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Inhibition of binding of 2-acetylaminofluorene to DNA by butylated hydroxytoluene and butylated hydroxyanisole in vitro
Cancer Letters, 47 (1.989) 211-216 Elsevier Scientific Publishers Ireland
Inhibition butylated vitro N. Richer,
of binding of Z-acetylaminofluorene to DNA by hydroxytoluene and butylated hydroxyanisole in
M. Marion
Department
211 Ltd.
of Chemistry,
and Universitk
F. Denizeau’ du Qukbec ii Monkhal,
Mont&l
(Canada)
(Received 10 June 1988) (Revision received 25 May 1989) (Accepted 26 May 1989)
Summary
with
DNA which may reflect on alteration
carcinogen Numerous antioxidants
studies have shown that the food butylated hydroxytoluene (BHT)
and butylated hydroxyanisole (BHA), specific exposure conditions, can inhibit tocarcinogenesis
induced
Keywords: 2-acetylaminofluorine; butylated hydroxyanisole; butylated hydroxytoluene; anticarcinogenic properties; liver.
under hepavarious
by
in
metabolic activation.
carcinogens. The purpose of the present work was to study the biochemical mechanisms
Introduction
responsible
Phenolic antioxidants are widely used as food additives to prevent the development of rancidity. Human consumption of compounds such as BHA and BHT has been estimated. For example, in Canada the daily intake would be of the order of 0.4 mg/kg of body wt. [ 161. Numerous studies have shown that BHA and BHT can modify the response to various carcinogens. Depending on the target organ and on exposure conditions, inhibitory as well as stimulatory effects have been observed [15,17]. In the liver, it was found that BHT, fed together with the experimental carcinogen 2-AAF, inhibited carcinogenicity [ 191. Aflatoxin B,-induced hepatocarcinogenesis was inhibited by BHA and BHT when these were present in the diet 1 week before carcinogen, during administration and 1 week after cessation [25]. Moreover, BHA and BHT reduced the development of y-glutamyl-transpeptidasepositive foci initiated in the liver by diethylnitrosamine [ 151.
for
the anticarcinogenic
actioity of
BHA and BHT using in uitro systems. The effects of BHA and BHT on the binding of Zacetylaminofluorene (Z-AAF) to DNA was determined
in
a microsomd
system
and
in
primary cultures of rat hepatocytes. It was found that both antioxidants reduce the binding of
and that of N-OH-Z-acetylami-
ZAAF
nofluorene
(N-OH-2-AAF)
to
caij
thymus
DNA in the presence of liver microsomes. inhibition
was h,owever more pronounced
the parent
binding
The with
compound.
Lower levels of DNA also detected in hepatocytes with 2-AAF along with BHA or
were
incubated
These results suggest that phenolic antioxidants can exert anticarcinogenic activity
BHT.
through
modulation
‘Correspondence Mont&al, C.P. Canada.
of carcinogen
interaction
to: F. Denizeau, Universitk du Qu&ec a 8888, Succ. A Mont&al, Quhbec H3C 3P8,
0 1989 Elsevier Scientific Publishers 0304-3835/89/$03.50 Published and Printed in Ireland
Ireland Ltd.
212
Formation of carcinogen-DNA adducts has also been followed upon treatment with antioxidants: addition of BHA to the diet inhibited the binding of benzo[a]pyrene to liver DNA in vivo [ 11; similarly, BHT feeding was shown to reduce the binding of 2-AAF to hepatic DNA [12]. In primary cultures of rat hepatocytes, selective reduction of nucleic acid binding of aflatoxin B, was seen when pretreatment with BHT was applied in vivo. More recently, the reduction of 2-AAF-induced DNA repair in human and rat hepatocytes by BHT has also been observed 151. Changes in the activity of enzymes responsible for xenobiotic metabolism have been proposed to be involved in the anticarcinogenic effects of antioxidants. Indeed, induction of the detoxifying enzymes glutathione S-transferase [2,9], epoxide hydratase and UDP-glucuronyltransferase [4], has been detected after in vivo exposure to BHA. Despite the numerous investigations on anticarcinogenic activity of antioxidants in the liver, the mechanisms underlying this phenomenon are still not fully understood. In an effort to extend the previous studies, the purpose of the present work was to investigate the anticarcinogenic properties of BHA and BHT towards the hepatocarcinogen (2-AAF) in in vitro systems. The effects of BHA and BHT on the binding of 2-AAF to DNA was determined in a microsomal system and in primary cultures of rat hepatocytes. Materials and methods Microsome-mediated binding of [3H]2-AAF and [3H]N-OH-2-AAF was performed using a procedure similar to that of Speier and Wattenberg [24]. The microsomes were prepared according to the method of Dallner [7] from livers of male SpragueDawley rats weighing 150-300 g. Each sample was made up of the following: 4 mg calf thymus DNA, 4 FM NADPH, 3.5 mg microsomal proteins and 3.5 VM sodium citrate buffer, pH 7.4 in a final volume of 6 ml. BHA, BHT, [3H]2-AAF and [3H]N-OH-2-AAF (312-341 mCi/mmol;
Chemsyn Science Laboratories) were dissolved in dimethyl sulfoxide (DMS) at concentrations of 1 M, 118 pCi/ml and 148 pCi/ml respectively. The reaction was initiated by adding the antioxidant and the radioactive substrate (final concentration: 2 &i/ml). Control incubations were carried out without the antioxidant. Non-specific binding was determined using samples in which heat-inactivated microsomal preparations were employed. After incubation at 37OC for 60 min, the reaction was stopped by placing the samples at - 40°C for 20 s. DNA was extracted with chloroform: isoamyl alcohol (24 : 1) and purified according to the method of Muller and Rajewsky [20] using hydroxyapatite (DNA-grade Bio-gel HTP). The amount of DNA was determined by means of the thiobarbituric acid assay [ll] and its radioactivity content was measured by liquid scintillation counting. Primary cultures of rat hepatocytes were prepared as we previously reported [8]. Male Sprague-Dawley rats weighing 150-300 g were used. The hepatocytes were isolated according to the technique described by Seglen 1231 with slight modifications [8]. Viable cells (4.5 x 106) were seeded in each 60-mm petri dish. The culture medium consisted of WME supplemented with 10% FBS, 100 units/ml penicillin and 100 pg/ml streptomycin (WME-10% FBS). The cells were allowed to attach for 2 h at 37OC, in an atmosphere of 95% air, 5% CO,, saturated with humidity. The incubation with the carcinogen was then initiated by adding fresh WME1% FBS containing [3H]2-AAF (4 $Zi/ml), with or without the antioxidant. After a 20-h exposure period at 37OC, the DNA was purified by a procedure inspired from those of Maniatis et al. [19] and Christie et al. [6]. It consisted of the following steps: the cells were suspended in Hank’s balanced salt solution, washed by centrifugation and lysed with 20 mM Tris-HCl buffer pH 8.0, containing 150 mM NaCl, 1% SDS and 50 pg/ml proteinase K. The samples were placed on a mobile plate and subjected to gentle agitation
213
(35 inversions/min) for 3 h at 37OC. Then, extractions were carried out with phenol-Tris, pH 7.9 and chloroform/isoamyl alcohol (24 : 1) ; the aqueous phase was re-extracted with chloroform and its DNA was precipitated with cold absolute ethanol. The samples were kept overnight .at - 20°C, the DNA pellets were washed wiith 70% ethanol and dissolved in buffer (10 mM Tris, pH 7.9). The quantitation was performed by measuring the absorbance at 260 nm. The ratios of absorbance AZi0 JA280 nm’ which provide an estimation of the purity of the material, were in the region of 1.80 & 0.5; this indicates the absence of significant protein contamination [13]. Aliquots of the same samples were used for radioactivity measurements. Results Figure 1 shows the effect of BHA and BHT on the binding (of [3H]2-AAF to DNA in the microsomal system. A significant inhibition of adduct formation, which becomes quite pronounced (> 75%) at the highest antioxidant levels, can be observed. Similarly, Fig. 2 presents the binding profiles obtained with N-OH2-AAF, a 2-AAF metabolite known to be
formed by liver microsomal P-450 mixedfunction oxidase. It can be seen that the binding of [3H]N-OH-2-AAF was lower in samples to which BHA or BHT was added; however, the effect was less marked than with the parent compound. The next series of experiments was aimed at testing whether the inhibition of the binding of 2-AAF to the DNA would be observed in a complete cellular system. Freshly isolated hepatocytes were exposed to [3H]2-AAF and to BHA or BHT for 20 h. The results are presented in Fig. 3. It is interesting to note that adduct formation is again lower in BHA- or BHT-treated samples. In this case however, it was not possible to obtain a more extended inhibition profile because maximum antioxidant concentration was limited by cytotoxicity. Indeed, at levels of BHA and BHT higher than those indicated, the release of lactate dehydrogenase was detectable and there was some loss of cell viability. Discussion The results of the microsome-mediated assay, as well as those obtained with freshly isolated hepatocytes, show that binding of
r ._ m
0
10
Antioxidant concentration (mM) Inhibition of [3H]2-AAF-DNA binding by BHA and BHT in the microsomal assay. The data represent means f S.E. for 3 experiments. The level of binding in untreated controls was 13.8 f 1.3 DPM/pg DNA. The values shown were corrected for non-specific binding.
Fig.1.
80 60
Antioxidant
concentration
(mM)
Inhibition of [3H]N-OH-2-AAF-DNA binding by BHA and BHT in the microsomal assay. The data represent Fig. 2. means f S.E. for 3 experiments. The level of binding in untreated controls was 45.2 + 4.5 DPM/pg DNA. The values shown were corrected for non-specific binding.
n
Control
5
BHA BHT
q
El
90
70
Antioxidant Fig. 3. means + DPM/pg untreated
concentration
(PM)
Inhibition of [3H]2-AAF binding to the DNA of rat hepatocytes exposed to BHA and BHT. The data represent S.E. for 3 experiments (3 different cell populations). The level of binding in untreated controls was 118 f 9 DNA. lP < 0.05 according to Student’s t-test for comparisons between antioxidant-treated samples and control samples.
215
2-AAF to DNA is inhibited by BHA and BHT. Several mechanisms could account for these observations. Inhibition of c:arcinogenesis by antioxidants could result from an interaction at the initial activation step i.nvolving cytochrome P-450. Indeed, it has been reported that BHA and BHT bind to cytochrome P-450 [22] and can undergo biotransformation through the action of this enzymatic: system. The protective effect of BHT on 2-AAF genotoxicity has already been postulated to be linked to the enzymatic activation of 2-AAF [5]. However, the reduction in DNA binding seen with IV-OH-2-AAF, cytochrome P-s450 metabolite of 2-AAF which is believed to be required for the activation of the parent compound, suggests that other target sites are possible. Deacetylases which are found in the 100,000 x g pellet represent such potential sites. Similarly, the increase in mutagenesis of N-OH-2-AAF in the presence of lOO..OOO X g rat liver supernatant was attributed to deacetylases and acetyltransferase [15]. Moreover, data have been obtained which s.upport the view that free radical species are formed in the process of N-OH2-AAF activation [ 10,141. The action of BHA or BHT in the case of this carcinogen could further be explained in terms of their radicalscavenging properties. The results obtained with the complete cellular system are in agreement with those accumulated with the microsome-mediated binding assay. This exemplifies the fact that in vitro models, with their convenience, reproducibility and speed, allow the screening of potential anticarcinogenic agents. This was previously suggested by Bhattacharya et al. [3] who used the microsome-mediated formation of DNA adduct by aflatoxin B, to test the anticarcinogenie potential of a variety of agents including BHA and BHT which, among others, were inhibitory such as in the present series of experiments. However, despite the fact that invaluable information about the action of putative anticarcinogens can be obtained with in vitro models, tlhese still have some important limitations. DNA-carcinogen adduct formation
is believed to be the initiating event in chemical carcinogenesis, but it is not sufficient for tumor induction. Therefore, anticarcinogenic activity could take place via mechanisms pertaining to tumor promotion and progression. Further progress has to be made for in vitro models to encompass such possibilities. The mechanisms underlying the modulation of the carcinogenesis process by antioxidants appear quite complex. The results that are reported here suggest that, in addition to the induction of detoxifying enzymes which occurs upon in vivo antioxidant treatment [&lo, 111, other mechanisms may be involved in the anticarcinogenic effects of BHA or BHT in the liver as postulated above: inhibition of bioactivation and radical-scavenging activity. Such mechanisms would depend on direct interactions of these compounds with liver cells and would not necessitate enzyme induction. References Anderson, M.W., Boroujerdi, M. and Wilson, A.G.E. (1981) Inhibition in vivo of the formation of adducts between metabolites of benzo[o]pyrene and DNA by butylated hydroxyanisole. Cancer Res., 41,4309-4315. Benson, A.M., Hunkeler, M.J. and Morrow, J.F. (1984) Kinetics of glutathione transferase, glutathione transferase messenger RNA, and reduced nicotinamide adenine dinucleotide (phosphate): quinone reductase induction by 2 (3)-tert-butyl-4-hydroxyanisole in mice. Cancer Res., 44, 5256-5261. Bhattacharya, R.K., Firozi, P.F. and V.S. Aboobaker, V.S. (1984) Factors modulating the formation of DNA adduct by aflatoxin B, in vitro. Carcinogenesis, 5, 13591362. Cha, Y-N. and Bueding, E. (1979) Effect of 2 (3)reti butyl-4-hydroxyanisole administration on the activities of several hepatic microsomal and cytoplasmic enzymes in mice. Biochem. Pharmacol., 28, 1917-1921. Chipman, J.K. and Davies, J.E. (1988) Reduction of 2acetylaminofluorene-induced unscheduled DNA synthesis in human and rat hepatocytes by butylated hydroxytoluene. Mutat. Res., 207, 193-198. Christie, N., Cantoni, O., Evans, R.M. Meyn, R.E. and Costa, M. (1984) Use of mammalian DNA repair-deficient mutants to assess the effects of toxic metal compounds on DNA. Biochem. Pharmacol., 33, 1661-1670. Dallner, G. (1974) isolation of rough and smooth microsomes. Methods Enzymol., 31, 191-201. Denbeau, F., Marion, M., Chevalier, G. and Cote, M.G. (1985) Inability of chrysotile asbestos fibers to modulate the
216
9
10
11
12
13
14
15
16
17
2-acetylaminofluorene-induced UDS in primary cultures of rat hepatocytes. Mutat. Res. 155, 83-90. Dock, L., Martinez, M. and Jernstrom, 9. (1984) Induction of hepatic glutathione S-transferase activity by butylated hydroxyanisole and conjugation of benzo[o]pyrene dial-epoxide. Carcinogenesis, 5, 841-844. Floyd, R.A. (1982) The role of free radicals in arylamine carcinogensis. In: Free Radicals and Cancer, pp. 361394. Editor: R.A. Floyd. Marcel Deckker, New York. Gold, D.V. and Shochat, D. (1980) A rapid calorimetric assay for the estimation of microgram quantities of DNA. Anal. Biochem., 105, 121-125. Goodman, J.I., Trosko, J.E. and Yager, J.D., Jr. (1976) Studies on the mechanism of inhibition of 2-acetylaminofluorene toxicity by butylated hydroxytoluene. Chem.Biol. Interact., 12, 171-182. Gupta, R.C. (1984) Nonrandom binding of the carcinogen N-hydroxy-2-acetylaminofluorene to repetitive sequences of rat liver DNA in viva, Proc. Natl. Acad. Sci., 81, 69436947. Hinson, J.A., Andrews, L.S. and Gillette, J.R. (1982) A possible role of free radicals in the mutagenicity of Nhydroxy-2-acetylaminofluorene. In: Free Radicals and Cancer, pp. 423-440. Editor: R.A. Floyd. Marcel Dekker, New York. Ito, N.. Fukushima, S. and Tsuda. H. (1986) Carcinogenicity and modification of the carcinogenic response by BHA, BHT and other antioxidants. CRC Crit. Rev. Toxicol., 15,109-127. Kirkpatrick, D.C. and Lauer, B.H. (1986) Intake of phenolit antioxidant from Foods in Canada. Food Chem. Toxic., 24, 1035-1037. Lindenschmidt, R.C., Tryka, A.F., Good, M.E. and Witschi, H.P. (1986) The effects of dietary butylated hydroxy-
18
19
20
21
22
23 24
25
toluene on liver and colon tumor development in mice. Toxicology, 38, 151-160. Maura, Y., Weisburger, J.H. and Williams, G.M. (1984) Dose-dependent reduction of N-2-fluorenylacetamideinduced liver cancer and enhancement of bladder cancer in rats by butylated hydroxytoluene. Cancer Res., 44, 1604 - 1610. Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982) Molecular Cloning, U.S.A. Cold Spring Harbor Laboratory Muller, R. and Rajewsky, M.F. (1980) Immunological quantification by high affinity antibodies of 06.ethyldeoxyguanosine in DNA exposed to N-ethyl-N-nitrosourea Cancer Res.. 40,887-896. Salocks, C.B., Hsieh, D.P.H. and Byard. J.L. (1984) Effects of butylated hydroxytoluene pretreatment on the metabolism and genotoxicity of aflatoxin 9, in primary cultures of adult rat hepatocytes: Selective reduction of nucleic acid binding. Toxicol. Appl. Pharmacol., 76, 498 -509. Schenkman, J.B., Remmer, H. and Estabrook, R.W. (1967) Spectral studies of drug interaction with hepatic microsomal cytochrome. Mol. Pharmacol., 3, 113-123. Seglen, P.O. (1976) Preparation of isolated rat liver cells. Methods Cell Biol., 13, 29-83. Speier, J.L. and Wattenberg, L.W. (1975) Alterations in microsomal metabolism of benzo[a]pyrene in mice fed butylated hydroxyanisole. J. Natl. Cancer Inst., 55, 469470. Williams, G.M., Tanaka, T. and Maeura, Y. (1986) Doserelated inhibition of aflatoxin 9, induced hepatocarcinogenesis by the phenolic antioxidants, butylated hydroxyanisole and butylated hydroxytoluene. Carcinogenesis, 7. 1043-1050.
Inhibition butylated vitro N. Richer,
of binding of Z-acetylaminofluorene to DNA by hydroxytoluene and butylated hydroxyanisole in
M. Marion
Department
211 Ltd.
of Chemistry,
and Universitk
F. Denizeau’ du Qukbec ii Monkhal,
Mont&l
(Canada)
(Received 10 June 1988) (Revision received 25 May 1989) (Accepted 26 May 1989)
Summary
with
DNA which may reflect on alteration
carcinogen Numerous antioxidants
studies have shown that the food butylated hydroxytoluene (BHT)
and butylated hydroxyanisole (BHA), specific exposure conditions, can inhibit tocarcinogenesis
induced
Keywords: 2-acetylaminofluorine; butylated hydroxyanisole; butylated hydroxytoluene; anticarcinogenic properties; liver.
under hepavarious
by
in
metabolic activation.
carcinogens. The purpose of the present work was to study the biochemical mechanisms
Introduction
responsible
Phenolic antioxidants are widely used as food additives to prevent the development of rancidity. Human consumption of compounds such as BHA and BHT has been estimated. For example, in Canada the daily intake would be of the order of 0.4 mg/kg of body wt. [ 161. Numerous studies have shown that BHA and BHT can modify the response to various carcinogens. Depending on the target organ and on exposure conditions, inhibitory as well as stimulatory effects have been observed [15,17]. In the liver, it was found that BHT, fed together with the experimental carcinogen 2-AAF, inhibited carcinogenicity [ 191. Aflatoxin B,-induced hepatocarcinogenesis was inhibited by BHA and BHT when these were present in the diet 1 week before carcinogen, during administration and 1 week after cessation [25]. Moreover, BHA and BHT reduced the development of y-glutamyl-transpeptidasepositive foci initiated in the liver by diethylnitrosamine [ 151.
for
the anticarcinogenic
actioity of
BHA and BHT using in uitro systems. The effects of BHA and BHT on the binding of Zacetylaminofluorene (Z-AAF) to DNA was determined
in
a microsomd
system
and
in
primary cultures of rat hepatocytes. It was found that both antioxidants reduce the binding of
and that of N-OH-Z-acetylami-
ZAAF
nofluorene
(N-OH-2-AAF)
to
caij
thymus
DNA in the presence of liver microsomes. inhibition
was h,owever more pronounced
the parent
binding
The with
compound.
Lower levels of DNA also detected in hepatocytes with 2-AAF along with BHA or
were
incubated
These results suggest that phenolic antioxidants can exert anticarcinogenic activity
BHT.
through
modulation
‘Correspondence Mont&al, C.P. Canada.
of carcinogen
interaction
to: F. Denizeau, Universitk du Qu&ec a 8888, Succ. A Mont&al, Quhbec H3C 3P8,
0 1989 Elsevier Scientific Publishers 0304-3835/89/$03.50 Published and Printed in Ireland
Ireland Ltd.
212
Formation of carcinogen-DNA adducts has also been followed upon treatment with antioxidants: addition of BHA to the diet inhibited the binding of benzo[a]pyrene to liver DNA in vivo [ 11; similarly, BHT feeding was shown to reduce the binding of 2-AAF to hepatic DNA [12]. In primary cultures of rat hepatocytes, selective reduction of nucleic acid binding of aflatoxin B, was seen when pretreatment with BHT was applied in vivo. More recently, the reduction of 2-AAF-induced DNA repair in human and rat hepatocytes by BHT has also been observed 151. Changes in the activity of enzymes responsible for xenobiotic metabolism have been proposed to be involved in the anticarcinogenic effects of antioxidants. Indeed, induction of the detoxifying enzymes glutathione S-transferase [2,9], epoxide hydratase and UDP-glucuronyltransferase [4], has been detected after in vivo exposure to BHA. Despite the numerous investigations on anticarcinogenic activity of antioxidants in the liver, the mechanisms underlying this phenomenon are still not fully understood. In an effort to extend the previous studies, the purpose of the present work was to investigate the anticarcinogenic properties of BHA and BHT towards the hepatocarcinogen (2-AAF) in in vitro systems. The effects of BHA and BHT on the binding of 2-AAF to DNA was determined in a microsomal system and in primary cultures of rat hepatocytes. Materials and methods Microsome-mediated binding of [3H]2-AAF and [3H]N-OH-2-AAF was performed using a procedure similar to that of Speier and Wattenberg [24]. The microsomes were prepared according to the method of Dallner [7] from livers of male SpragueDawley rats weighing 150-300 g. Each sample was made up of the following: 4 mg calf thymus DNA, 4 FM NADPH, 3.5 mg microsomal proteins and 3.5 VM sodium citrate buffer, pH 7.4 in a final volume of 6 ml. BHA, BHT, [3H]2-AAF and [3H]N-OH-2-AAF (312-341 mCi/mmol;
Chemsyn Science Laboratories) were dissolved in dimethyl sulfoxide (DMS) at concentrations of 1 M, 118 pCi/ml and 148 pCi/ml respectively. The reaction was initiated by adding the antioxidant and the radioactive substrate (final concentration: 2 &i/ml). Control incubations were carried out without the antioxidant. Non-specific binding was determined using samples in which heat-inactivated microsomal preparations were employed. After incubation at 37OC for 60 min, the reaction was stopped by placing the samples at - 40°C for 20 s. DNA was extracted with chloroform: isoamyl alcohol (24 : 1) and purified according to the method of Muller and Rajewsky [20] using hydroxyapatite (DNA-grade Bio-gel HTP). The amount of DNA was determined by means of the thiobarbituric acid assay [ll] and its radioactivity content was measured by liquid scintillation counting. Primary cultures of rat hepatocytes were prepared as we previously reported [8]. Male Sprague-Dawley rats weighing 150-300 g were used. The hepatocytes were isolated according to the technique described by Seglen 1231 with slight modifications [8]. Viable cells (4.5 x 106) were seeded in each 60-mm petri dish. The culture medium consisted of WME supplemented with 10% FBS, 100 units/ml penicillin and 100 pg/ml streptomycin (WME-10% FBS). The cells were allowed to attach for 2 h at 37OC, in an atmosphere of 95% air, 5% CO,, saturated with humidity. The incubation with the carcinogen was then initiated by adding fresh WME1% FBS containing [3H]2-AAF (4 $Zi/ml), with or without the antioxidant. After a 20-h exposure period at 37OC, the DNA was purified by a procedure inspired from those of Maniatis et al. [19] and Christie et al. [6]. It consisted of the following steps: the cells were suspended in Hank’s balanced salt solution, washed by centrifugation and lysed with 20 mM Tris-HCl buffer pH 8.0, containing 150 mM NaCl, 1% SDS and 50 pg/ml proteinase K. The samples were placed on a mobile plate and subjected to gentle agitation
213
(35 inversions/min) for 3 h at 37OC. Then, extractions were carried out with phenol-Tris, pH 7.9 and chloroform/isoamyl alcohol (24 : 1) ; the aqueous phase was re-extracted with chloroform and its DNA was precipitated with cold absolute ethanol. The samples were kept overnight .at - 20°C, the DNA pellets were washed wiith 70% ethanol and dissolved in buffer (10 mM Tris, pH 7.9). The quantitation was performed by measuring the absorbance at 260 nm. The ratios of absorbance AZi0 JA280 nm’ which provide an estimation of the purity of the material, were in the region of 1.80 & 0.5; this indicates the absence of significant protein contamination [13]. Aliquots of the same samples were used for radioactivity measurements. Results Figure 1 shows the effect of BHA and BHT on the binding (of [3H]2-AAF to DNA in the microsomal system. A significant inhibition of adduct formation, which becomes quite pronounced (> 75%) at the highest antioxidant levels, can be observed. Similarly, Fig. 2 presents the binding profiles obtained with N-OH2-AAF, a 2-AAF metabolite known to be
formed by liver microsomal P-450 mixedfunction oxidase. It can be seen that the binding of [3H]N-OH-2-AAF was lower in samples to which BHA or BHT was added; however, the effect was less marked than with the parent compound. The next series of experiments was aimed at testing whether the inhibition of the binding of 2-AAF to the DNA would be observed in a complete cellular system. Freshly isolated hepatocytes were exposed to [3H]2-AAF and to BHA or BHT for 20 h. The results are presented in Fig. 3. It is interesting to note that adduct formation is again lower in BHA- or BHT-treated samples. In this case however, it was not possible to obtain a more extended inhibition profile because maximum antioxidant concentration was limited by cytotoxicity. Indeed, at levels of BHA and BHT higher than those indicated, the release of lactate dehydrogenase was detectable and there was some loss of cell viability. Discussion The results of the microsome-mediated assay, as well as those obtained with freshly isolated hepatocytes, show that binding of
r ._ m
0
10
Antioxidant concentration (mM) Inhibition of [3H]2-AAF-DNA binding by BHA and BHT in the microsomal assay. The data represent means f S.E. for 3 experiments. The level of binding in untreated controls was 13.8 f 1.3 DPM/pg DNA. The values shown were corrected for non-specific binding.
Fig.1.
80 60
Antioxidant
concentration
(mM)
Inhibition of [3H]N-OH-2-AAF-DNA binding by BHA and BHT in the microsomal assay. The data represent Fig. 2. means f S.E. for 3 experiments. The level of binding in untreated controls was 45.2 + 4.5 DPM/pg DNA. The values shown were corrected for non-specific binding.
n
Control
5
BHA BHT
q
El
90
70
Antioxidant Fig. 3. means + DPM/pg untreated
concentration
(PM)
Inhibition of [3H]2-AAF binding to the DNA of rat hepatocytes exposed to BHA and BHT. The data represent S.E. for 3 experiments (3 different cell populations). The level of binding in untreated controls was 118 f 9 DNA. lP < 0.05 according to Student’s t-test for comparisons between antioxidant-treated samples and control samples.
215
2-AAF to DNA is inhibited by BHA and BHT. Several mechanisms could account for these observations. Inhibition of c:arcinogenesis by antioxidants could result from an interaction at the initial activation step i.nvolving cytochrome P-450. Indeed, it has been reported that BHA and BHT bind to cytochrome P-450 [22] and can undergo biotransformation through the action of this enzymatic: system. The protective effect of BHT on 2-AAF genotoxicity has already been postulated to be linked to the enzymatic activation of 2-AAF [5]. However, the reduction in DNA binding seen with IV-OH-2-AAF, cytochrome P-s450 metabolite of 2-AAF which is believed to be required for the activation of the parent compound, suggests that other target sites are possible. Deacetylases which are found in the 100,000 x g pellet represent such potential sites. Similarly, the increase in mutagenesis of N-OH-2-AAF in the presence of lOO..OOO X g rat liver supernatant was attributed to deacetylases and acetyltransferase [15]. Moreover, data have been obtained which s.upport the view that free radical species are formed in the process of N-OH2-AAF activation [ 10,141. The action of BHA or BHT in the case of this carcinogen could further be explained in terms of their radicalscavenging properties. The results obtained with the complete cellular system are in agreement with those accumulated with the microsome-mediated binding assay. This exemplifies the fact that in vitro models, with their convenience, reproducibility and speed, allow the screening of potential anticarcinogenic agents. This was previously suggested by Bhattacharya et al. [3] who used the microsome-mediated formation of DNA adduct by aflatoxin B, to test the anticarcinogenie potential of a variety of agents including BHA and BHT which, among others, were inhibitory such as in the present series of experiments. However, despite the fact that invaluable information about the action of putative anticarcinogens can be obtained with in vitro models, tlhese still have some important limitations. DNA-carcinogen adduct formation
is believed to be the initiating event in chemical carcinogenesis, but it is not sufficient for tumor induction. Therefore, anticarcinogenic activity could take place via mechanisms pertaining to tumor promotion and progression. Further progress has to be made for in vitro models to encompass such possibilities. The mechanisms underlying the modulation of the carcinogenesis process by antioxidants appear quite complex. The results that are reported here suggest that, in addition to the induction of detoxifying enzymes which occurs upon in vivo antioxidant treatment [&lo, 111, other mechanisms may be involved in the anticarcinogenic effects of BHA or BHT in the liver as postulated above: inhibition of bioactivation and radical-scavenging activity. Such mechanisms would depend on direct interactions of these compounds with liver cells and would not necessitate enzyme induction. References Anderson, M.W., Boroujerdi, M. and Wilson, A.G.E. (1981) Inhibition in vivo of the formation of adducts between metabolites of benzo[o]pyrene and DNA by butylated hydroxyanisole. Cancer Res., 41,4309-4315. Benson, A.M., Hunkeler, M.J. and Morrow, J.F. (1984) Kinetics of glutathione transferase, glutathione transferase messenger RNA, and reduced nicotinamide adenine dinucleotide (phosphate): quinone reductase induction by 2 (3)-tert-butyl-4-hydroxyanisole in mice. Cancer Res., 44, 5256-5261. Bhattacharya, R.K., Firozi, P.F. and V.S. Aboobaker, V.S. (1984) Factors modulating the formation of DNA adduct by aflatoxin B, in vitro. Carcinogenesis, 5, 13591362. Cha, Y-N. and Bueding, E. (1979) Effect of 2 (3)reti butyl-4-hydroxyanisole administration on the activities of several hepatic microsomal and cytoplasmic enzymes in mice. Biochem. Pharmacol., 28, 1917-1921. Chipman, J.K. and Davies, J.E. (1988) Reduction of 2acetylaminofluorene-induced unscheduled DNA synthesis in human and rat hepatocytes by butylated hydroxytoluene. Mutat. Res., 207, 193-198. Christie, N., Cantoni, O., Evans, R.M. Meyn, R.E. and Costa, M. (1984) Use of mammalian DNA repair-deficient mutants to assess the effects of toxic metal compounds on DNA. Biochem. Pharmacol., 33, 1661-1670. Dallner, G. (1974) isolation of rough and smooth microsomes. Methods Enzymol., 31, 191-201. Denbeau, F., Marion, M., Chevalier, G. and Cote, M.G. (1985) Inability of chrysotile asbestos fibers to modulate the
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