Induction of sister-chromatid exchanges by indirect mutagens/carcinogens in cultured rat hepatoma and esophageal tumor cells and in Chinese hamster Don cells co-cultivated with rat cells

2 rat cell lines originated from ascites hepatoma AH66-B and esophageal tumor R1 were examined for their inducibility of sister-chromatid exchanges (SCEs) after treatment with 14 kinds of indirect mutagens/carcinogens, including 6 amine derivatives, 4 azo compounds, 3 aromatic hydrocarbons and 1 steroid. Of the 14 chemicals tested, 2-acetylaminofluorene (AAF), butylbutanolnitrosamine (BBN), dimethylnitrosamine (DMN), cyclophosphamide (CP), urethane, 2-methyl-4-dimethylaminoazobenzene (2-MeDAB), 3′-methyl-4-dimethylaminoazobenzene (3′-MeDAB), 4-o-tolylazo-o-toluidine (4-TT), benzo[a]pyrene (BP), 7,12-dimethyl-benz[a]anthracene (DMBA) and diethylstilbestrol (DES) were estimated to be effective inducers of SCEs in AH66-B and/or R1 cells, without the use of exogenous activating systems. Cell-mediated SCE tests with 6 selected chemicals, CP, 2-MeDAB, 4-TT, BP, DMBA and DES, showed a significant increase of SCEs in Chinese hamster Don-6 cells co-cultivated with AH66-B or R1 cells, depending on the number and sensitivity of AH66-B or R1 cells, as well as on the dose of chemicals tested, whereas singly cultured Don-6 cells were much less sensitive or almost insensitive to these chemicals. The above findings suggest that AH66-B and R1 cells may retain metabolic activities to convert a wide range of indirect mutagens/carcinogens into their active forms to induce SCEs, and that these cell lines provide simple and reliable screening systems in vitro, including the cell-mediated SCE assay, for detection of genotoxic agents, without the use of exogenous activation systems.     

Effect of retinoids on carcinogen-induced mutagenesis in Salmonella tester strains

The ability of retinol (Rol) in altering mutation frequencies induced by 7 carcinogens was studied in Salmonella/microsome assay using 4 tester strains namely TA98, TA100, TA102 and TA1535. The 7 carcinogens used were aflatoxin B1 (AFB), cyclophosphamide (CPP), 3-methylcholanthrene (MCA), benzo[a]pyrene (BP), benz[a]anthracene (BA), 9,10-dimethyl-1,2-benz[a]anthracene (DMBA) and mitomycin C (MMC). As reported previously, Rol significantly reduced the number of His+ revertants induced by AFB. It also reduced mutations induced by CPP or MCA but not that by BP, BA, DMBA or MMC. The abilities of Rol, retinoic acid, retinyl acetate and a known inhibitor for certain P-450 isozymes, 7,8-benzoflavone (BF) in inhibiting mutations caused by AFB and BP were studied and compared. All the 3 retinoids caused significant reduction of AFB-induced His+ revertants in a dose-dependent manner, but there was no effect on BP-induced mutation. BF strongly inhibited both AFB- and BP-induced revertants. The possibility of retinoids in exerting their effects on mutagenesis of precarcinogens by inhibiting only certain forms of cytochrome P-450 enzymes is discussed.     

Effects of arachidonic acid and indomethacin on sister-chromatid exchange induction by polycyclic aromatic hydrocarbons in mammalian cell lines

Arachidonic acid (AA), a prostaglandin precursor, significantly potentiated sister-chromatid exchange (SCE) induction in vitro by benzo[a]pyrene (BP) and 7,12-dimethylbenz[a]anthracene (DMBA) in the aryl hydrocarbon hydroxylase (AHH)-inducible human hepatoma C-HC-4 cells, and to a lesser extent in the non-inducible rat tumor AH66-B and R1 and Chinese hamster Don-6 cells, all of which were less sensitive to these compounds than C-HC-4 cells. Indomethacin (IM), an inhibitor of prostaglandin endoperoxide synthetase (PES), moderately suppressed SCE induction by BP or DMBA in AH66-B and R1 cells, but it exerted no such effect in C-HC-4 and Don-6 cells. In C-HC-4 cells, however, IM completely eliminated the potentiating effect of AA on SCE induction by both BP and DMBA. The above findings suggest that PES in prostaglandin biosynthesis may also be involved in the metabolic activation of polycyclic aromatic hydrocarbons to genotoxic forms capable of inducing SCEs, in addition to AHH system.     

The effects of pretreatment with cytochrome P-450 inducers and preincubation with a cytochrome P-450 effector on the mutagenicity of genotoxic carcinogens mediated by hepatic and renal S9 from two species of marine fish

A series of experiments was designed to characterize the cytochrome P-450-dependent activation of 7 genotoxic carcinogens in the Salmonella preincubation assay by hepatic postmitochondrial fractions (S9) from the oyster toadfish and the Americal eel and by renal S9 from the toadfish. Significant S9-dependent mutagenicity was observed for benzo[a]pyrene (BAP), 2-aminoanthracene (2AA), aflatoxin B1 (AFB1), 7,12-dimethylbenz[a]anthracene (DMBA) and cyclophosphamide (CP) with hepatic S9 from untreated fish (UI S9) of both species and with renal S9 from untreated toadfish, although renal UI S9 was only marginally effective for activating AFB1. Neither UI S9 from toadfish liver or kidney nor that from eel liver consistently affected the direct mutagenicity of ethylene dibromide (EDB) or substantially activated dimethylnitrosamine (DMN). Pretreatment of toadfish with 3-methylcholanthrene (MC) decreased the mutagenicity of 2AA and increased the mutagenicities of BAP, AFB1 and DMBA, whereas, pretreatment of eels with MC increased the mutagenicities of BAP, 2AA and AFB1. Pretreatment of toadfish with Aroclor 1254 (AC) decreased the mutagenicity of AFB1 and increased the mutagenicity of 2AA, whereas, pretreatment of eels with AC increased the mutagenicities of BAP and DMBA. Pretreatment of toadfish with beta-napthoflavone (BNF) effected changes similar to those by pretreatment with MC except that the mutagenicity of AFB1 was not increased. Coincubation with 10(-4) M alpha-napthoflavone (ANF) decreased the mutagenicity of BAP mediated by toadfish MC and BNF S9 and eel AC S9 and decreased the mutagenicity of AFB1 mediated by toadfish MC and BNF S9 and by eel MC S9. Coincubation with ANF increased the mutagenicity of AFB1 mediated by toadfish and eel AC S9 and increased the mutagenicity of 2AA mediated by eel AC S9. Pretreatment of toadfish with MC, BNF and AC decreased the mutagenicity of 2AA mediated by renal S9 and ANF decreased the mutagenicity of 2AA mediated by renal UI and BNF S9. MC pretreatment of toadfish and eels and BNF pretreatment of toadfish induced BAP monooxygenase activity in hepatic microsomes. ANF (10(-4) M) inhibited the BAP monooxygenase activity of MC microsomes from toadfish and eels and of BNF microsomes from toadfish. The conjugation effectors diethyl maleate and salicylamide alone or combined had little or no effect on the mutagenicities of BAP and 2AA mediated by toadfish and eel UI and MC S9.(ABSTRACT TRUNCATED AT 400 WORDS)     

Hepatocyte-mediated SCE induction by indirect mutagens: importance of hepatocyte density and cell-to-cell contact

The SCE-inducing effects of the indirectly acting mutagens cyclophosphamide (CP), dimethylnitrosamine (DMN) and aflatoxin B1 (AFB1) were analysed in hepatocyte (hpc)/mammalian cell coculture systems with regard to the importance of the hpc density. V79 cells and human lymphocytes served as target cells. For all 3 compounds steadily increasing genetic effects were observed when the hpc density was increased from 3.2 X 10(4) up to 3.2 X 10(6) viable hpc per culture (25-cm2 flask), i.e. the more hpc available for metabolisation, the more genetic effects induced. The frequency distributions of the CP-induced SCE values were clearly different from those obtained with DMN, especially when high hpc densities were used: distribution patterns obtained for the mutagen with stable metabolites (CP) are characterized by the presence of distinct maxima and the absence of cells with SCE control values, whereas distribution patterns for the mutagen with very short-lived metabolites (DMN) can be described by the absence of maxima and the presence of cells with SCE control values. The frequency distributions of the AFB1-induced SCE values were more similar to the CP type than to the DMN type. From these results it is deduced that close contact between metabolising and target cells is necessary for the detection of the genotoxic effect of DMN. For CP and AFB1 a direct contact seems not to be essential, i.e. reactive intermediates may also be transported via the culture medium to the target cells.     

Differences in the induction of SCEs between human whole blood cultures and purified lymphocyte cultures and the effect of an S9 mix

Studies for SCE induction are frequently performed on human blood cultures. Either whole blood cultures (WBC) or purified lymphocyte cultures (PLC) are employed. However, it has been shown that fundamental differences with respect to metabolic activity exist between these two systems. In order to further characterize the whole blood culture and the purified lymphocyte culture, differently acting substances were studied comparatively with and without an Aroclor-1254-induced S9 mix. Treatment with ethyl methanesulfonate (EMS), a direct mutagen, produced distinct SCE induction in both systems. Cyclophosphamide (CP) and benzo[a]pyrene (BP), two indirect mutagens, also led to a significant increase of SCEs both in WBC and PLC without S9 mix. Only with CP was this effect more pronounced after addition of S9 mix. Sodium selenite (Na2SeO3), which induced SCEs in WBC, did not show this effect in the PLC. After S9 mix was added to purified lymphocytes, an increase of SCEs by sodium selenite was observed as in WBC. H2O2, a radical former, led to SCE induction in purified lymphocytes but not in the whole blood culture. By adding S9 mix, a distinct reduction of the SCEs induced by H2O2 was established. These results show that human lymphocytes can metabolize indirect mutagens and that it should be kept in mind when using S9 mix that, besides mixed-function oxygenases, it also contains enzymes which influence the SCE-inducing effects of substances.     

Anticlastogenic effects of black tea (World blend) and its two active polyphenols theaflavins and thearubigins in vivo in Swiss albino mice.

This study investigated the inhibition of cyclophosphamide (CP) and dimethylbenz(a)anthracene (DMBA) induced genetic damage by black tea (World blend) and its two active polyphenols theaflavins (TF) and thearubigins (TR) in Swiss albino mice as measured by chromosome aberrations (CA) and sister chromatid exchanges (SCE). Three different concentrations (5, 10 and 20%) of tea and a single dose of TF and TR were tested for their anticlastogenic effects against DMBA (50 mg/kg body weight) and CP (20 mg/kg for CA and 10 mg/kg for SCE). A significant decrease in CA was observed in all the three concentrations of tea extract plus DMBA treated groups when compared with the respective DMBA treated group alone. Similarly a significant decrease in CA was observed in all the three concentrations of tea extracts plus CP treated series when compared with the group treated with CP alone. In SCE assay, a significant decrease in SCE was observed in 5, 10 and 20% black tea extract plus CP and 10 and 20% tea extracts plus DMBA treated groups when compared with the CP or DMBA treated group alone. In the single dose of TF and TR treated groups a significant decrease in both CA and SCE was observed in both the TF and TR plus both the carcinogen treated groups when compared with their positive controls. The protective effects of black tea extracts were more significant than that of its two polyphenols. This study indicates that both black tea and its active polyphenols TF and TR have significant anticlastogenic effects in bone marrow cells of mice.     

Mutagenicity of benzo[a]pyrene 7,8-dihydrodiol and 7,12-dimethylbenz[a]anthracene 3,4-dihydrodiol in S. typhimurium mediated by microsomes from rat liver and mouse skin

The mutagenic activities of trans-7,8-dihydro-7,8-dihydroxybenzo[a]-pyrene (BP 7,8-diol) and of trans-3,4-dihydroxy-7,12-dimethylbenz[a]-anthracene (DMBA 3,4-diol) towards S. typhimurium TA100 were measured in assays that were carried out on a micro-scale in liquid medium in the presence of microsomal fractions prepared from mouse skin or rat liver. In the presence of an NADPH-generating system, microsomal enzymes converted both diols into mutagens that were probably the respective 'bay-region' diol-epoxides. The rate of the enzyme-catalysed conversion of the BP 7,8-diol into mutagens by microsomal preparations from mouse epidermis was similar to that occurring with microsomes from rat liver. Pretreatment of mice by the topical application of benz[a]anthracene (BA) or 7,12-dimethylbenz[a]-anthracene (DMBA) increased the mutagenic activity of BP 7,8-diol mediated by mouse skin microsomal preparations by 2-fold and this was paralleled by a 4-fold increase in epidermal aryl hydrocarbon (benzo[a]pyrene) hydroxylase (AHH) activity. The results are discussed in relation to the high susceptibility of mouse skin to polycyclic aromatic hydrocarbon (PAH) carcinogenesis.     

Inhibitory effects of coffee on the genotoxicity of carcinogens in mice

The mouse bone marrow micronucleus test was carried out to evaluate the possible inhibitory effects of 3 doses (125, 250 and 500 mg/kg) of standard instant coffee on the in vivo genotoxicity of 7,12-dimethylbenz[a]anthracene (DMBA), benzo[a]pyrene (BP), aflatoxin B1 (AFB1) and urethane (UR). Coffee was orally administered twice, 2 and 20 h before the carcinogens were injected intraperitoneally. From the results obtained, it was evident that the administration of 250 and 500 mg coffee/kg body weight could significantly inhibit the in vivo genotoxicity of these carcinogens. A linear dose response was observed for the inhibitory effect of coffee. Furthermore, inhibition of genotoxicity by coffee was observed in bone marrow cells which were sampled at 6-h intervals (48, 54, 60, 66 and 72 h) from the time of peak induction of micronuclei by DMBA.     

The Liverbeads as a tool for the comet assay

Liverbeads, cryopreserved hepatocytes entrapped within an alginate matrix, were examined for their relevance in the comet assay. It was estimated by their capacity to activate the indirectly acting mutagens, cyclophosphamide (CP), benzo[a]pyrene (BP), dimethylbenzanthracene (DMBA) and 2-acetylaminofluorene (2-AAF), into DNA reactive metabolites. The comet assay performed in alkaline condition is a sensitive method for detecting strand breaks at the level of individual cells and allows use of quiescent cells. Experimental conditions as treatment time, cell density, beads dissociation and viability were investigated. Significant statistical positive results assessed by the tail extent moment (TEM) were observed with both human and rat Liverbeads after 12h duration incubation compared to metabolic non-competent cells, HeLa S3. Due to the maintenance of specific functions assessed by the observed capacity to metabolize xenobiotics, Liverbeads represent a suitable tool system, easy to handle, for the detection of promutagens using the comet assay.     

The effect of agent treatment time on the induction of sister-chromatid exchanges in mouse bone marrow cells in vivo

The effect of time of agent administration, via intraperitoneal injection, on the yield of SCEs in bone marrow cells of male B6C3F1 mice was determined for cyclophosphamide (CP), 7,12-dimethylbenz[a]anthracene (DMBA) and mitomycin C (MMC). Animals were treated with several doses of each carcinogen/mutagen at 3 different treatment times: -1, +1 and +8 h in relation to the onset of BrdUrd administration. The results of these studies indicate that the optimal treatment time for inducing a maximal SCE response is agent-specific. For CP, the slope of the SCE response was greatest at the +8 h treatment time while the maximal response for DMBA occurred at the -1 h treatment time. For MMC, the slope of the SCE response was independent of treatment time and of the method of bromodeoxyuridine administration (intravenous infusion vs. tablet implantation) but dependent on the laboratory conducting the study (Brookhaven National Laboratory vs. Oak Ridge Associated Universities). Based on the results of these studies, the +1 h acute treatment time is considered optimal for the in vivo cytogenetic evaluation of suspect chemicals for genotoxic activity when bone marrow is used as the target cell population.     

Survival and mutagenesis of bacterial plasmids with localized carcinogen adducts

Induction of 6-thioguanine (TG) resistance by chemical mutagens was examined in a line of cells derived from a human epithelial teratocarcinoma cell clone. The cells, designated as P3 cells, have a stable diploid karyotype with 46(XX) chromosomes, including a translocation between chromosomes 15 and 20. Efficient recovery of TG-resistant mutants induced by the direct-acting mutagens: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG); 7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10 -tetrahydrobenzo[a]pyrene (BPDE); and benzo[a]pyrene (B[a]P); activated in a cell-mediated assay, required an expression time of 7 days and a saturation density of 2 X 10(4) cells/60-mm petri dish. The TG-resistant mutant cells induced by MNNG and BPDE maintained their resistant phenotype 4-6 weeks after isolation. This mutant phenotype was associated with a more than 10-fold reduction in hypoxanthine-guanine phosphoribosyl transferase (HGPRT) activity relative to that of the parental P3 cell line, which was shown to catalyze the formation of 4.6 pmoles inosine-5'-monophosphate (IMP)/min/microgram protein. Induction of TG resistance was also observed in P3 cells cocultivated in a cell-mediated assay with human breast carcinoma cells, which are capable of polycyclic aromatic hydrocarbon (PAH) metabolism, after treatment with the carcinogenic PAHs: B[a]P, chrysene, 7,12-dimethylbenz[a]anthracene (DMBA), and 3-methylcholanthrene (MCA). The degree of mutant induction in this assay was related to the carcinogenic potency of these PAHs in experimental animals. The most potent mutagen was DMBA, followed in decreasing order by MCA, B[a]P, and chrysene. DMBA, at 0.4 microM, increased the frequency of mutants for TG resistance from 2 for the control to about 200 TG-resistant mutants/10(6) colony-forming cells (CFC). Benzo[e]pyrene (B[e]P) and pyrene, which are not carcinogenic, were not effective in the assay. None of the PAHs was mutagenic in the P3 cells cultivated in the absence of the PAH-metabolizing cells. These results indicate that the P3 cells can be useful for the study of mutagenesis at the HGPRT locus by direct-acting chemical mutagens, as well as by chemicals activated in a cell-mediated assay.     

The effect of agent dose and treatment time on the intercellular distribution of sister-chromatid exchanges induced by genotoxic agents in mouse bone marrow cells in vivo

Using two methods of bromodeoxyuridine (BrdUrd) administration and three genotoxic chemicals, the effects of dose and treatment time on the intercellular distribution of sister-chromatid exchanges (SCE) in the bone marrow of male B6C3F1 mice were evaluated. The dispersion of SCE among solvent control mice infused intravenously with BrdUrd or implanted subcutaneously with a BrdUrd tablet partially coated with paraffin was largely consistent with a Poisson model. Intraperitoneal treatment with cyclophosphamide (CP; solvent = phosphate-buffered saline), 7,12-dimethylbenzanthracene (DMBA; solvent = corn oil) and, in mice infused with BrdUrd, mitomycin C (MMC; solvent = phosphate-buffered saline) induced a significant increase in SCE, the distribution of which was not distributed as a Poisson. For CP and MMC, the increase in dispersion was dose-dependent and independent of treatment time (-1, +1 or +8 h in relation to the start of the BrdUrd treatment). The lack of a treatment time effect suggests that there were no significant differences among treatment times in the distribution of the reactive forms of these two chemicals, no variation in cell-stage sensitivity, and no cellular toxicity to modulate the response. For DMBA, the increased dispersion of induced SCE depended on treatment time and was not simply related to dose. The increase in dispersion was agent-specific; at equal levels of SCE induction, the distribution of SCE in mice treated with DMBA exhibited greater dispersion than SCE in mice treated with either CP or MMC. These differences between DMBA and CP/MMC are probably due to DMBA's slower absorption/distribution kinetics, its requirement for metabolic activation to genotoxic metabolites and its extended half-life. These data suggest that analyzing the distribution of SCE, in addition to mean frequency, is a useful method for evaluating agent specific patterns in SCE induction.     

Comparative genotoxicity of 3 procarcinogens in V79 cells as related to glutathione S-transferase activity of hepatocytes from untreated rats and those fed 2% butylated hydroxyanisole

When 7,12-dimethylbenz[a]anthracene (DMBA) and aflatoxin B1 (AFB1) were activated by hepatocytes from Fischer 344 rats fed a diet containing 2% butylated hydroxyanisole (BHA), frequencies of mutation to 6-thioguanine resistance (TGR) at the HGPRTase gene locus and to ouabain resistance (OuR) at the Na+,K(+)-ATPase gene locus in V79 cells were 30-70% less than those obtained with hepatocytes from untreated controls. A difference in the mutation frequency did not occur when dimethylnitrosamine (DMN) was activated by BHA induced- rather than control-hepatocytes. Analysis of hepatocytes from rats fed 2% BHA showed a small (1.5-fold), but significant, increase in glutathione levels over that in the controls but no change in activity of cytochrome P450. Cytosolic glutathione S-transferase (GST) activity was increased 2-3-fold in hepatocytes from rats fed the 2% BHA diet. These results suggest that mutagenic response to DMBA and AFB1 is reduced, at least in part, because of BHA-induction of hepatocyte GST activity; while activation of DMN can occur by pathway(s) unaffected by BHA-induction of these liver enzymes. In contrast to mutation frequencies, significant differences between BHA- and control-activation in the production of sister-chromatid exchange (SCE) and micronucleus formation (MN) were not detected with any of the genotoxins. It was concluded that the mechanism(s) by which SCE and MN occur are likely unrelated to the capacity of BHA to induced activity of hepatic enzymes, e.g. the GSH S-transferases, that directly or indirectly affect mutation end-points.     

Effects of caffeine on sister-chromatid exchanges (SCE) in vivo.

Chinese hamsters were twice treated with caffeine via stomach tube. The single doses were either 20, 100, 200 or 400 mg per kg body weight. A dose-dependent increase was observed in the frequencies of SCE induced in vivo in bone-marrow cells. Two intraperitoneal injections of the chemical mutagens, cyclophosphamide or benzo[a]pyrene, led to a pronounced increase of the frequency of SCE. Simultaneous applications of the chemical mutagens and caffeine decreased the rate of SCE. The effect of caffeine per se to induce SCE, and the mechanisms by which caffeine reduces the level of SCE induced by chemical mutagens are discussed.     

Skin cytogenetic assay for the detection of clastogens-carcinogens topically administered to mice.

A method for assessing the effect of clastogens on mouse skin epidermal cells was devised and applied. Toxic and mutagenic responses in epidermal cells were tested using two known mutagens and carcinogens, urethane (URE) and 7,12-dimethylbenz[a]anthracene (DMBA). Cell generation time, sister-chromatid exchanges (SCE) and chromosomal aberrations (CA) after topical and intraperitoneal (i.p.) treatment were measured in epidermal and bone marrow cells. After topical administration both tissues responded similarly, whereas after i.p. treatment skin cells were less responsive than bone marrow cells. However, the results indicate the validity of this new cytogenetic approach for the assessment of the genotoxicity of compounds applied directly to skin.     

Mechanisms of regulation of rat ovarian 7,12-dimethylbenz[a]anthracene hydroxylase

The mechanisms of regulation of ovarian 7,12-dimethylbenz[a]anthracene (DMBA) hydroxylase were investigated with respect to hormonal requirements and effects of the antiestrogen tamoxifen and known inducers of cytochrome P-450. The DMBA hydroxylase is increased endogenously about 3-fold in the proestrus phase as compared to the metestrus/diestrus phases (M. Bengtsson and J. Rydstrom, Science, 219 (1983) 1437-1438). A similar effect was caused by the gonadotropins follicle-stimulating hormone (FSH) and luteinizing hormone (LH) whereas pregnant mare's serum gonadotropin (PMSG) brought about a 3-7-fold increase, suggesting that the estrus cycle-dependence of the DMBA hydroxylase was due directly or indirectly to gonadotropins. In contrast, differentiation of granulosa/theca cells to corpus luteum cells after ovulation, caused by administration of human chorionic gonadotropin (hCG), led to a marked decrease in activity. The activity was not specific for DMBA since substitution of this hydrocarbon for benzo[a]pyrene (BP) as substrate gave similar results. A possible role of estrogens in this context was investigated by the administration of tamoxifen simultaneous with gonadotropin treatment, which caused a partial inhibition of the hydroxylase activity. Both estradiol and 3-methyl-cholanthrene (MC) increased DMBA hydroxylase but the effects of these agents were not additive. In contrast, the effects of estradiol and MC were partially additive to that of gonadotropin. On the basis of these results, it is proposed that the rat ovary contains one or several aryl hydrocarbon hydroxylases located in the granulosa/theca cells which are regulated by estrogens, MC and beta-naphthoflavone (BNF) and that the role of gonadotropins is to proliferate granulosa/theca cells.     

Antagonizing effect of 3-aminoharman on induction of sister-chromatid exchanges by mutagens

3-Aminoharman (3AH, 3-amino-1-methyl-9H-pyrido[3,4-b]indole), which has been reported as a novel substance with an antagonistic effect on induction of sister-chromatid exchange (SCE) by polycyclic mutagens in the presence of the metabolic activation system, was examined with a cultured human lymphoblastoid cell line, NL3, for its effect on SCE induction by direct-acting mutagens such as mitomycin C (MMC), nitrogen mustard N-oxide (NMO), methyl methanesulfonate (MMS), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 4-nitroquinoline 1-oxide (4NQO) and 3-hydroxyamino-1-methyl-5H-pyrido[4,3-b]indole (OH-Trp-P-2), and also by ultraviolet light (UV) irradiation. The results obtained on simultaneous treatment with 3AH and mutagens were as follows: (1) 3AH suppressed more than 50% of SCEs induced by MMC, NMO and OH-Trp-P-2; (2) 4NQO- and MNNG-induced SCEs were also suppressed by 3AH but to a lesser degree; (3) MMS-induced SCEs were not, however, altered by 3AH; and (4) the suppression of SCE by 3AH was dose-dependent. Treatment of cells with 3AH for 2 h immediately before MMC exposure suppressed SCE induction to a significant degree similar to the simultaneous treatment, but post-treatment with 3AH was much less effective. 3AH inhibited SCE induction by NMO when 3AH treatment was carried out either before or after NMO treatment, to an extent similar to the simultaneous treatment. Treatments with 3AH either before or after UV exposure did not change the UV-induced SCEs. Results with these direct-acting mutagens ruled out the relevance of metabolic activation as a necessary step for the antagonizing effect of 3AH.     

Modifying effects of the enzyme inducers, phenobarbital and 3-methylcholanthrene, on dominant lethal events induced by 7, 12-dimethylbenz[a]anthracene in mice

The effects of pretreatment with either phenobarbital (PB) or 3-methylcholanthrene (MC) on the induction of dominant lethal events by 7, 12-dimethylbenz[a]anthracene (DMBA) in male mice were studied. DMBA induced dominant lethals in both post- and pre-meiotic germ cells of mice. The incidences of DMBA-induced dominant lethals were markedly reduced in pre-meiotic germ cells by the pretreatment with PB, whereas a significant reduction of the lethal events in post-meiotic germ cells was observed with MC. No significant reduction of living implants was detected in pre-meiotic germ cells on pretreatment with PB. The contents of liver microsomal cytochrome p-450 of mice pretreated with PB or MC were about twice those of non-treated mice.     

Mutagenicity and metabolism of dimethylnitrosamine and benzo[alpha]pyrene in tissue homogenates from inbred Syrian hamsters treated with phenobarbital, 3-methylcholanthrene or polychlorinated biphenyls.

There are significant differences between mice and hamsters in polycyclic hydrocarbon and nitrosamine metabolism. Homogenates of liver, lung and intestinal mucosa from 6 strains of Syrian golden hamster were compared for their ability to metabolize benzo[alpha]pyrene (BP) and dimethylnitrosamine (DMN) to mutagens. Females of strains MHA/SSLak, LSH/SlLak, CB/SsLak, PD4/Lak LHC/Lak and Lak:LVG (SYR) were either untreated or received phenobarbital (PB), 3-methylcholanthrene (MC) or polychlorinated biphenyls (AR) to induce drug-metabolizing enzymes. Salmonella typhimurium TA92 and TA98 were used as indicators of the formation of mutagans. Dimethylnitrosamine demethylase (DMND) was assayed using 1 mM DMN as substrate. Aryl hydrocarbon hydroxylase (AHH) was measured using benzo[alpha]pyrene as substrate. MC does not induced AHH activity in hamster liver, but is an excellent inducer of enzymes converting BP to mutagens. This lack of correlation between increased AHH activity and increased metabolism of BP to mutagen in liver is in marked contrast to correlations seen in mice. MC induces AHH in hamster lung and intestinal mucosa. AR induces AHH in liver, lung and intestinal mucosa. Activity of DMND in liver is not affected by treatment of hamsters with BP or AR, but is repressed approx. 30% by treatment with MC. Activity of DMND and conversion of DMN to mutagen are correlated (r = 0.59) in hamster liver. Microsomes of hamster liver are more effective than those from mouse in converting DMN to mutagen, despite similar DMND activities in livers from the two species.