body weight Duration (days)
0.75
1.5
0.06
180
and tissue weight
of rata
Group
-_-
Control Experimental Control Experimental
_.-_-.-__l--
Body weight .--_l__l-..____Initial
Final
148 157 142 136
I78 136 399 283
2 X.0 f 0.9 i 9.1, IS.0
_-
--.-__---.-l-._-
Lt t ” It
X.0 LF 29 18
_._._-
2.9 3.9 3.0 3. I
+ i. rt 5
0. I 0.Y 0.2 0.08 --..
-_.-.--__“.
Results are expressed as mean t SEM of four analyses m each group *P < 0.5 is considered significant
modulation of AFBI-DNA adduct formation, can cause pathological effects in different organs ] X,9]. In this study an attempt was made to examine the influence of low-dose dietary BHT, i.e 0.06% fed for a period of 6 months to rats on microsomemediated AFB 1-DNA adduct formation and related enzymes.
2. Materials
solved in 0.02 ml dimethyl sulphoxide, microsomes equivalent to I .O mg protein, and 0.1 mg calf thymus DNA. After incubation 2 ml of phenol mixture were added along with 0.9 mg calf thymus DNA as a carriet to each tube. DNA was extracted and measured as described earlier /lo]. Finally, the radioactivity bound to DNA was determined by liquid scintillation counting.
and methods 3. Results
2.1. Animul treatment
Male albino Wistar rats were used for all experiments. One group was fed a basal diet supplemented with 0.75% BHT for 2 weeks and the other was fed a diet containing 0.06% BHT for 6 months. Two groups of rats served as controls and were fed only the basal diet. Each experimental group consisted of four animals. Liver microsomal and cytosolic fractions were prepared as described previously [ 1O]. Protein was measured by Lowry’s method with bovine serum albumin as standard [ 111. Microsomal cytochrome P-450 levels were determined spectrophotometrically [ 121. GSH S-transferase activity was assayed by the method of Habig et al. [ 131. DNA was measured colorimetritally by the diphenylamine reaction 1141.
There was no difference in the growth of rats fed the basal diet or the supplemented diets. There was a significant (34.4%) increase in liver weight in the high-dose BHT group as compared to low-dose and control animals (Table 1j. The microsomal cytochrome P-450 content remained unchanged due to both low and high dose BHT-treatment (Table 2). This was further confirmed I‘able 2 Effect of dietary enzyme activities.
BHT
on rat liver
BHT
Duration
Group
dOSC
(days)
Cyfochrome P-450 (nmoh mg protein)
~GSH S-transFeraae (units/ mg protein)
Cuntrol Experimental Control Experimental
0.78 0.65 0.56 0.42
1096 It 46 1659 t 72” 1207+75 1272 i: 99
f(h) 0.75
! .5
microsomat
f 0.05 + 0.05 zk 0.08 f. 0.10
and cytosolic
2.2. Microsome mediatedAFBl -DNA binding
lml
Incubations were carried out for 30 min in 0.1 M phosphate buffer (pH = 7.4) at 37’C and contained in a total volume of 1.O ml 2 mM NADPH, 2 nmol AFB 1 containing [3H]AFB1 (spec. act. 300 gCi/Fmol) dis-
The results are expressed as mean zk SEM of four determinations. Units of enzyme activity are expressed as nmol CDNB conjugate! min/mg protein. “1’ c: 0.5 is considered significant.
I 80
A. Allameh
/Cancer
when microsomes were used in the AFBl metabolic activation system in the presence of calf thymus DNA (Table 3). BHT intake at a dose of 0.75% for 2 weeks significantly induced the cytosolic GSH S-transferase activity. However, low dose BHT pre-treatment (0.06% for Gmonths) failed to increase the activity of this enzyme (Table 2). Addition of the cytosolic fraction prepared from control- and BHT-treated rats caused a differential inhibition of AFB 1-DNA adduct formation. Addition of liver cytosol from high dose BHT-treated rats to the incubation system inhibited the binding by about 48%, as compared to that of control- and low-dose BHT-treated animals (Table 4).
4. Discussion AFBl is metabolised to a number of metabolites including the highly reactive 8,9-epoxide. AFB lepoxide binding to DNA, RNA and/or proteins is essential for the mutagenic and carcinogenic effects of the toxin [ 15,161. A major detoxification pathway for AFBl is via conjugation of its epoxide with cellular GSH [17]. AFB l-DNA binding is largely controlled by the activities of liver GSH S-transferase and indeed inversely correlated with the formation of AFBl-GSH conjugate formation [ 18,191. AFBl-GSH conjugation is achieved upon addition of hepatic cytosolic fraction and GSH to the in vitro cytochrome P-450dependent AFB 1 metabolic activation system. Dietary BHT and BHA (butylated hydroxyanisole) are usually effective in both cytosolic GSH S-transferase induction and inhibition of AFBl-DNA bindTable 3 Influence of BHT on the microsome thymus DNA BHT dose (%) 0.75 0.06%
Duration (days)
mediated
AFB I binding
Group
r3H]AFB l-DNA (pm01 AFB l/mg
Control Experimental Control Experimental
261 308 276 278
to calf
Letters
114
(1997)
219
217-220
Table 4 Cytosolic
inhibition
of AFBl
Microsome
Cytosol
Control Control Control
BHT (high) BHT (low)
binding
GSH (5 mM) + +
to calf thymus
DNA
[3H]AFBIDNA (pmol)
Binding (% of control)
21 267 139 249
7.8 100 52 93
+6 + 14 If: 8 f 11
Incubation medium and other experimental details are as described in Section 2. Where indicated, samples contained liver microsomes equivalent to 1 mg protein cytosol equivalent to 30 mg fresh tissue from either control or BHT low- or high dose-pretreated rats. AFBl-DNA binding in the absence of cytosol is considered to be 100%. Data am expressed as [3H]AFBl bound per mg DNA per 30 min. Results are mean + SEM of three duplicate determinations.
ing at a very high dose (OS-0.75% in diet for a period of l-2 weeks). This dose is associated with a significant increase in liver weight (Table l), together with other pathological effect [8,9]. In this study the effect of a low dose of dietary BHT on the enzyme system controlling AFB 1-DNA binding was compared with the conventional high dose. Hepatic microsomal cytochrome P-450 was unaffected by low-dose BHT pretreatment of rats (Table 2). Similarly, the level of hepatic GSH S-transferase activity remained unchanged due to low-dose BHT intake. Induced hepatic cytosolic GSH S-transferase activity in rats fed a diet containing 0.75% BHT, showed that only high doses of dietary antioxidants influence the biotransformation of AFB 1. The human daily intake of BHT is 0.6 mg/kg body weight [ 161 which is at most only about 1% of the low dose used in our experiments. It is concluded that the permitted dose of BHT added to processed food as preservative plays no role in the metabolic activation of AFBl.
binding DNA)
References 15 180
zk f f +
21 23 8.9 12
Incubation medium and other experimental details are as described in Section 2. Results are expressed as mean + SEM of four analysis carried out with duplicates.
[I]
Wattenberg, L.W. (1980) Inhibition of chemical carcinogenesis by antioxidants. In: Carcinogenesis, Vol.5, Modification of Chemical Carcinogens, p. 85. Editor: T.J. Slaga. Raven Press, New York. [2] Williams, G.M., Tanaka, T. and Maeura, Y. (1986) Dose related inhibition of aflatoxin Bl induced hepatocarcinogenesis by the phenolic antioxidants,butylated hydroxyanisole
[3]
[S)
]5]
[6]
171
[X]
[9]
1lo]
and butylated hydroxytolucnc. Carcinogen&a. ‘7. 104310.50. Benson. A.M., Cha, Y.N., Bueding, E., Heine, H.S. and Talalay, P. (1970) Elevation of extrahepatic glutathione S-transferase and epoxide hydratase activities by 2(3)-t-butyl-4hydroxyanisole. Fed. hoc.. 38, 506. Fukayama, M.Y. and Hsieh. D.P.H. (1984) The effect 01 butylated hydroxytoluene on the in viva metabolism, DNA binding and mutagenesis of aflatoxin Bl in the rat. Food Chum. Toxicol., 22. 335-360. Salocks, C.B., Hsieh, D.P.H. and Byard. J.L. (1981) Butylated hydroxytoluene pretreatment protect against cytotoxicity and reduces covalent binding of aflatoxin Bl in primary hepatocyte cul~urcs. Toxicol. Appl. Pharmacol., 59, 331-34s. Kensler, T.W., Egner, P.A.. Davidson, NE., Roebuck, B.D., Pikul, A. and Groopman, J.D. (1986) Modulation of aflatoxin metabolism, aflatoxin-N7-guanine formation and hepatic tumorigenesis in rats fed ethoxyquine: role of induction of glutathione S-transferases. Cancer Res., 46. 3924-393 1. Lotlikar, P.D., Clearheld, MS. and Jhee, EC. (1984) Effect of butylated hydroxyanisole on in vivo and in vitro hepatic aflatoxin Bl-DNA binding in rats. Cancer Lett., 24. 241250. Takahashi, 0, and Hiraga, K. ( 1978) Dose-responce study 01 hemorrhagic death by dietary butylated hydroxytoluene in male rats. Toxicol. Appl. Pharmacol., 43, 134- 136. Takahashi, 0. and Hiraga. K. (1978) The relationship between hemorrhage induced by butylated hydroxytoluene and its antioxidants properties or structural characteristics. Toxicol. Appl. Pharmacol., 46, 811-814. Allameh, A., Saxena, M. and Raj, H.G. (1988) Differential effects of butylated hydroxyanisole on metabolism of afla-
11 I]
1121
1131
] 141
IiSl
1161
] 171
[ 181
1191
toxm Bl m vitro by liver and lung microsomes. Cancer Lett.. 40, 49-57. Lowry, O.H.. Rosebrough, N.J., Farr, A.L. and Randall, R.J. ( 19.5 1) Protein measurement with the Folin phenol reagent. J. Biol. Chcm., 193, 26.5-275. Omura. T. and Sato, R. (1964) The carbon monoxide-binding pigment of liver microsomes. I. Evidence for its hemoprotein. .I. Biol. Chem., 239, 2370-2378. Habig, W.H., Pabst, M.J. and Jakoby, W.B. (1974) Giutathione S-transferases. The first enzymatic step in mercapturic acid formation. J. Bid. Chem., 7130-7139. Burton, K. (1956) A study of the conditions and mechanisms of the diphenylamine reaction for the colotimetric estimation of deoxyribonucleic acid. B&hem. J., 62, 315-323. Swenson, D.H.. Lin. J.-K., Miller, EC. and Miller, J.A. (1977) Aflatoxin B I-2,3-oxide as a probable intermediate in the covalent binding of aflatoxin B 1 and 82 to rat livei DNA and ribosomal RNA in viva. Cancer Res., 37, 172 -1X I. Essigman, J.M., Green, CL., Croy. R.G., Fowler, K.W. Buchi, G.H. and Wogan, G.N. (1983) Interactions of aflatoxin Bl and alkylating agents with DNA: structural and functional studies. Cold Spring Harbor Quant. Biol., 47, 327-337. Degen, G.H. and Newmann, H.G. (1981) Differences in aliatoxin susceptibility of rat and mouse are correlated with the capability in vitro to inactivate aflatoxin Bl-epoxide. Carcinogenesis, 2, 299-306. Raj, H.G., Clearfield, M.S. and Lotlikar, PD. (1984) Comparativc kinetic studies on afiatoxin Bl-DNA binding on allatoxin Bl--glutathione conjugation with rat and hamster livers in vitro. Carcinogenesis, 5, 879-884. FAO/WHO Expert Committee on Food Additives (1980) WHO Food Additive Series. No. 15, 106 pp.
ELSEVIER
Cancer
Letters
114 (1997)
217-220
Comparison of the effect of low- and high-dose dietary butylated hydroxytoluene on microsome-mediated aflatoxin B 1-DNA binding A. Allameh* Department
of Biochemistry,
Faculty
of Medical
Sciences,
Tarbiat
Moafaress
University,
PO Box 14155-4838,
Tehran,
I.R. Iran
Abstract Butylated hydroxytoluene (BHT) is known to inhibit tumor formation due to several chemical carcinogens including aflatoxin Bl (AFBl). Pre-treatment of laboratory animals with high doses of BHT (0.75% for 15 days in diet) is associated with pathological effects. The mechanism of action of BHT against AFB 1 carcinogenesis is by induction of liver glutathione (GSH) S-transferases. As a result, the formation of AFB 1-DNA binding is effectively inhibited. In the present study, effects of low and high doses of dietary BHT on in vitro activation and inactivation of AFB 1 was examined. BHT feeding to rats at a dose of 0.75% for 15 days caused a significant increase in the GSH S-transferase activity. Addition of liver cytosolic fractions prepared from the rats pre-treated with high dose BHT to the cell free system caused a 48% inhibition in AFBl-DNA binding. In contrast, low dose BHT feeding (0.06% for 6 months) had little influence on GSH S-transferase activity. This was corroborated when addition of liver cytosol from low dose BHT-treated rats failed to inhibit microsome-mediated AFBlDNA binding as compared to that of control. It is concluded that the permitted dose of BHT, added to processed food as preservative, plays no role in the biotransformation of AFBI. 0 1997 Elsevier Science Ireland Ltd. Keywords:
Butylated hydroxytoluene; Aflatoxin B 1; Glutathione transferases; DNA adduct
1. Introduction A variety of naturally occurring compounds which are present in the human diet have been shown to inhibit experimental carcinogenesis [l]. Examples of such compounds are retinoids, which appear to act at the initiation stage, and selenium, phenolic antioxidants and ethoxyquin, which may act either at the initiation or promotion stages [2]. The exact mechanism of action of these compounds as anti-carcinogens is not well understood; however, it has been shown that they produce changes in mixed function oxidase and phase II drug metabolising enzymes [3]. * Tel.: +98 21 8007830;
fax: +98 21 8006544.
0304-3835/97/$17.00 0 1997 Elsevier PII SO304-3835(97)04667-3
Science
Ireland
Butylated hydroxytoluene (BHT) is one of the several antioxidants commonly used as a preservative in processed food. The effective dose of BHT is known to be between 0.5 and 0.75% for a period of 1-2 weeks. BHT inhibits the carcinogenicity and mutagenicity of many chemicals including aflatoxin Bl (AFBl) in various laboratory animals [4,5]. Dietary BHT inhibits the binding of carcinogens to cellular macromolecules, particularly DNA [6]. AFB 1 conjugate formation to liver GSH, catalysed by various hepatic cytosolic GSH S-transferases, is one of the detoxification pathways of the ultimate carcinogen, AFB 1-epoxide. Cytosolic GSH S-transferases play an important role in the inhibition of AFB 1-DNA binding, in vitro and in vivo [7]. High-dose BHT treatment, although effective in
Ltd. All rights reserved
218 Table
1
Effect of dietary BHT dose (c/c)
BHT on body weight Duration (days)
0.75
1.5
0.06
180
and tissue weight
of rata
Group
-_-
Control Experimental Control Experimental
_.-_-.-__l--
Body weight .--_l__l-..____Initial
Final
148 157 142 136
I78 136 399 283
2 X.0 f 0.9 i 9.1, IS.0
_-
--.-__---.-l-._-
Lt t ” It
X.0 LF 29 18
_._._-
2.9 3.9 3.0 3. I
+ i. rt 5
0. I 0.Y 0.2 0.08 --..
-_.-.--__“.
Results are expressed as mean t SEM of four analyses m each group *P < 0.5 is considered significant
modulation of AFBI-DNA adduct formation, can cause pathological effects in different organs ] X,9]. In this study an attempt was made to examine the influence of low-dose dietary BHT, i.e 0.06% fed for a period of 6 months to rats on microsomemediated AFB 1-DNA adduct formation and related enzymes.
2. Materials
solved in 0.02 ml dimethyl sulphoxide, microsomes equivalent to I .O mg protein, and 0.1 mg calf thymus DNA. After incubation 2 ml of phenol mixture were added along with 0.9 mg calf thymus DNA as a carriet to each tube. DNA was extracted and measured as described earlier /lo]. Finally, the radioactivity bound to DNA was determined by liquid scintillation counting.
and methods 3. Results
2.1. Animul treatment
Male albino Wistar rats were used for all experiments. One group was fed a basal diet supplemented with 0.75% BHT for 2 weeks and the other was fed a diet containing 0.06% BHT for 6 months. Two groups of rats served as controls and were fed only the basal diet. Each experimental group consisted of four animals. Liver microsomal and cytosolic fractions were prepared as described previously [ 1O]. Protein was measured by Lowry’s method with bovine serum albumin as standard [ 111. Microsomal cytochrome P-450 levels were determined spectrophotometrically [ 121. GSH S-transferase activity was assayed by the method of Habig et al. [ 131. DNA was measured colorimetritally by the diphenylamine reaction 1141.
There was no difference in the growth of rats fed the basal diet or the supplemented diets. There was a significant (34.4%) increase in liver weight in the high-dose BHT group as compared to low-dose and control animals (Table 1j. The microsomal cytochrome P-450 content remained unchanged due to both low and high dose BHT-treatment (Table 2). This was further confirmed I‘able 2 Effect of dietary enzyme activities.
BHT
on rat liver
BHT
Duration
Group
dOSC
(days)
Cyfochrome P-450 (nmoh mg protein)
~GSH S-transFeraae (units/ mg protein)
Cuntrol Experimental Control Experimental
0.78 0.65 0.56 0.42
1096 It 46 1659 t 72” 1207+75 1272 i: 99
f(h) 0.75
! .5
microsomat
f 0.05 + 0.05 zk 0.08 f. 0.10
and cytosolic
2.2. Microsome mediatedAFBl -DNA binding
lml
Incubations were carried out for 30 min in 0.1 M phosphate buffer (pH = 7.4) at 37’C and contained in a total volume of 1.O ml 2 mM NADPH, 2 nmol AFB 1 containing [3H]AFB1 (spec. act. 300 gCi/Fmol) dis-
The results are expressed as mean zk SEM of four determinations. Units of enzyme activity are expressed as nmol CDNB conjugate! min/mg protein. “1’ c: 0.5 is considered significant.
I 80
A. Allameh
/Cancer
when microsomes were used in the AFBl metabolic activation system in the presence of calf thymus DNA (Table 3). BHT intake at a dose of 0.75% for 2 weeks significantly induced the cytosolic GSH S-transferase activity. However, low dose BHT pre-treatment (0.06% for Gmonths) failed to increase the activity of this enzyme (Table 2). Addition of the cytosolic fraction prepared from control- and BHT-treated rats caused a differential inhibition of AFB 1-DNA adduct formation. Addition of liver cytosol from high dose BHT-treated rats to the incubation system inhibited the binding by about 48%, as compared to that of control- and low-dose BHT-treated animals (Table 4).
4. Discussion AFBl is metabolised to a number of metabolites including the highly reactive 8,9-epoxide. AFB lepoxide binding to DNA, RNA and/or proteins is essential for the mutagenic and carcinogenic effects of the toxin [ 15,161. A major detoxification pathway for AFBl is via conjugation of its epoxide with cellular GSH [17]. AFB l-DNA binding is largely controlled by the activities of liver GSH S-transferase and indeed inversely correlated with the formation of AFBl-GSH conjugate formation [ 18,191. AFBl-GSH conjugation is achieved upon addition of hepatic cytosolic fraction and GSH to the in vitro cytochrome P-450dependent AFB 1 metabolic activation system. Dietary BHT and BHA (butylated hydroxyanisole) are usually effective in both cytosolic GSH S-transferase induction and inhibition of AFBl-DNA bindTable 3 Influence of BHT on the microsome thymus DNA BHT dose (%) 0.75 0.06%
Duration (days)
mediated
AFB I binding
Group
r3H]AFB l-DNA (pm01 AFB l/mg
Control Experimental Control Experimental
261 308 276 278
to calf
Letters
114
(1997)
219
217-220
Table 4 Cytosolic
inhibition
of AFBl
Microsome
Cytosol
Control Control Control
BHT (high) BHT (low)
binding
GSH (5 mM) + +
to calf thymus
DNA
[3H]AFBIDNA (pmol)
Binding (% of control)
21 267 139 249
7.8 100 52 93
+6 + 14 If: 8 f 11
Incubation medium and other experimental details are as described in Section 2. Where indicated, samples contained liver microsomes equivalent to 1 mg protein cytosol equivalent to 30 mg fresh tissue from either control or BHT low- or high dose-pretreated rats. AFBl-DNA binding in the absence of cytosol is considered to be 100%. Data am expressed as [3H]AFBl bound per mg DNA per 30 min. Results are mean + SEM of three duplicate determinations.
ing at a very high dose (OS-0.75% in diet for a period of l-2 weeks). This dose is associated with a significant increase in liver weight (Table l), together with other pathological effect [8,9]. In this study the effect of a low dose of dietary BHT on the enzyme system controlling AFB 1-DNA binding was compared with the conventional high dose. Hepatic microsomal cytochrome P-450 was unaffected by low-dose BHT pretreatment of rats (Table 2). Similarly, the level of hepatic GSH S-transferase activity remained unchanged due to low-dose BHT intake. Induced hepatic cytosolic GSH S-transferase activity in rats fed a diet containing 0.75% BHT, showed that only high doses of dietary antioxidants influence the biotransformation of AFB 1. The human daily intake of BHT is 0.6 mg/kg body weight [ 161 which is at most only about 1% of the low dose used in our experiments. It is concluded that the permitted dose of BHT added to processed food as preservative plays no role in the metabolic activation of AFBl.
binding DNA)
References 15 180
zk f f +
21 23 8.9 12
Incubation medium and other experimental details are as described in Section 2. Results are expressed as mean + SEM of four analysis carried out with duplicates.
[I]
Wattenberg, L.W. (1980) Inhibition of chemical carcinogenesis by antioxidants. In: Carcinogenesis, Vol.5, Modification of Chemical Carcinogens, p. 85. Editor: T.J. Slaga. Raven Press, New York. [2] Williams, G.M., Tanaka, T. and Maeura, Y. (1986) Dose related inhibition of aflatoxin Bl induced hepatocarcinogenesis by the phenolic antioxidants,butylated hydroxyanisole
[3]
[S)
]5]
[6]
171
[X]
[9]
1lo]
and butylated hydroxytolucnc. Carcinogen&a. ‘7. 104310.50. Benson. A.M., Cha, Y.N., Bueding, E., Heine, H.S. and Talalay, P. (1970) Elevation of extrahepatic glutathione S-transferase and epoxide hydratase activities by 2(3)-t-butyl-4hydroxyanisole. Fed. hoc.. 38, 506. Fukayama, M.Y. and Hsieh. D.P.H. (1984) The effect 01 butylated hydroxytoluene on the in viva metabolism, DNA binding and mutagenesis of aflatoxin Bl in the rat. Food Chum. Toxicol., 22. 335-360. Salocks, C.B., Hsieh, D.P.H. and Byard. J.L. (1981) Butylated hydroxytoluene pretreatment protect against cytotoxicity and reduces covalent binding of aflatoxin Bl in primary hepatocyte cul~urcs. Toxicol. Appl. Pharmacol., 59, 331-34s. Kensler, T.W., Egner, P.A.. Davidson, NE., Roebuck, B.D., Pikul, A. and Groopman, J.D. (1986) Modulation of aflatoxin metabolism, aflatoxin-N7-guanine formation and hepatic tumorigenesis in rats fed ethoxyquine: role of induction of glutathione S-transferases. Cancer Res., 46. 3924-393 1. Lotlikar, P.D., Clearheld, MS. and Jhee, EC. (1984) Effect of butylated hydroxyanisole on in vivo and in vitro hepatic aflatoxin Bl-DNA binding in rats. Cancer Lett., 24. 241250. Takahashi, 0, and Hiraga, K. ( 1978) Dose-responce study 01 hemorrhagic death by dietary butylated hydroxytoluene in male rats. Toxicol. Appl. Pharmacol., 43, 134- 136. Takahashi, 0. and Hiraga. K. (1978) The relationship between hemorrhage induced by butylated hydroxytoluene and its antioxidants properties or structural characteristics. Toxicol. Appl. Pharmacol., 46, 811-814. Allameh, A., Saxena, M. and Raj, H.G. (1988) Differential effects of butylated hydroxyanisole on metabolism of afla-
11 I]
1121
1131
] 141
IiSl
1161
] 171
[ 181
1191
toxm Bl m vitro by liver and lung microsomes. Cancer Lett.. 40, 49-57. Lowry, O.H.. Rosebrough, N.J., Farr, A.L. and Randall, R.J. ( 19.5 1) Protein measurement with the Folin phenol reagent. J. Biol. Chcm., 193, 26.5-275. Omura. T. and Sato, R. (1964) The carbon monoxide-binding pigment of liver microsomes. I. Evidence for its hemoprotein. .I. Biol. Chem., 239, 2370-2378. Habig, W.H., Pabst, M.J. and Jakoby, W.B. (1974) Giutathione S-transferases. The first enzymatic step in mercapturic acid formation. J. Bid. Chem., 7130-7139. Burton, K. (1956) A study of the conditions and mechanisms of the diphenylamine reaction for the colotimetric estimation of deoxyribonucleic acid. B&hem. J., 62, 315-323. Swenson, D.H.. Lin. J.-K., Miller, EC. and Miller, J.A. (1977) Aflatoxin B I-2,3-oxide as a probable intermediate in the covalent binding of aflatoxin B 1 and 82 to rat livei DNA and ribosomal RNA in viva. Cancer Res., 37, 172 -1X I. Essigman, J.M., Green, CL., Croy. R.G., Fowler, K.W. Buchi, G.H. and Wogan, G.N. (1983) Interactions of aflatoxin Bl and alkylating agents with DNA: structural and functional studies. Cold Spring Harbor Quant. Biol., 47, 327-337. Degen, G.H. and Newmann, H.G. (1981) Differences in aliatoxin susceptibility of rat and mouse are correlated with the capability in vitro to inactivate aflatoxin Bl-epoxide. Carcinogenesis, 2, 299-306. Raj, H.G., Clearfield, M.S. and Lotlikar, PD. (1984) Comparativc kinetic studies on afiatoxin Bl-DNA binding on allatoxin Bl--glutathione conjugation with rat and hamster livers in vitro. Carcinogenesis, 5, 879-884. FAO/WHO Expert Committee on Food Additives (1980) WHO Food Additive Series. No. 15, 106 pp.