Search

This paper describes the total synthesis of thelephantin G, thus revising the proposed structure 1 to 2. The key steps involved a double Suzuki-Miyaura coupling and an esterification reaction. By a similar strategy, ganbajunins D and E (3 and 4) were also prepared. Compound 2 strongly inhibited TNF (tumor necrosis factor)-alpha production in rat basophilic leukemia (RBL-2H3) cells: IC(50) = 3.5 nM, while a mixture of 1 and its regioisomer 15 showed no such activity.

Biosynthetic feeding studies of [1- 13 C], [2- 13 C], and [1,2- 13 C 2 ]-labeled sodium acetates into 5-epihydroxyvertinolide, a new sorbicillinoid, gives an incorporation pattern that proves the γ-lactone ring formation associated with a ring cleavage reaction of the precursor, a potential intermediate of sorbicillinol biosynthesis.

Vialinin A, a powerful inhibitor (IC50 90 pM) of TNF-alpha production, was synthesized from sesamol in 11 steps with 28% overall yield. The key reactions include a double Suzuki coupling of electron-rich aryl triflate with phenylboronic acid and an oxidative deprotection of bis-MOM ether. In addition, the related synthetic studies also suggest the necessity for structural revision of ganbajunin C, a positional isomer of vialinin A.

The retention of deoxynivalenol in noodles and bread made from naturally-contaminated flour was examined by a chemical analysis (HPLC) and bioassays. The retention level of deoxynivalenol obtained from both assays was reduced by boiling process, although only the bioassays showed it to have been reduced by baking. This study is the first to estimate the exposure to deoxynivalenol from the consumption of the final products of wheat flour in Japan.

A post-treatment assay of the umu test was performed to detect genotoxicity of 10 phenylenediamine (PDA) derivatives using Salmonella typhimurium TA1535/pSK1002 with/without S9 mix. Seven chemicals (o-PDA, 4-chloro-o-PDA, 4-nitro-o-PDA, p-PDA, 2-chloro-p-PDA, 2-nitro-p-PDA, and 2,5-diaminotoluene) showed positive results with S9 mix, but three chemicals (m-PDA, 4-chloro-m-PDA, and 2,4-diaminotoluene) were negative with and without S9 mix. Four of 7 chemicals (o-PDA, 4-chloro-o-PDA, 4-nitro-o-PDA, and 2-nitro-p-PDA) that gave positive results with S9 mix were also positive without S9 mix. These results indicate that the genotoxicity of PDA derivative possessing m-position amino substituents was not detected in the umu post-treatment assay using TA1535/pSK1002.

While screening for bioactive compounds from edible mushrooms, a new potent antioxidant, vialinin A (1), together with a known compound, ganbajunin B (2), and a mixture of ganbajunins D (3) and E (4), were isolated from the dry fruiting bodies of Thelephora vialis. The structure of 1, 5',6'-bis(phenylacetoxy)-1,1':4',1''-terphenyl-2',3',4,4''-tetraol, was elucidated by spectroscopic and chemical methods. This compound had strong 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical-scavenging activity with an EC(50) value of 14.0 microM, nearly equal to that of butylated hydroxytoluene (BHT; EC(50) = 10.0 microM). A radical scavenging experiment using 1 and DPPH radicals indicated that 1 donated two hydrogen atoms to two molecules of the DPPH radical under hydrophobic conditions.

Fungi growing on domestic rice were examined from April to June, 2003. One hundred samples of rice, which had been harvested in the autumn of 2002, were collected from the local market, and 15 samples of stored rice, which had been harvested in 2001 and stored in warehouses under government control, were used as samples. From each sample, 50 grains (100 grains in total) were plated on potato-dextrose agar (PDA) and malt yeast 40% sucrose agar (M40YA) containing chloramphenicol after being washed with sterile distilled water to remove any microorganisms on the surface, and incubated at 25 degrees C for a week. For most of the rice samples harvested in the preceding year, the proportion of grains infected with fungi was less than 20% of the total grains tested. In about half the samples of rice stored for one and half years, more than 80% of the grains were infected with fungi that grew on M40YA. The major genera of fungi isolated from the rice harvested in the preceding year were Penicillium and Alternaria, and those from the rice stored for one and a half years were Aspergillus, Penicillium and Eurotium. P. islandicum, A. versicolor, A. ochraceus and others were isolated as possible mycotoxin-producers in the mycoflora of domestic rice. P. islandicum was isolated from 3 samples, and 82% of the grains were infected with this fungus in one sample. All three isolates from these samples appeared to produce luteoskyrin on Czapek yeast extract agar, based on TLC and HPLC analysis.

There is a need for simple rapid tests for evaluating the carcinogenic potential of the thousands of chemical compounds that are developed each year. The DNA-damaging effects of 83 National Toxicology Program (NTP) chemicals, including noncarcinogens and carcinogens, were examined in the umu test by measuring the expression of the umuDC-lacZ genes in Salmonella typhimurium TA1535/pSK1002. Salmonella were exposed to individual NTP chemicals at 37°C for 2 hr both with and without a rat liver S9 mix; the treated cells were then diluted and incubated for a further 2 hr (posttreatment assay). O-nitrophenyl-β-D-galactoside was added to the cultures and the β-galactosidase activity driven by the Salmonella umuDC-lacZ genes was determined by measurement of the OD420 nm and OD550 nm of the cultures. Salmonella cell number was simultaneously determined by measurement of OD600 nm. The overall concordance between genotoxicity in the umu test and carcinogenicity was 67%, which was similar to the concordance between Ames' test results and carcinogenicity (63%) using the same 83 NTP chemicals. The results of this study indicate that the umu test with a single Salmonella strain is a simple rapid system, with accuracy comparable to existing, more time-consuming assays. Environ. Mol. Mutagen. 44:329–345, 2004. © 2004 Wiley-Liss, Inc.

To clarify whether di (2-ethylhexyl) phthalate (DEHP) has immunotoxic effects on both the expression of surface molecules (CD3, CD4, CD8 and CD28) on T cells of the thymus and spleen in male C57BL/6 mice.Animals were orally administered a 0.1% or 0.2% DEHP-containing diet for 10 or 20 days. Dietary corn oil was used as the vehicle for DEHP in preparing the diet.Significant hepatic hypertrophy was clearly observed in the DEHP-exposed groups, while no atrophy was seen in the thymus or spleen in any treatment groups. In the thymus and spleen, no variation in the proportions of both T cells expressing CD3, CD4 and CD8 was shown with cytometry analysis. The surface expression of CD3, CD4, CD8 and CD28 on both T cells was also invariable in all analyzed stages of thymic differentiation and in the spleen. No effect of DEHP on mitogenesis was shown in the splenic T cells with anin vitro [(3)H]-thymidine-incorporation technique.DEHP is probably not an immunosuppressor, particularly for T cells.

Summary 1,4-Dioxane is a nongenotoxic hepatocarcinogen but in our previous replicative DNA synthesis (RDS) studies with the [ 3 H]thymidine (TdR)-technique, it failed to increase hepatocyte RDS values when given by gavage to male F344 rats as a single 2000 mg/kg body weight dose. However, in a current series of trials with TdR, it showed equivocal responses 24 or 48 hr following treatment with 2000 mg/kg in time-course experiments, and positive responses 24 hr following 1000, 1500 and 2000 mg/kg in dose-response experiments. An increased RDS incidence was also observed at the dose of 2000 mg/kg with data for 5-bromo-2′-deoxyuridine (BrdU)-incorporation. These present findings thus support the hypothesis that a capacity to induce cell proliferation may play a key role in 1,4-dioxane hepatocarcinogenesis.

A novel dibenzofuran compound designated vialinin B was isolated from dry fruiting bodies of an edible mushroom, Thelephora vialis, and potently inhibits TNF-alpha production in RBL-2H3 cells (IC(50)=0.02nM) and is a promising anti-allergic agent.

The hypothesis has been proposed that cell proliferation, or replicative DNA synthesis (RDS) in S-phase cells, is a nongenotoxic (Ames-negative) mechanism involved in tumorigenesis, providing a very useful conceptual basis for carcinogen testing. In this present study, hepatocyte RDS experiments were conducted using 5-bromo-2'-deoxyuridine (BrdU) labeling in combination with histopathological observation, comparing our results with earlier findings for in situ [3H]thymidine (TdR) labeling. The present BrdU data proved to be consistent with the previous TdR data in all but one case. Hepatocyte RDS induction was observed for some chemicals without hepatotoxicity. BrdU labeling in combination with histopathological observation is therefore a reliable approach to assessment of test compound effects in vivo.

According to current data, the capacity to cause nonprogrammed or unscheduled cell proliferation in target tissues, a common characteristic of chemical carcinogens, may play a more important role in the development of tumors than does genotoxicity. This paper provides strong support for the validity of this conclusion. Ames-negative nongenotoxicants may be considered to be carcinogenic primarily because of their ability to induce cell proliferation in animal tissues and organs. In addition, such nongenotoxic carcinogens may also provide latent and modest DNA (equivocal) modifications that never lead to Ames-positive events. Conversely, noncarcinogenesis by Ames-positive agents is likely to be linked to a lack of stimulation of cell division. Nongenotoxic and genotoxic carcinogens rely on both cell proliferation and equivocal DNA modification for their full carcinogenicity. Such equivocal DNA modifications do not appear to be formed by tumor promoters. The role of cell proliferation may provide a favorable milieu for the occurrence of genetic instability, give rise to selective "apoptosis-resistant abnormal cells," and then affect clonal expansion of these cells. Therefore, understanding the influence of nongenotoxic and genotoxic carcinogens on cell proliferation capability is a key point in determining the mechanisms of chemical carcinogenesis. Considering the contradictory and common features of genotoxicants and carcinogens, early detection of nonprogrammed cell proliferation is the most effective approach to predict human and rodent carcinogenicity.

In vivo-in vitro hepatocyte replicative DNA synthesis (RDS) tests using male F344 rats and B6C3F1 mice were conducted in the present study applying single treatments of 56 nongenotoxic (Ames-negative) hepatocarcinogens, 7 genotoxic (Ames-positive) hepatocarcinogens, 31 nongenotoxic and genotoxic noncarcinogens, and the correlation between early increased RDS events leading to cell proliferation and hepatocarcinogenesis was then reviewed. In the RDS test, 51 of the 63 nongenotoxic and genotoxic hepatocarcinogens clearly induced increased RDS events, whereas it was very rare for noncarcinogens to cause such increase. A good correlation between early RDS induction and hepatocarcinogenicity was thus achieved. The results suggest that nongenotoxic hepatocarcinogens always give equivocal DNA modifications which never lead to Ames-positive events, such as extremely low levels of oxidative DNA adducts and/or alteration of DNA methylation levels (hypomethylation). Genotoxic hepatocarcinogens might simultaneously act as tumor promoters in multistage carcinogenesis and genetic instability-inducing agents. That early disruption of cell cycle controls, resulting in induction of RDS events by nongenotoxic and genotoxic hepatocarcinogens, is postulated to contribute to carcinogenesis in line with a process of increasing genetic instability. In the present review, the current status of the correlation between cell proliferation and hepatocarcinogenesis is also discussed.

Transgenic Drosophila which carry a canine cytochrome P-450 were established and evaluated for its value on toxicology testing. Dah1, a cDNA clone for the canine hepatic P-4501A1, was ligated between the promoter of Drosophila heat-shock protein gene and the terminator of actin 5C gene, and then microinjected into Drosophila embryos with a transposon P element. The injected DNA was stably integrated into chromosomal DNA in germ line cells. After heat-shock treatments, transgenic larvae produced mRNA and a detectable amount of protein that cross-reacted with antibodies against rat P-4501A1. The ability of transgenic flies to activate procarcinogens was monitored in the DNA repair test with the endpoint being preadult mortality of repair-defective mei-9a mei-41D5/Y males. Heat-shock treatments of larvae showed that the repair-defective males carrying the P-4501A1 transgene were killed by lower concentrations of 7,12-dimethylbenz[a]anthracene than their non-transgenic or non heat-shocked counterparts. Coadministration of alpha-naphthoflavone, one of the inhibitors of P-4501A subfamily, cancelled the enhanced mortality of transgenic males, supporting that 7,12-dimethylbenz[a]anthracene was metabolized to its genotoxic form by the expressed P-4501A1 enzyme. This study indicates a potential utility of transgenic Drosophila for screening mammalian promutagens and procarcinogens.

This paper describes the total synthesis of thelephantin G, thus revising the proposed structure 1 to 2. The key steps involved a double Suzuki−Miyaura coupling and an esterification reaction. By a similar strategy, ganbajunins D and E (3 and 4) were also prepared. Compound 2 strongly inhibited TNF (tumor necrosis factor)-α production in rat basophilic leukemia (RBL-2H3) cells: IC50 = 3.5 nM, while a mixture of 1 and its regioisomer 15 showed no such activity.

A first total synthesis of vialinin B, a powerful inhibitor (IC(50) 20 pM) of TNF-alpha production, is described. The key reactions include a double Suzuki-Miyaura coupling of electron-rich aryl bromide with a couple of phenylboronic acids, a Cu-mediated Ullmann reaction, and a LHMDS-promoted phenylacetylation. This synthesis proceeded in 11 steps with 18% overall yield from a known sesamol derivative.

Increasing attention has been focused on food-drug interactions. We have investigated the inhibitory effect of Chinese edible mushrooms, Boletus calopus and Suillus bovinus, on cytochrome P450 (CYP) 1A2, 2C9, 2D6, and 3A4, the main drug-metabolizing enzymes. Three pulvinic acid derivatives, atromentic acid (1), variegatic acid (2), and xerocomic acid (3), isolated from Boletus calopus and Suillus bovinus, revealed nonspecific inhibitory effects on all four CYPs. Using these compounds, the maximum IC50 values obtained with CYP3A4 in vitro were atromentic acid (1), 65.1+/-3.9 microM; variegatic acid (2), 2.2+/-0.1 microM; and xerocomic acid (3), 2.4+/-0.1 microM. Variegatic acid (2) and xerocomic acid (3) were effective inhibitors, comparable to cimetidine, dicoumarol, erythromycin, safrole, and uniconazole. Variegatic acid (2) and xerocomic acid (3) efficiently reduced ferryl myoglobin in CYPs. Reduction of ferryl heme to ferric heme is likely the mechanism of the nonspecific inhibitory effects of these compounds on CYPs.

Thelephora vialis is a mushroom that grows in symbiosis with pine trees in Yunnan, China, a place known to have some of the richest and most diverse bioresources in the world. This is one of the most favored edible mushrooms, due to its flavor. Our screening for bioactive compounds from these mushrooms isolated a novel potent antioxidant, vialinin A, together with known compounds, from the dry fruiting bodies of T. vialis. Vialinin A is a terphenyl derivative and was elucidated by spectroscopic and chemical methods. Vialinin A showed anti-allergic activities, inhibition of beta-hexosaminidase, tumor necrosis factor (TNF)-alpha, interleukin 4 and monocyte chemotactic protein 1 release from RBL-2H3 cells, whereas atromentin and an inseparable mixture of ganbajunins D and E showed no such effects. Vialinin A displayed potent inhibition of TNF-alpha production from RBL-2H3 cells (IC50, 0.09+/-0.01 nM), indicating stronger inhibition than tacrolimus for organ transplantation (IC50, 0.25+/-0.03 nM). The potent inhibitory activities of these compounds against TNF-alpha production indicate promising new candidates for anti-allergic agents.

In order to examine the ability of the umu test detecting system, 18 antitumor drugs were tested using the Salmonella umu test. The tested antitumor drugs were selected so as to produce different biochemical actions, and they were classified into three categories; five agents of group I (antimetabolites), eight agents of group II (alkylating agents), and five agents of group III (antibiotics). The results showed that all antimetabolites, all alkylating agents, and three of the antibiotics had positive responses, but the antibiotics aclarubicin (ACR) and chromomycin A3 (CHR) had negative responses. Both antibiotics that gave negative responses were anthracyclines, but daunomycin (DNR), which was one of the anthracyclines, had a positive result in the umu test. These results suggest that it is possible for the umu test to detect genotoxicity of chemicals regardless of the types of DNA damage (inhibition of DNA synthesis relative enzyme, DNA base alkylating, DNA strand-break, and DNA adduct), but difficult for it to detect genotoxicity of any anthracyclines.

Soy-protein isolate (SPI) enhances liver cell damage in Long-Evans rats with a cinnamon-like coat color (LEC rats), which have a defect in Atp7b, the Wilson disease gene. Animals administered an SPI-diet from an age of six weeks died significantly earlier than those administered a control-diet, AIN-93G, from severe liver cell damage associated with jaundice. Since the liver copper level was higher with the SPI-diet than the control-diet, one of the reasons for SPI-toxicity to LEC rats might be due to the higher uptake of copper into liver cells. In the present study, liver levels of glutathione, and liver and intestinal mRNA and protein levels were determined for metallothionein, MT-1 and MT-2. Furthermore, liver and intestinal mRNA expression for the high affinity copper transporter, Ctr1, was determined. None of the parameters showed any significant differences between the SPI-diet and control-diet groups, except for Ctr1 mRNA levels in the liver. It is thus suggested that SPI enhances liver cell copper uptake through induction of Ctr1 expression and this might be the mechanism underlying increased liver damage in LEC rats.

Griseofulvin (GF), a carcinogenic spindle poison, was tested in two types of somatic-cell assays of Drosophila melanogaster, one of which detects the induction of DNA damage and the other mutation/mitotic recombination. In both assays, GF was fed to tester larvae and genetic endpoints examined after emergence. In the wing spot test, trans-heterozygous flies carrying mwh and flr3 wing-hair mutations produced both significant and dose-dependent increases in the frequency of mwh single spots over the control level but no increase of twin spots. In the DNA repair test, double-mutant larvae carrying both mei-9(a) (excision repair-defective) and mei-41(D5) (postreplication repair-defective) mutations showed hypersensitivity to killing by GF compared with their DNA repair-proficient counterparts, suggesting that GF caused potentially lethal DNA damages which were efficiently repaired by the DNA repair-proficient but not -defective larvae. These lines of evidence clearly demonstrate that GF is genotoxic in somatic cells of Drosophila. It is noted that (1) GF-fed larvae showed a developmental delay and (2) surviving adult flies had morphological abnormalities in their eyes and wings.

To evaluate the applicability of an in vivo-in vitro replicative DNA synthesis (RDS) test using rat hepatocytes, we conducted the RDS test with 22 nongenotoxic (Ames-negative) hepatocarcinogens and 25 noncarcinogens under our standardized conditions and judgement criteria. Compared to controls (RDS incidence of under 1.0%), the RDS test gave positive results for 18 hepatocarcinogens (positive sensitivity: 82%), and negative results for 20 noncarcinogens (negative specificity: 80%), and thus the overall concordance was 81%. These findings strongly suggest that the RDS test is an extremely useful method for early detection of nongenotoxic hepatocarcinogens.

Distilled styrene showed mutagenic activity towards Salmonella typhimurium TA 100 after activ ation with hepatic 9,000 X g supernatant fractions (S-9). The S-9 fractions ,had been obtained from rats treated with 3-methylcholanthrene or phenobarbital and fortified with an NADPH-generating system in the presence of 3,3,3-trichloropropene oxide (TCPO), an hepatic microsomal epoxide hydratase inhibitor. No mutagenic activity was observed when either the TCPO was omitted or rats were treated with a polychlorinated biphenyl mixture (PCBs). The S-9 fraction from 3-methylcholanthrene-treated rats activated styrene more effectively than the liver homogenate from phenobarbital-treated animals. As has already been pointed out, phenyloxirane, an intermediate in the hepatic microsomal biotransformation of styrene, induced the mutagenesis of TA 100 cells in the absence of 8-9 and TCPO, but the mutagenesis was totally abolished by the addition of S-9. A further addition of TCPO partially restored the mutagenetic activity of phenyloxirane. Gas chromatographic data on phenyloxirane and I-phenyl1,2-ethanediol in the testing system for styrene ind icat ed that phenyloxirane could not account for the mutagenesis induced by the hepatic activation of styrene since sums of phenyloxirane and its hydrolytic product, I-phenyl-l,2-ethanediol (formed from styrene during incubation with 8-9 in the presence and absence of TCPO) were much smaller than the amount which induced mutagenesis; they also showed that PCBs were the most potent inducers for hepatic rnonooxygenase, which catalyzes the conversion of styrene to phenyloxirane. PCBs induced hepatic monooxygenase approximately 1.4 times as effectively as 3-methylcholanthrene, in spite of its inability to increase the hepatic activation system for the mutagenicity of styrene. There is therefore a possibility that at least one more unknown active metabolite with an epoxide structure is present. The ar ene oxide I-vinvlbenzene 3,4-oxide, which was synthesized in our laboratory and was assumed to be a precusor to 4-vinylphenol (a previously isolated urinary metabolite of styrene), had a potent killing effect but no mutagenic activity towards TA 100 cells. A search for the ultimate mutagen is now in progress in our laboratory. l4C-styrene bound covalently to microsomal protein of rat liver when incubated in the presence of an NADPH-generating system. The absence of the cofactor decreased the binding remarkably. The NADPH-dependent protein-binding was increased by TCPO in vitro and by the phenobarbital pretreatment of rats in vivo. Pretreatment of rats with carbon tetrachloride decreased the amount of radioactivity bound to hepatic microsomal protein.

Phenacetin is mutagenic in Salmonella typhimurium TA 100 when liver 9,000 X g supernatant fractions from PCB-treated hamsters instead of rats are used. A mechanism of the species difference in phenacetin mutagenicity was investigated. By high-performance liquid chromatography analysis, it was found that phenacetin is activated to direct-acting mutagens through N-hydroxylation and deacetylation by hamster liver microsomes. Although no significant species difference was observed in N-hydroxylation, rates of deacetylation were 9 to 150 times higher in hamsters than in rats. The results indicate that the marked species difference in phenacetin mutagenicity is due to the difference in deacetylation activity between rat and hamster liver microsomes.

Four isogenic strains of Escherichia coli with the same auxotrophic marker ( arg F am — namely wild-type, uvrA − , polA − and recA − ) were used for testing the lethalities and mutagenicities of of 1-naphthyl N -methyl- N -nitrosocarbamate (nitroso-NAC), 3-methylphenyl N -methyl- N -nitrosocarbamate (nitroso-MTMC), and 3,4-dimethylphenyl N -methyl- N -nitrosocarbonate (nitroso-MPMC). The strains recA − and polA − showed a similarly higher sensitivity to killing than wild-type and uvrA − after treatments wuth each of the three chemicals, whereas the strains wild-type , uvrA − , and polA − were equally mutable by these compounds at equal doses. The strain recA − was hardly mutable by nitroso-NAC, but significant levels of Arg + mutations were observed after treatments with nitroso-MTMC and nitroso-MPMC. These and previous results suggest that both nitroso-MTMC and nitroso-MPMC are similar in their mutagenicity pattern to N -methyl- N ′-nitro- N -nitrosoguanidine whereas nitroso-NAC is similar to methyl methanesulfonate or X-rays, and that the major damage to DNA of the three agents is not excisable by the uvrA + -dependent excision repair, probably methylation in DNA.

The nitrosation of methyl N-methylanthranilate (MMA), which is permitted to be used to foods as flavor in Japan, with equimolar nitrite was measured at 37°C at the concentrations of 10-1-10mM of both reactants. MMA was nitrosated very easily, and when 0.1mM of MMA was reacted with equimolar sodium nitrite at pH 2.0 fbr 5min and at pH 3.0 for 20min, the yields of methyl N-methyl-N-nitrosoanthranilate (NO-MMA) were 89.5% and 82.6%, respectively. The rate of nitrosation was found to be dependent on the acidity of reaction mixture and the highest yields of NO-MMA was obtained below pH 2.0 at the MMA concentration of 0.2mM for 5min. A mutation test of NO-MMA was carried out, but NO-MMA was non-mutagenic to Salmonella typhimurium tester strain TA 100 and TA 98 in the presence and absence of the metabolic activation system (S9 mix).

Mutagenicity of 6 aminobenzene derivatives against Salmonella typhimurium TA98 was studied in the presence of various S9 fractions. S9, which has been prepared form the livers of rats, hamsters and mice after pretreatment with different types of inducers; polychlorinated biphenyls, phenobarbital and 3-methylcholanthrene, was used as the methabolic activating enzyme in this mutation assay. The S9 fractions from 3-methylcholanthrene-treated rats and mice are most useful for mutation induction by the all aminobenzenes used. The mutagenic activity of the compounds was clearly correlated to 3-methylcholanthrene-induced cytochrome P-450. However, any significant correlation between aniline hydroxylase activity and the mutagenesis was not observed.

Comparison studies for detecting differences between liver microsome and S9 preparations from 4 strains (Donryu, Fischer, Sprague-Dawley, Wistar) of young male rats were carried out with pretreatment of the animals by inducers such as PCBs and PB plus 5,6-BF. Each microsome fraction was assayed for the enzymic activity of metabolism of model substrates such as aniline, benzophetamine, BP, DMN and 7-ethoxycoumarin. The hepatic S9 sample was also compared, as regards its metabolizing ability to activate 9 pre-mutagens (2AA, AAF, o-AAT, BP, DAB, DMBA, DMN, m-PDA, quinoline) to directly acting mutagens in the Salmonella/hepatic S9 activation test by using TA98, TA100 and TA1537 strains with or without cytochrome P450 inhibitors (SKF-525A, metyrapone, 7,8-benzo-flavone). In the enzymic assay with PCBs-induced microsomes, BP hydroxylation a strain-specific difference: the microsomes from Fischer and Wistar rats were more effective for metabolizing BP than those from the other strains of rat. The effect of induction by BP plus 5,6-BF for Fischer rats showed relatively higher enzymic activity in the same induction group. Other microsomes prepared from rats with and without induction by PB plus, 5,6-BF did not show a clear-cut strain dependency in the enzymic activities assayed. In the mutation experiments with hepatic S9 samples, the examination of DAB and quinoline revealed a marked strain difference when S9 samples prepared from PCBs-pretreated and PB-plus-5,6-BF-induced rats were used: the S9 sample from Fischer rats was available for activating the two pre-mutagens to directly acting mutagens. No marked difference in the metabolic activation of the remaining 7-pre-mutagens was observed on other S9 preparations. In examinations of mutagenicity activities with the use of three inhibitors, the two S9 preparations made with the two induction methods showed inhibition profiles closely similar to each other. However, there were minor differences in the profiles by these inhibitors. From these findings it was concluded that Fischer rat-liver S9 is useful for detecting mutagens in the metabolic activation test, when induction by PB plus 5,6-BF was used in the Ames Salmonella test.

The comparative cytogenetic and mutagenic effects between trivalent and hexavalent chromium were investigated. Five chromium compounds, K 2 Cr 2 O 7 and K 2 CrO 4 containing Cr 6+ , and Cr(CH 3 COO) 3 , Cr(NO 3 ) 3 and CrCl 3 containing Cr 3+ , were examined for their ability to induce chromosomal damage in cultures of human leukocytes, for their reactivity with DNA by a rec-assay system and for mutagenicity in the E. coli Hs30R test system. Chromosome-breaking activity was significantly higher for the compounds with hexavalent than trivalent chromium, the efficiency being in the decreasing order K 2 Cr 2 O 7 > K 2 CrO 4 >> Cr(CHCOO) 3 > Cr(NO 3 ) 3 , CrCl 3 . In the rec-assay and mutation assay, hexavalent (K 2 Cr 2 O 7 and K 2 CrO 4 ) and trivalent Cr(CH 3 COO) 3 ) compounds gave positive results, their mutagenic potential being higher in the same order of clastogenic magnitude.

The alcohol extracts freshly prepared from gamma-irradiated potatoes were examined for their mutagenic activity in bacterial and mammalian cell systems. Negative results were obtained from all following test systems: Mutation assays with Salmonella typhimurium His- strains such as TA 100, TA 98, TA 1535, TA 1537, and streptomycin-dependent mutant (SMd) strain, TA 100-10, inductests with Escherichia coli strains, K 12 GY 5027 and K 12 C600, chromosomal aberration tests with Chinese hamster cells in culture, as well as micronucleus tests in mice. In addition, no difference in the mutagenic activities was found between extracts prepared from the irradiated and the unirradiated potatoes, suggesting that no mutagenic substance was produced in potatoes following gamma-irradiation.

The mechanism of species difference in N-hydroxyphenacetin mutagenicity was investigated by using the Salmonella/microsome mutagenicity test and high-performance liquid chromatographic (HPLC) analysis. Mutagenicity of N-hydroxyphenacetin in Salmonella typhimurium TA100 was about 10 times more efficiently detected with a liver 9000g supernatant fraction (S9) from hamsters than with the corresponding fraction from rats in the absence of a reduced nicotinamide adenine dinucleotide phosphate-generating system. Paraoxon and sodium fluoride, both inhibitors of microsomal amidase, not only inhibited the mutagenicity of N-hydroxyphenacetin, but also retarded the formation of the deacetylation product, p-nitrosophenetole, a strong intrinsic mutagen. The N-hydroxyphenacetin-deacetylation activity was about 20 times higher in liver microsomes from the hamster than in those from the rat.

A comparative study on enzymic factors influencing the metabolic inactivation of phenyloxirane (styrene oxide), a major mutagenic metabolite of styrene in the liver, was carried out with respect to soluble glutathione S-transferase and microsomal epoxide hydratase in the 9000 X g supernatant fraction (S9) from a rat-liver homogenate. The mutagenic activity of phenyloxirane to Salmonella typhimurium TA100 was markedly reduced by S9 in the presence of glutathione but to a smaller extent in its absence. The retarding effect of glutathione on the inherent mutagenic activity of phenyloxirane was exerted by the soluble supernatant of S9 but not by microsomes. A gas-liquid chromatographic study indicated that the effect of glutathione was attributable to the disappearance of the mutagen from the microbial assay system. The rate of the disappearance was 10-20 times as fast in the soluble supernatant fraction as in the microsomes when fortified with more than 4 mM glutathione. Our results strongly suggest that in hepatic cells of the rat, cytosol glutathione S-transferase plays a much more important role than microsomal epoxide hydratase in the detoxication of the metabolite, phenyloxirane.

Styrene showed a weak mutagenic activity towards Salmonella typhimurium TA 100 after being activated by the hepatic 9000g supernatant fraction (S9) obtained from rats pretreated with 3-methylcholanthrene or phenobarbital and fortified with an NADPH-generating system, in the presence of the epoxide hydratase inhibitor, 3, 3, 3-trichloropropene oxide (TCPO). The 3-methylcholanthrene-pretreated rat liver S9 activated styrene more effectively than the phenobarbital treated one. No mutagenic activity was observed when either TCPO or S9 was omitted. As has already been pointed out, phenyloxirane, an intermediate in the major metabolic pathway of styrene by hepatic microsomes, induced mutations in TA 100 and TA 1535 cells in the absence of both S9 and TCPO. Analytical data of phenyloxirane and its hydrolytic product, 1-phenyl-1, 2-ethanediol, formed in the mutation assay system for styrene indicated that the inducers used for hepatic drug-metabolizing enzymes enhanced both microsomal monooxygenase and epoxide hydratase activities. Relative ratios of the enhanced monooxygenase to hydratase activities were twice as high in 3-methylcholanthrene as the control and 1.8 and 1.2 times in phenobarbital and PCB, respectively, although the last enhanced both activities to the highest extent. Phenyloxirane was accumulated in the presence of TCPO in the styrene-activating system most significantly when 3-methylcholanthrene-pretreated rat liver S9 was used. However, the mutagenicity exerted by the metabolic activation of styrene could not necessarily be explained only by phenyloxirane since sums of both metabolites were smaller than the amount of the epoxide required for inducing the mutation in Salmonella and suggested a possibility of the presence of at least one more unknown mutagenic metabolite with an epoxide structure.

Biotransformation of styrene (ethenylbenzene) into styrene glycol (1-phenyl-1, 2-ethanediol) via styrene oxide (phenyloxirane), a mutagen and skin carcinogen, by rat liver microsomes in the presence of an NADPH-generating system has been investigated. Both metabolites were identified by gas-chromatography-mass spectroscopy. In the microsomal system, styrene oxide appeared only after a brief incubation and disapeared thereafter in spite of the continued formation of styrene glycol. This phenomenon was rationally interpreted on the basis of previously obtained evidence that during aerobic incubations, NADPH-dependent microsomal lipid peroxidation decreased P-450 activities selectively to a remarkable extent compared with epoxide hydratase activities. Addition of 3, 3, 3-trichloropropene oxide, a potent epoxide hydratase inhibitor, to the incubation medium prolonged the biological half-life of styrene oxide significantly, indicating one of major factors determining the biological level of the mutagen to be the relative ratio of these enzyme activities.