- Email: [email protected]
Selenium inhibition of benzo[a]pyrene, 3-methylcholanthrene, and 3-methylcholanthrylene mutagenicity in Salmonella typhimurium strains TA98 and TA100
Mutation Research, 190 (1987) 101-105
101
Elsevier MTRL 0959
Selenium inhibition of benzo[a]pyrene, 3-methylcholanthrene, and 3-methylcholanthrylene mutagenicity in Salmonella typhimurium strains TA98 and TA100 P. Prasanna1, M.M. Jacobs2 and S.K. Yang 1Department of Pharmacology, F. Edward Hebert School of Medicine, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814-4799 (U.S.A.) and 2The MITRE Corporation/Metrek Division, 1820 Dolley Madison Boulevard, McLean, VA 22102 (U.S.A.)
(Accepted 14 October 1986)
Keywords: Benzo[a]pyrene,inhibition; Selenium; 3-Methylcholanthrene;3-Methylcholanthrylene;(Salmonella typhimurium).
Summary Selenium (Se) decreased the mutagenicity of benzo[a]pyrene (BP), 3-methylcholanthrene (3MC), and 3-methylcholanthrylene (3MCE) in Salmonella t y p h i m u r i u m strains TA98 and TA100. Metabolism of BP, 3MC and 3MCE to mutagens was accomplished with the liver $9 fraction from Aroclor 1254-treated male Sprague-Dawley rats. Exposure of the bacteria to 4 nmoles BP, 10 nmoles 3MC, or 10 nmoles 3MCE in the presence of $9, and up to 200 nmoles Se as Na2SeO3 resulted in decreased mutagenicities up to 39, 66 and 60%0 of their respective control activities without Se in TA98 and up to 46, 52 and 64% of their respective control activities without Se in TA100. Se (200 nmoles) alone was not mutagenic in strains TA98 or TA100 with or without $9. BP, 3MC and 3MCE were not mutagenic in either strain without $9. None of the tested concentrations of BP, 3MC, 3MCE and Se were cytotoxic. Assays of the aryl hydrocarbon hydroxylase (AHH) activity in the $9 preparation revealed decreased A H H activity with increase in Se concentration. The decreased mutagenicity and A H H activity were Se (as Na2SeO3) dependent and could not be duplicated by sulfur (S as NaeSO3). Inhibition of A H H activity by Se provides an explanation of the mechanism of Se inhibition of BP, 3MC and 3MCE mutagenicities in S. t y p h i m u r i u m TA98 and TA100.
Selenium compounds have exhibited antitumorigenic properties in experimental animals, antimutagenic properties in microorganisms, and Correspondence: Dr. P. Prasanna, Department of Pharmacology, F. Edward Hebert School of Medicine, Uniformed Services Universityof the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814-4799(U.S.A.).
anticlastogenic properties in cell cultures. Recently, two comprehensive reviews have been published on the properties of Se influencing carcinogenesis (Vernie, 1984) and on the properties of Se influencing mutagenesis and genotoxicity (Shamberger, 1985). In animals, Se supplements to diet or drinking water have decreased the incidence of colon tumor induction by 1,2-dimethylhydrazine and
0165-7992/87/$ 03.50 © 1987ElsevierSciencePublishers B.V. (BiomedicalDivision)
102
methylazoxymethanol acetate (Jacobs et al., 1977a); of spontaneous and 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary tumors (Ip, 1981; Medina and Shephard, 1981; Schrauzer and Ishmael, 1974); and of hepatic tumor induction by 3'-methyl-4-dimethylaminoazobenzene (Clayton and Baumann, 1949; Griffin and Jacobs, 1977), DMBA (Shamberger, 1970) and by 2-acetylaminofluorene (AAF) (Harr et al., 1973; Marshall et al., 1979). In Ames SalmoneUa/microsomal assays, Se has decreased the mutagenicity of AAF and its metabolites (Jacobs et al., 1977b; Rosin and Stitch, 1979), of BP (Lin et al., 1984), and of DMBA (Martin et al., 1981). In human cell cultures, Se has decreased methyl methanesulfonate- and N-hydroxy-2-acetylaminofluorene (N-OH-AAF)-induced sister-chromatid exchanges (Ray et al., 1978), and caused DNA fragmentation, DNA-repair synthesis, and chromosome aberrations (Lo et al., 1978). In separate studies Se has been demonstrated to inhibit AHH activity (Lin et al., 1984; Rasco et al., 1977) and N-hydroxylase activity (Marshall et al., 1979), thereby suppressing metabolic activation of BP and AAF, respectively. The experimental results presented in this communication show decreased mutagenicities of BP, 3MC and 3MCE in the Ames Salmonella/microsomal assay and attribute these decreases, at least in part, to Se inhibition of AHH activity in the metabolic activation of the polycyclic aromatic hydrocarbons (PAHs). Materials and methods $9 preparation The $9 fraction (29.6 mg protein/ml) for metabolic activation was prepared from the livers of Aroclor 1254-treated male Sprague-Dawley rats (100-120 g body weight). The rats were injected intraperitoneaUy once with 0.5-0.6 ml of corn oil containing 0.05-0.06 g Aroclor 1254 (0.5 g Aroclor 1254/kg body weight). After 4 days, the rats were decapitated and the livers were removed and homogenized in 0.15 M KCI at 4°C. The homogenate was centrifuged at 9000 × g for 10 min to yield the supernatant $9 fraction.
Mutagenicity assay BP and 3MC were purchased from Sigma Chemical Co. (St. Louis, MO). 3MCE was a generous gift of Dr. Peter P. Fu of the National Center for Toxicological Research. The mutagenicities of BP, 3MC and 3MCE were tested in S. typhimurium strain TA98, which detects frameshift mutagens, and strain TAI00, which detects base-pair substitution mutagens, according to the modified test developed by Ames and collaborators (Ames et al., 1975; Maron and Ames, 1983). A cofactor mixture was prepared which consisted of 5.7 mM NADP, 7.1 mM glucose 6-phosphate, 4.7 mM KCI, and 11.4 mM MgCI2 in a 0.14 M sodium phosphate buffer, pH 7.4. An appropriate volume of rat-liver $9 fraction was added so that the final cofactor mixture contained 30 #1 per 0.5 ml of cofactor mixture. The mutagenicity test was carried out by incubation at 37°C in a dry water bath of a test tube containing 0.5 ml of cofactor mixture, 0.1 ml bacteria culture containing l0 s cells, 0.05 ml of DMSO containing 0-10 nmoles of BP, 3MC, or 3MCE and 0.05 ml of 0.14 M sodium phosphate buffer (pH 7.4) containing 0-200 nmoles of Na2SeO3 or 0-200 nmoles of Na2SO3. After incubation for I0 min, 2 ml of top agar, containing the histidine/biotin solution described in Marin and Ames (1983), was added to each test tube. After thorough mixing, the mixture was poured onto a plate containing 25 ml of solidified minimal glucose agar and the His + revertants were scored after incubation at 37°C for 48 h. All assays were performed in triplicate. A H H assay The assay for AHH activity was carried out according to a modification of the fluorometric procedure developed by Nebert and Gelboin (1968). The assay was carried out in a l-ml mixture containing 50 nmoles of Tris-HCl (pH 7.5), 3 nmoles of MgCI2, 0.5 mg of NADPH, an appropriate volume of rat-liver $9 fraction containing 0.1-0.2 mg of protein, 80 nmoles of BP (added in 40/zl of acetone), and 0-200 nmoles of Na2SeO3 (Alfa Products, Ventron Corporation) or 0-200 nmoles of
103
Na2SO3 (Sigma Chemical Co.). The volume of the mixture was adjusted to 1 ml by the addition of water. Protein was determined by the colorimetric method of Lowry et al. (1951). A unit of AHH activity was expressed in pmoles of phenolic products formed/min/mg protein using 3-hydroxybenzo[alpyrene (from Chemical Repository of National Cancer Institute) as the fluorescence standard.
Results
In S. typhimurium strains TA98 and TA100, $9 was required to convert BP, 3MC, and 3 MCE to mutagens (Table 1). In the absence of $9, these 3
PAHs were not mutagenic in either strain and yielded the background number of spontaneous revertants observed in the DMSO solvent control. The DMSO solvent control and up to 200 nmoles of Se were not mutagenic in TA98 and TA100 with or without $9 present. A viable cell count of 10s cells per plate with no apparent cytotoxicity was observed in all assays of Se alone, each pAH alone, and Se in combination with each PAH. The mutagenicity of BP, 3MC and 3 MCE in S. typhimurium TA98 in the presence of $9 was decreased by Se. The mutagenic response of 4 nmoles BP to 20, 100 and 200 nmoles Se was reduced to 71, 52 and 39070, respectively, of that with BP alone. The mutagenic responses of 10
TABLE 1 EFFECTS OF S E L E N I U M (Se) AND SULFUR (S) ON T H E M U T A G E N I C I T Y OF BP, 3MC A N D 3MCE T O W A R D S. t y p h i m u r i u m TA98 AND TA100 Chemical
DMSO DMSO
Se or S (nmoles/
$9 a
H i s + revertants per plate ( ± S D ) (0/0 of control)
TA98/Se b
TA100/Se b
TA 98/ S c
TAI00/S c
0 0
+
25 _+ 7 21 ± 5
110 ± 29 124 ± 19
39 ± 7 40 ± 4
110 ± 29 108 ± 6
Se (or S) a Se (or S)d
200 200
+
21 ± 2 17 ± 6
144 ± 21 156 ± 7
24 ± 5 32 ± 5
110 + 25 112 ± 11
BP (4 nmoles)
0 0 20 100 200
+ + + +
22 245 174 128 96
7 21 26 16 13
(100070) (71070) (52%) (390/0)
136 922 630 410 420
4 49 31 20 38
(100%) (6807o) (44°70) (46%)
46 280 271 270 265
0 0 20
+ +
39 + 4 134 + 16 113 ± 13
(100070) (84%)
100 ± 10 956 ± 37 716 ± 91
(1000/0) (75%)
39 ± 4 130 ± 9 167 ± 32
109 ± 4 1050 ± 39 1066 ± 127
100 200
+ +
93 + 6 89 ± 12
(69070) (66070)
489 ± 99 497 ± 42
(51%) (52070)
148 ± 40 145 + 27
968 ± 19 870 ± 5
0 0
+
50 _+ 4 1546 ± 75
(1000/0)
20 100 200
+ + +
1036 _+ 38 914 ± 156 935 ± 152
3MC (10 nmoles)
3MCE (10 nmoles)
± ± + ± ±
± ± ± ± ±
± ± + ± ±
2 13 16 25 16
122 _+ 10 2164 ± 241
50 + 4 (100070) 2702 ± 24 e
(670/0) 2124 ± 37 (590/o) 1667 ± 44 ( 6 0 ° 7 0 ) 1390 ± 116
(980/0) 2717 ± 185 (770/0) 2441 ± 212 ( 6 4 0 7 o ) 2414 ± 494
83 966 1056 987 1026
± ± ± ± ±
22 40 39 60 21
182 + 35 2592 ± 298 e 2509 ± 181 2773 ± 64 2450 ± 50
a In the presence ( + ) or absence ( - ) of $9. b Assays in the presence or absence of Se. Assays in the presence or absence of S. 0 Assays in the presence or absence of either Se or S as noted in footnotes b and ~ e These two sets of experiments were carried out several months after the other experiments were completed. The H i s ÷ revertants scored were reproducible for each time period.
104 nmoles 3MC and of 10 nmoles 3MCE, a minor metabolite of 3MC (Thakker et al., 1978), were reduced by 20, 100 and 200 nmoles Se to 84, 69 and 66070 respectively, of 3MC alone, and to 67, 59 and 60070, respectively, of 3MCE alone. The mutagenicity of BP, 3MC and 3MCE in S. typhimurium TAI00 in the presence of $9 was also decreased by Se. The mutagenic response of 4 nmoles BP to 20, 100 and 200 nmoles Se was reduced to 68, 44 and 46070, respectively, of that with BP alone. The mutagenic responses of 10 nmoles each of 3MC and 3MCE were reduced by 20, 100 and 200 nmoles Se to 75, 51 and 52070, respectively of 3MC alone, and to 98, 77 and 64070, respectively, of 3MCE alone. Assays of the AHH activity in the S9-preparation were reduced by 20, 100 and 200 nmoles Se to 94, 71 and 59070, respectively, of the AHH activity in the absence of Se. No decrease in AHH activity was observed with Na2SO3. A representative set of control data from parallel experiments carried out with S as Na2SO3 in TA98 is illustrated in Table 1. For these experiments the background number of spontaneous revertants in TA98 for the DMSO solvent control was 40 ± 4 revertants per plate. Addition of 200 nmoles S as Na2SO3 yielded 32 ± 5 revertants per plate, which was comparable with the spontaneous level. The data in Table 1 show that BP, 3MC and 3MCE yielded the spontaneous number of revertants in assays without $9 present in both TA98 and TA100. Increased numbers of revertants (280 + 13 revertants per plate for BP, 130 + 9 revertants per plate for 3MC, and 2702 ± 24 revertants per plate for 3MCE in Table 1) were observed in assays with $9 activation in TA98. Addition of 20-200 nmoles S as Na2SO3 to BP, 3MC or 3MCE assays in TA98 and in TA100 did not markedly decrease the mutagenicity as did Se; however a slight decrease in mutagenicity of 3MC was observed with 200 nmoles S in TA100.
Discussion The results in this study suggest that Se may play
an important role as an inhibitor of the metabolic activation of BP, 3MC and 3MCE. The doses of PAH selected for these studies induced the optimal mutagenic response in the dose-response curves and were 4 nmoles BP, 10 nmoles 3MC and 10 nmoles 3MCE. Addition of Se decreased the mutagenicity of all 3 PAHs in strain TA98, which detects frameshift mutagens, and in strain TA100, which detects base-pair substitution mutagens. Se (200 nmoles) decreased the mutagenicity of BP, 3MC and 3MCE to 39, 66 and 60°70 of their respective controls in strain TA98, and to 46, 52 and 64°70 of their respective controls in strain TAI00. The effective molar ratios of Se to PAH yielding these decreases in each strain were 50:1 (Se:BP), 20:1 (Se:3MC), and 20:1 (Se:3MCE) and were similar to ratios of Se to AAF and Se to N-OH-AAF reported for decreased mutagenicity (Jacobs, Matney, and Griffin, 1977). Addition of 200 nmoles Se to AHH assays decreased this enzyme activity to 59°70 of the control assay in the absence of Se. Because Se and S are related elements that can form isologous compounds such as selenomethionine and methionine, the Se effects were compared with the S controls in this study. The decreases in mutagenicities and AHH activity were apparently a specific effect of Se, since Na2SO3, added to parallel bacterial and enzyme assay mixtures in the same concentrations as NaESeO3, did not decrease either activity. The inhibition of AHH activity by Se suggests the interactions between Se and PAH-metabolizing enzyme systems which caused reductions in mutagenicities in strains TA98 and TA100 by BP, 3MC and 3 MCE.
Acknowledgement This work was supported by an Independent Research and Development award from The MITRE Corporation through the Henry M. Jackson Foundation for the Advancement of Military Medicine Protocol No. G17586 at the Uniformed Services University of the Health Sciences.
105
References Ames, B.N., J. McCann and E. Yamasaki (1975) Methods for detecting carcinogens and mutagens with the Salmonella/ mammalian-microsome mutagenicity test, Mutation Res., 31, 347-364. Clayton, C.C., and C.A. Baumann (1949) Diet and azo dye tumors: effect of diet during a period when the dye is not fed, Cancer Res., 9, 575-582. Griffin, A.C., and M.M. Jacobs (1977) Effects of selenium on azo dye hepatocarcinogenesis, Cancer Lett., 3, 177-181. Harr, J.R., J.H. Exon, P.H. Weswig and P.D. Whanger (1973) Relationship of dietary selenium concentration; chemical cancer induction, and tissue concentration of selenium in rats, Clin. Toxicol., 6, 287-293. Ip, C. (1981) Factors influencing the anticarcinogenic efficacy of selenium in dimethylbenzanthracene-induced mammary tumorigenesis in rats, Cancer Res., 41, 2683-2686. Jacobs, M.M., B. Jansson and A.C. Griffin (1977a) Inhibitory effects of selenium on 1,2-dimethylhydrazine and methylazoxymethanol acetate induction of colon tumors, Cancer Lett., 2, 133-138. Jacobs, M.M., T.S. Matney and A.C. Griffin (1977b) Inhibitory effects of selenium on the mutagenicity of 2-acetylaminofluorene (AAF) and AAF derivatives, Cancer Lett., 2, 319-322. Lin, W.S., C. Scrimshaw and M. Kapoor (1984) Selenium suppresses the metabolism of benzo[a]pyrene by rat-liver extracts, and exerts a dual effect on its mutagenicity, Xenobiotica, 14, 893-902. Lo, L.W., J. Koropatnick and H.F. Stich (1978) The mutagenicity and cytotoxicity of selenite, 'activated' selenite and selenate for normal and DNA repair-deficient human fibroblasts, Mutation Res., 49, 305-312. Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall (1951 ) Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193, 265-275. Maron, D.M., and B.N. Ames (1983) Revised methods for the Salmonella mutagenicity test, Mutation Res., 113, 173-215. Marshall, M.V., M.A. Rasco, M.M. Jacobs and A.C. Griffin (1979) Selenium effects on the carcinogenicity and metabolism of 2-acetylaminofluorene, Cancer Lett., 7, 331-338.
Martin, S.E., G.H. Adams, M. Schillaci and J.A. Milner (1981) Antimutagenic effects of selenium on acridine orange and 7,12-dimethylbenzanthracene in the Ames Salmonella/ microsomal system, Mutation Res., 82, 41-46. Medina, D., and F. Shephard (1981) Selenium-mediated inhibition of 7,12-dimethylbenzanthracene-induced mouse mammary tumorigenesis, Carcinogenesis, 2, 451-455. Nebert, D.W., and H.V. Gelboin (1968) Substrate-inducible microsomal aryl hydroxylase in mammalian cell culture, I. Assay and properties of induced enzyme, J. Biol. Chem., 243, 6242-6249. Rasco, M.A., M.M. Jacobs and A.C. Griffin (1977) Effects of selenium on aryl hydrocarbon hydroxylase activity in cultured human lymphocytes, Cancer Lett., 3, 295-301. Ray, J.A., L.C. Altenburg and M.M. Jacobs (1978) Effect of sodium selenite and methyl methanesulfonate or Nhydroxy-2-acetyl-aminofluorene co-exposure on sister chromatid exchange production in human whole blood cultures, Mutation Res., 57, 359-368. Rosin, M.P., and H.F. Stitch (1979) Assessment of the use of the Salmonella mutagenesis assay to determine the influence of antioxidants on carcinogen-induced mutagenicity, Int. J. Cancer, 23, 722-727. Schrauzer, G.N., and D. Ishmael (1974) Effects of selenium and of arsenic on the genesis of spontaneous mammary tumors in inbred C3H mice, Ann. Clin. Lab. Sci., 2, 441-447. Shamberger, R.J. (1970) Relationship of selenium to cancer, I. Inhibitory effect of selenium on carcinogenesis, J. Natl. Cancer Inst., 44, 931-936. Shamberger, R.J. (1985) The genotoxicity of selenium, Mutation Res., 154, 29-48. Thakker, D.R., W. Levin, T.A. Stoning, A.H. Conney and D.M. Jerina (1978) Metabolism of 3-methycholanthrene by rat liver microsomes and a highly purified monooxygenase system with and without epoxide hydrolase, in: P.W. Jones and R.I. Freudenthal (Eds.), Carcinogenesis, Vol. 3, Polynuclear Aromatic Hydrocarbons, Raven, New York, pp. 253-264. Vernie, L.N. (1984) Selenium in carcinogenesis, Biochim. Biophys. Acta, 738, 203-217. Communicated by R.J. Preston
101
Elsevier MTRL 0959
Selenium inhibition of benzo[a]pyrene, 3-methylcholanthrene, and 3-methylcholanthrylene mutagenicity in Salmonella typhimurium strains TA98 and TA100 P. Prasanna1, M.M. Jacobs2 and S.K. Yang 1Department of Pharmacology, F. Edward Hebert School of Medicine, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814-4799 (U.S.A.) and 2The MITRE Corporation/Metrek Division, 1820 Dolley Madison Boulevard, McLean, VA 22102 (U.S.A.)
(Accepted 14 October 1986)
Keywords: Benzo[a]pyrene,inhibition; Selenium; 3-Methylcholanthrene;3-Methylcholanthrylene;(Salmonella typhimurium).
Summary Selenium (Se) decreased the mutagenicity of benzo[a]pyrene (BP), 3-methylcholanthrene (3MC), and 3-methylcholanthrylene (3MCE) in Salmonella t y p h i m u r i u m strains TA98 and TA100. Metabolism of BP, 3MC and 3MCE to mutagens was accomplished with the liver $9 fraction from Aroclor 1254-treated male Sprague-Dawley rats. Exposure of the bacteria to 4 nmoles BP, 10 nmoles 3MC, or 10 nmoles 3MCE in the presence of $9, and up to 200 nmoles Se as Na2SeO3 resulted in decreased mutagenicities up to 39, 66 and 60%0 of their respective control activities without Se in TA98 and up to 46, 52 and 64% of their respective control activities without Se in TA100. Se (200 nmoles) alone was not mutagenic in strains TA98 or TA100 with or without $9. BP, 3MC and 3MCE were not mutagenic in either strain without $9. None of the tested concentrations of BP, 3MC, 3MCE and Se were cytotoxic. Assays of the aryl hydrocarbon hydroxylase (AHH) activity in the $9 preparation revealed decreased A H H activity with increase in Se concentration. The decreased mutagenicity and A H H activity were Se (as Na2SeO3) dependent and could not be duplicated by sulfur (S as NaeSO3). Inhibition of A H H activity by Se provides an explanation of the mechanism of Se inhibition of BP, 3MC and 3MCE mutagenicities in S. t y p h i m u r i u m TA98 and TA100.
Selenium compounds have exhibited antitumorigenic properties in experimental animals, antimutagenic properties in microorganisms, and Correspondence: Dr. P. Prasanna, Department of Pharmacology, F. Edward Hebert School of Medicine, Uniformed Services Universityof the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814-4799(U.S.A.).
anticlastogenic properties in cell cultures. Recently, two comprehensive reviews have been published on the properties of Se influencing carcinogenesis (Vernie, 1984) and on the properties of Se influencing mutagenesis and genotoxicity (Shamberger, 1985). In animals, Se supplements to diet or drinking water have decreased the incidence of colon tumor induction by 1,2-dimethylhydrazine and
0165-7992/87/$ 03.50 © 1987ElsevierSciencePublishers B.V. (BiomedicalDivision)
102
methylazoxymethanol acetate (Jacobs et al., 1977a); of spontaneous and 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary tumors (Ip, 1981; Medina and Shephard, 1981; Schrauzer and Ishmael, 1974); and of hepatic tumor induction by 3'-methyl-4-dimethylaminoazobenzene (Clayton and Baumann, 1949; Griffin and Jacobs, 1977), DMBA (Shamberger, 1970) and by 2-acetylaminofluorene (AAF) (Harr et al., 1973; Marshall et al., 1979). In Ames SalmoneUa/microsomal assays, Se has decreased the mutagenicity of AAF and its metabolites (Jacobs et al., 1977b; Rosin and Stitch, 1979), of BP (Lin et al., 1984), and of DMBA (Martin et al., 1981). In human cell cultures, Se has decreased methyl methanesulfonate- and N-hydroxy-2-acetylaminofluorene (N-OH-AAF)-induced sister-chromatid exchanges (Ray et al., 1978), and caused DNA fragmentation, DNA-repair synthesis, and chromosome aberrations (Lo et al., 1978). In separate studies Se has been demonstrated to inhibit AHH activity (Lin et al., 1984; Rasco et al., 1977) and N-hydroxylase activity (Marshall et al., 1979), thereby suppressing metabolic activation of BP and AAF, respectively. The experimental results presented in this communication show decreased mutagenicities of BP, 3MC and 3MCE in the Ames Salmonella/microsomal assay and attribute these decreases, at least in part, to Se inhibition of AHH activity in the metabolic activation of the polycyclic aromatic hydrocarbons (PAHs). Materials and methods $9 preparation The $9 fraction (29.6 mg protein/ml) for metabolic activation was prepared from the livers of Aroclor 1254-treated male Sprague-Dawley rats (100-120 g body weight). The rats were injected intraperitoneaUy once with 0.5-0.6 ml of corn oil containing 0.05-0.06 g Aroclor 1254 (0.5 g Aroclor 1254/kg body weight). After 4 days, the rats were decapitated and the livers were removed and homogenized in 0.15 M KCI at 4°C. The homogenate was centrifuged at 9000 × g for 10 min to yield the supernatant $9 fraction.
Mutagenicity assay BP and 3MC were purchased from Sigma Chemical Co. (St. Louis, MO). 3MCE was a generous gift of Dr. Peter P. Fu of the National Center for Toxicological Research. The mutagenicities of BP, 3MC and 3MCE were tested in S. typhimurium strain TA98, which detects frameshift mutagens, and strain TAI00, which detects base-pair substitution mutagens, according to the modified test developed by Ames and collaborators (Ames et al., 1975; Maron and Ames, 1983). A cofactor mixture was prepared which consisted of 5.7 mM NADP, 7.1 mM glucose 6-phosphate, 4.7 mM KCI, and 11.4 mM MgCI2 in a 0.14 M sodium phosphate buffer, pH 7.4. An appropriate volume of rat-liver $9 fraction was added so that the final cofactor mixture contained 30 #1 per 0.5 ml of cofactor mixture. The mutagenicity test was carried out by incubation at 37°C in a dry water bath of a test tube containing 0.5 ml of cofactor mixture, 0.1 ml bacteria culture containing l0 s cells, 0.05 ml of DMSO containing 0-10 nmoles of BP, 3MC, or 3MCE and 0.05 ml of 0.14 M sodium phosphate buffer (pH 7.4) containing 0-200 nmoles of Na2SeO3 or 0-200 nmoles of Na2SO3. After incubation for I0 min, 2 ml of top agar, containing the histidine/biotin solution described in Marin and Ames (1983), was added to each test tube. After thorough mixing, the mixture was poured onto a plate containing 25 ml of solidified minimal glucose agar and the His + revertants were scored after incubation at 37°C for 48 h. All assays were performed in triplicate. A H H assay The assay for AHH activity was carried out according to a modification of the fluorometric procedure developed by Nebert and Gelboin (1968). The assay was carried out in a l-ml mixture containing 50 nmoles of Tris-HCl (pH 7.5), 3 nmoles of MgCI2, 0.5 mg of NADPH, an appropriate volume of rat-liver $9 fraction containing 0.1-0.2 mg of protein, 80 nmoles of BP (added in 40/zl of acetone), and 0-200 nmoles of Na2SeO3 (Alfa Products, Ventron Corporation) or 0-200 nmoles of
103
Na2SO3 (Sigma Chemical Co.). The volume of the mixture was adjusted to 1 ml by the addition of water. Protein was determined by the colorimetric method of Lowry et al. (1951). A unit of AHH activity was expressed in pmoles of phenolic products formed/min/mg protein using 3-hydroxybenzo[alpyrene (from Chemical Repository of National Cancer Institute) as the fluorescence standard.
Results
In S. typhimurium strains TA98 and TA100, $9 was required to convert BP, 3MC, and 3 MCE to mutagens (Table 1). In the absence of $9, these 3
PAHs were not mutagenic in either strain and yielded the background number of spontaneous revertants observed in the DMSO solvent control. The DMSO solvent control and up to 200 nmoles of Se were not mutagenic in TA98 and TA100 with or without $9 present. A viable cell count of 10s cells per plate with no apparent cytotoxicity was observed in all assays of Se alone, each pAH alone, and Se in combination with each PAH. The mutagenicity of BP, 3MC and 3 MCE in S. typhimurium TA98 in the presence of $9 was decreased by Se. The mutagenic response of 4 nmoles BP to 20, 100 and 200 nmoles Se was reduced to 71, 52 and 39070, respectively, of that with BP alone. The mutagenic responses of 10
TABLE 1 EFFECTS OF S E L E N I U M (Se) AND SULFUR (S) ON T H E M U T A G E N I C I T Y OF BP, 3MC A N D 3MCE T O W A R D S. t y p h i m u r i u m TA98 AND TA100 Chemical
DMSO DMSO
Se or S (nmoles/
$9 a
H i s + revertants per plate ( ± S D ) (0/0 of control)
TA98/Se b
TA100/Se b
TA 98/ S c
TAI00/S c
0 0
+
25 _+ 7 21 ± 5
110 ± 29 124 ± 19
39 ± 7 40 ± 4
110 ± 29 108 ± 6
Se (or S) a Se (or S)d
200 200
+
21 ± 2 17 ± 6
144 ± 21 156 ± 7
24 ± 5 32 ± 5
110 + 25 112 ± 11
BP (4 nmoles)
0 0 20 100 200
+ + + +
22 245 174 128 96
7 21 26 16 13
(100070) (71070) (52%) (390/0)
136 922 630 410 420
4 49 31 20 38
(100%) (6807o) (44°70) (46%)
46 280 271 270 265
0 0 20
+ +
39 + 4 134 + 16 113 ± 13
(100070) (84%)
100 ± 10 956 ± 37 716 ± 91
(1000/0) (75%)
39 ± 4 130 ± 9 167 ± 32
109 ± 4 1050 ± 39 1066 ± 127
100 200
+ +
93 + 6 89 ± 12
(69070) (66070)
489 ± 99 497 ± 42
(51%) (52070)
148 ± 40 145 + 27
968 ± 19 870 ± 5
0 0
+
50 _+ 4 1546 ± 75
(1000/0)
20 100 200
+ + +
1036 _+ 38 914 ± 156 935 ± 152
3MC (10 nmoles)
3MCE (10 nmoles)
± ± + ± ±
± ± ± ± ±
± ± + ± ±
2 13 16 25 16
122 _+ 10 2164 ± 241
50 + 4 (100070) 2702 ± 24 e
(670/0) 2124 ± 37 (590/o) 1667 ± 44 ( 6 0 ° 7 0 ) 1390 ± 116
(980/0) 2717 ± 185 (770/0) 2441 ± 212 ( 6 4 0 7 o ) 2414 ± 494
83 966 1056 987 1026
± ± ± ± ±
22 40 39 60 21
182 + 35 2592 ± 298 e 2509 ± 181 2773 ± 64 2450 ± 50
a In the presence ( + ) or absence ( - ) of $9. b Assays in the presence or absence of Se. Assays in the presence or absence of S. 0 Assays in the presence or absence of either Se or S as noted in footnotes b and ~ e These two sets of experiments were carried out several months after the other experiments were completed. The H i s ÷ revertants scored were reproducible for each time period.
104 nmoles 3MC and of 10 nmoles 3MCE, a minor metabolite of 3MC (Thakker et al., 1978), were reduced by 20, 100 and 200 nmoles Se to 84, 69 and 66070 respectively, of 3MC alone, and to 67, 59 and 60070, respectively, of 3MCE alone. The mutagenicity of BP, 3MC and 3MCE in S. typhimurium TAI00 in the presence of $9 was also decreased by Se. The mutagenic response of 4 nmoles BP to 20, 100 and 200 nmoles Se was reduced to 68, 44 and 46070, respectively, of that with BP alone. The mutagenic responses of 10 nmoles each of 3MC and 3MCE were reduced by 20, 100 and 200 nmoles Se to 75, 51 and 52070, respectively of 3MC alone, and to 98, 77 and 64070, respectively, of 3MCE alone. Assays of the AHH activity in the S9-preparation were reduced by 20, 100 and 200 nmoles Se to 94, 71 and 59070, respectively, of the AHH activity in the absence of Se. No decrease in AHH activity was observed with Na2SO3. A representative set of control data from parallel experiments carried out with S as Na2SO3 in TA98 is illustrated in Table 1. For these experiments the background number of spontaneous revertants in TA98 for the DMSO solvent control was 40 ± 4 revertants per plate. Addition of 200 nmoles S as Na2SO3 yielded 32 ± 5 revertants per plate, which was comparable with the spontaneous level. The data in Table 1 show that BP, 3MC and 3MCE yielded the spontaneous number of revertants in assays without $9 present in both TA98 and TA100. Increased numbers of revertants (280 + 13 revertants per plate for BP, 130 + 9 revertants per plate for 3MC, and 2702 ± 24 revertants per plate for 3MCE in Table 1) were observed in assays with $9 activation in TA98. Addition of 20-200 nmoles S as Na2SO3 to BP, 3MC or 3MCE assays in TA98 and in TA100 did not markedly decrease the mutagenicity as did Se; however a slight decrease in mutagenicity of 3MC was observed with 200 nmoles S in TA100.
Discussion The results in this study suggest that Se may play
an important role as an inhibitor of the metabolic activation of BP, 3MC and 3MCE. The doses of PAH selected for these studies induced the optimal mutagenic response in the dose-response curves and were 4 nmoles BP, 10 nmoles 3MC and 10 nmoles 3MCE. Addition of Se decreased the mutagenicity of all 3 PAHs in strain TA98, which detects frameshift mutagens, and in strain TA100, which detects base-pair substitution mutagens. Se (200 nmoles) decreased the mutagenicity of BP, 3MC and 3MCE to 39, 66 and 60°70 of their respective controls in strain TA98, and to 46, 52 and 64°70 of their respective controls in strain TAI00. The effective molar ratios of Se to PAH yielding these decreases in each strain were 50:1 (Se:BP), 20:1 (Se:3MC), and 20:1 (Se:3MCE) and were similar to ratios of Se to AAF and Se to N-OH-AAF reported for decreased mutagenicity (Jacobs, Matney, and Griffin, 1977). Addition of 200 nmoles Se to AHH assays decreased this enzyme activity to 59°70 of the control assay in the absence of Se. Because Se and S are related elements that can form isologous compounds such as selenomethionine and methionine, the Se effects were compared with the S controls in this study. The decreases in mutagenicities and AHH activity were apparently a specific effect of Se, since Na2SO3, added to parallel bacterial and enzyme assay mixtures in the same concentrations as NaESeO3, did not decrease either activity. The inhibition of AHH activity by Se suggests the interactions between Se and PAH-metabolizing enzyme systems which caused reductions in mutagenicities in strains TA98 and TA100 by BP, 3MC and 3 MCE.
Acknowledgement This work was supported by an Independent Research and Development award from The MITRE Corporation through the Henry M. Jackson Foundation for the Advancement of Military Medicine Protocol No. G17586 at the Uniformed Services University of the Health Sciences.
105
References Ames, B.N., J. McCann and E. Yamasaki (1975) Methods for detecting carcinogens and mutagens with the Salmonella/ mammalian-microsome mutagenicity test, Mutation Res., 31, 347-364. Clayton, C.C., and C.A. Baumann (1949) Diet and azo dye tumors: effect of diet during a period when the dye is not fed, Cancer Res., 9, 575-582. Griffin, A.C., and M.M. Jacobs (1977) Effects of selenium on azo dye hepatocarcinogenesis, Cancer Lett., 3, 177-181. Harr, J.R., J.H. Exon, P.H. Weswig and P.D. Whanger (1973) Relationship of dietary selenium concentration; chemical cancer induction, and tissue concentration of selenium in rats, Clin. Toxicol., 6, 287-293. Ip, C. (1981) Factors influencing the anticarcinogenic efficacy of selenium in dimethylbenzanthracene-induced mammary tumorigenesis in rats, Cancer Res., 41, 2683-2686. Jacobs, M.M., B. Jansson and A.C. Griffin (1977a) Inhibitory effects of selenium on 1,2-dimethylhydrazine and methylazoxymethanol acetate induction of colon tumors, Cancer Lett., 2, 133-138. Jacobs, M.M., T.S. Matney and A.C. Griffin (1977b) Inhibitory effects of selenium on the mutagenicity of 2-acetylaminofluorene (AAF) and AAF derivatives, Cancer Lett., 2, 319-322. Lin, W.S., C. Scrimshaw and M. Kapoor (1984) Selenium suppresses the metabolism of benzo[a]pyrene by rat-liver extracts, and exerts a dual effect on its mutagenicity, Xenobiotica, 14, 893-902. Lo, L.W., J. Koropatnick and H.F. Stich (1978) The mutagenicity and cytotoxicity of selenite, 'activated' selenite and selenate for normal and DNA repair-deficient human fibroblasts, Mutation Res., 49, 305-312. Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall (1951 ) Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193, 265-275. Maron, D.M., and B.N. Ames (1983) Revised methods for the Salmonella mutagenicity test, Mutation Res., 113, 173-215. Marshall, M.V., M.A. Rasco, M.M. Jacobs and A.C. Griffin (1979) Selenium effects on the carcinogenicity and metabolism of 2-acetylaminofluorene, Cancer Lett., 7, 331-338.
Martin, S.E., G.H. Adams, M. Schillaci and J.A. Milner (1981) Antimutagenic effects of selenium on acridine orange and 7,12-dimethylbenzanthracene in the Ames Salmonella/ microsomal system, Mutation Res., 82, 41-46. Medina, D., and F. Shephard (1981) Selenium-mediated inhibition of 7,12-dimethylbenzanthracene-induced mouse mammary tumorigenesis, Carcinogenesis, 2, 451-455. Nebert, D.W., and H.V. Gelboin (1968) Substrate-inducible microsomal aryl hydroxylase in mammalian cell culture, I. Assay and properties of induced enzyme, J. Biol. Chem., 243, 6242-6249. Rasco, M.A., M.M. Jacobs and A.C. Griffin (1977) Effects of selenium on aryl hydrocarbon hydroxylase activity in cultured human lymphocytes, Cancer Lett., 3, 295-301. Ray, J.A., L.C. Altenburg and M.M. Jacobs (1978) Effect of sodium selenite and methyl methanesulfonate or Nhydroxy-2-acetyl-aminofluorene co-exposure on sister chromatid exchange production in human whole blood cultures, Mutation Res., 57, 359-368. Rosin, M.P., and H.F. Stitch (1979) Assessment of the use of the Salmonella mutagenesis assay to determine the influence of antioxidants on carcinogen-induced mutagenicity, Int. J. Cancer, 23, 722-727. Schrauzer, G.N., and D. Ishmael (1974) Effects of selenium and of arsenic on the genesis of spontaneous mammary tumors in inbred C3H mice, Ann. Clin. Lab. Sci., 2, 441-447. Shamberger, R.J. (1970) Relationship of selenium to cancer, I. Inhibitory effect of selenium on carcinogenesis, J. Natl. Cancer Inst., 44, 931-936. Shamberger, R.J. (1985) The genotoxicity of selenium, Mutation Res., 154, 29-48. Thakker, D.R., W. Levin, T.A. Stoning, A.H. Conney and D.M. Jerina (1978) Metabolism of 3-methycholanthrene by rat liver microsomes and a highly purified monooxygenase system with and without epoxide hydrolase, in: P.W. Jones and R.I. Freudenthal (Eds.), Carcinogenesis, Vol. 3, Polynuclear Aromatic Hydrocarbons, Raven, New York, pp. 253-264. Vernie, L.N. (1984) Selenium in carcinogenesis, Biochim. Biophys. Acta, 738, 203-217. Communicated by R.J. Preston