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The nuclear receptor constitutive androstane receptor (CAR) regulates the expression of enzymes involved in endobiotic and xenobiotic metabolism. CAR is essential for liver tumor promotion by phenobarbital, a prototype tumor promoter of mice. The mechanism by which chronic microbial infection influences tumor progression is unknown. However, previous studies have suggested a role of endobiotics including certain bile acids in tumor promotion. We investigated the mechanism of liver tumor promotion by Helicobacter hepaticus (Hh), the prototype carcinogenic bacterium of mice. Twenty two CAR-/(KO) and 23 CAR+/+ (wild-type, WT) male mice (C3H/HeN) received a single intraperitoneal injection of the genotoxic carcinogen diethylnitrosamine (DEN) at 5 weeks of age. Another group consisting of 21 KO and 20 WT male mice did not receive DEN. The two groups (with and without DEN) were subsequently stratified into four subgroups (two KO and two WT) and orally inoculated with either Hh or sterile media at 8 weeks of age. Mice were euthanized at 50 weeks postinoculation, complete necropsies were performed, and samples were collected for histopathological, microbiological, molecular, and biochemical analyzes. Hepatic Hh colonization as well as hepatic expression of selected nuclear receptors, cytokines, and innate immune mediators were measured by real-time quantitative PCR. The serum concentrations of selected mono-, di-, and tri-hydroxylated bile acids were measured by liquid chromatography tandem mass spectrometry. Chronic infection with Hh induced hepatitis in WT and KO mice with or without DEN. However, relative to Hh-infected WT mice with DEN, Hhinfected KO mice with DEN exhibited the following statistically significant differences: Increased number of liver lobes with dysplasia/neoplasia (P

The effect of dose rate on radiation-induced mutations in two somatic tissues, the spleen and liver, was examined in transgenic gpt delta mice. These mice can be used for the detection of deletion-type mutations, and these are the major type of mutation induced by radiation. The dose rates examined were 920 mGy/min, 1 mGy/min and 12.5 microGy/min. In both tissues, the number of mutations increased with increasing dose at each of the three dose rates examined. The mutation induction rate was dependent on the dose rate. The mutation induction rate was higher in the spleen than in the liver at the medium dose rate but was similar in the two tissues at the high and low dose rates. The mutation induction rate in the liver did not show much change between the medium and low dose rates. Analysis of the molecular nature of the mutations indicated that 2- to 1,000-bp deletion mutations were specifically induced by radiation in both tissues after high- and low-dose-rate irradiation. The occurrence of deletion mutation without any sequence homology at the break point was elevated in spleen after high-dose-rate irradiation. The results indicate that the mutagenic effects of radiation in somatic tissues are dependent on dose rate and that there is some variability between tissues.

A sensitive umu test system for the detection of mutagenic nitroarenes has been developed using a new tester strain Salmonella typhimurium NM1011 having a high nitroreductase activity. The new strain was constructed by subcloning the bacterial nitroreductase gene into a plasmid pACYC184 and introducing the plasmid into the original strain S. typhimurium TA1535/pSK1002 harboring a fusion gene umuC′-′lacZ (pSK1002). Thus, the tester strain enabled us to monitor the genotoxic activities of various nitroarene compounds by measuring the β-galactosidase activity in the cells. The sensitivity of strain NM1011 was compared with that of the parent tester strain S. typhimurium TA1535/pSK1002 or a nitroreductase-deficient strain S. typhimurium NM1000 with respect to the induction of umuC gene expression by 17 mutagenic nitroarenes. The newly developed strain with high nitroreductase activity had about 3 times higher nitrofurazone-reductase activvity than the parent strain and was highly sensitive to the compounds 2-nitrofluorene, 1-nitronaptthalene, 2-nitronaphthalene, 1-nitropyrene, m-dinitrobenzene, 4,4′-dinitrobiphenyl, 3-nitrofluoranthene, 3,7-dinitrofluoranthene, 3,9-dinitrofluoranthene, 5-nitroacenaphthene and 2,4-dinitrotoluene. By contrast, the enzyme-deficient strain did not show any considerable response to 2-nitrofluorene, m-dinitrobenzene,, 1-nitronaphthalene, 2-nitronaphthalene, 1-nitropyrene, 4,4′-dinitrobiphenyl, 3-nitrofluoranthene, 3,7-dinitrofluoranthene, 2,4-dinitrotoluene and 5-nitroacenaphthene. These results suggest that the newly developed tester strain with high nitroreductase activity is very useful for the detection of potent mutagenic nitroarene compounds.

1,4-Dioxane is primarily used as industrial solvent for paints, surface-treating agent for artificial leather and is a stabilizer for chlorinated solvent. However, little is known about the in vivo mutagenic effects of this rat liver carcinogen. The purpose of the present study is to investigate the dose-response relationship for in vivo mutagenicity and carcinogenicity of 1,4-dioxane in the livers of gpt delta transgenic rats, an animal model for in vivo mutagenicity analysis. Thirty, 6-week-old male gpt delta rats were divided into four groups and received 1,4-dioxane in drinking water at the concentrations of 0, 200, 1000, 5000 ppm up to 16 weeks, respectively. After the completion of treatment, all the rats were killed under ether anesthesia and livers were excised. Halves of the livers were fixed in 10% buffered formalin followed by paraffin embedding for histological analysis and the rests were frozen for molecular analysis. The number of glutathione S-transferase placental form (GST-P)-positive foci, a rat liver preneoplastic marker and PCNA-positive cell index, a marker for cell proliferation, were determined immunohistochemically, and mutation frequency followed by DNA sequencing was examined using frozen liver tissue. The number of GST-P-positive foci and PCNA-positive cell index were significantly (p Mutation analysis revealed that treatment with 5000 ppm significantly (p

Background Recently, manufactured nano/microparticles such as fullerenes (C60), carbon black (CB) and ceramic fiber are being widely used because of their desirable properties in industrial, medical and cosmetic fields. However, there are few data on these particles in mammalian mutagenesis and carcinogenesis. To examine genotoxic effects by C60, CB and kaolin, an in vitro micronuclei (MN) test was conducted with human lung cancer cell line, A549 cells. In addition, DNA damage and mutations were analyzed by in vivo assay systems using male C57BL/6J or gpt delta transgenic mice which were intratracheally instilled with single or multiple doses of 0.2 mg per animal of particles. Results In in vitro genotoxic analysis, increased MN frequencies were observed in A549 cells treated with C60, CB and kaolin in a dose-dependent manner. These three nano/microparticles also induced DNA damage in the lungs of C57BL/6J mice measured by comet assay. Moreover, single or multiple instillations of C60 and kaolin, increased either or both of gpt and Spi- mutant frequencies in the lungs of gpt delta transgenic mice. Mutation spectra analysis showed transversions were predominant, and more than 60% of the base substitutions occurred at G:C base pairs in the gpt genes. The G:C to C:G transversion was commonly increased by these particle instillations. Conclusion Manufactured nano/microparticles, CB, C60 and kaolin, were shown to be genotoxic in in vitro and in vivo assay systems.

The umuDC locus of Escherichia coli is required for most mutagenesis by UV and many chemicals. Mutations in E. coli umuDC genes cloned on pBR322-derived plasmids wer e isolated by two methods. First, spontaneously-arising mutant umuDC plasmids that failed to confe cold-sensitive growth on a lexA51(Def) strain were isolated by selection. Second, mutant umuDC plasmids that affected apparent mutant yield after UV-irradiation in a strain carrying umuD+C+ in the chromosome were isolated by screening hydroxylamine-mutagenized umuD+C+ plasmids. pBR322-derived umuD+C+ plasmids inhibited the induction of the SOS response of lexA+ strains as measured by expression of din::Mu dl(lac) Ap) fusionsbut most mutant plasmids did not. Mutant plasmids defective in complementation of chromosomal umuD44, umuC36, or both were found among those selected for failure to confer cold-sensitivity, whereas those identified by the screening procedure yielded mostly mutant plasmids with more complex phenotypes. We studied in greater detail a plasmid pLM109, carrying the umuC125 mutation. This plasmid increased the sensitivity of lexA+ strainsto killing by UV-irradiation but was able to complement the deficiencies of umuC mutants in UV mutagenesis. pLM109 failed to confer cold-sensitive growth on lexA(Def) strains but inhibited SOS induction in lexA+ strains. The effect of pLM109 on the UV sensitivity of lexA(Def)strains was similar to that of the parental umuD+C+ plasmid. The mutation responsible for the phenotypes of pLM109 was localized to a 615-bp fragment. DNA sequencing revealed that the umuC125 mutation was a G:C → A:T transition that changed codon 39 of umuC from GCC → GTC thus changing Ala39 to Val39. The implications of the umuC125 mutation for umuDC-dependent effects on UV-mutagenesis and cell survival after UV damage are discussed.

Expression of the umuDC operon is required for UV and most chemical mutagenesis in Escherichia coli. The DNA which can restore UV mutability to a umuD44 strain and to a umuC122::Tn5 strain of E. coli has been cloned from Salmonella typhimurium TA1538. DNA sequence analysis indicated that the cloned DNA potentially encoded proteins with calculated molecular weights of 15,523 and 47,726 and was an analog of the E. coli umuDC operon. We have termed this cloned DNA the samAB (for Salmonella mutagenesis) operon and tentatively referred to the umuDC operon of S. typhimurium LT2 (C. M. Smith, W. H. Koch, S. B. Franklin, P. L. Foster, T. A. Cebula, and E. Eisenstadt, J. Bacteriol. 172:4964-4978, 1990; S. M. Thomas, H. M. Crowne, S. C. Pidsley, and S. G. Sedgwick, J. Bacteriol. 172:4979-4987, 1990) as the umuDCST operon. The samAB operon is 40% diverged from the umuDCST operon at the nucleotide level. Among five umuDC-like operons so far sequenced, i.e., the samAB, umuDCST, mucAB, impAB, and E. coli umuDC operons, the samAB operon shows the highest similarity to the impAB operon of TP110 plasmid while the umuDCST operon shows the highest similarity to the E. coli umuDC operon. Southern hybridization experiments indicated that (i) S. typhimurium LT2 and TA1538 had both the samAB and the umuDCST operons and (ii) the samAB operon was located in a 60-MDa cryptic plasmid. The umuDCST operon is present in the chromosome. The presence of the two homologous but different umuDC operons may be involved in the poor mutability of S. typhimurium by UV and chemical mutagens.

Salmonella typhimurium YG1021, YG1024, YG1026 and YG1029 are new derivatives of the Ames tester strains TA98 and TA100, with elevated ‘classical’ nitroreductase or acetyl-CoA: N-hydroxyarylamine O-acetyltransferase level. Thirty mutagens with different structures were tested using these strains and the sensitivities were compared with those of the conventional strains and of the enzyme-deficient strains. Elevated O-acetyltransferase activity of the indicator strains specifically increased their ability to detect the mutagenicity of aromatic nitro, amino and hydroxylamino compounds, whereas the strains with high nitroreductase activity were very sensitive to some nitroaromatics. The combined use of the isogenic tester strains with different metabolic capacities was quite useful to assess the intracellular metabolic activation and detoxification mechanisms of chemical mutagens.

Previous genetic studies have indicated that most UV mutagenesis in Escherichia coli requires the participation of the umuD and umuC gene products. However the mechanism of UV mutagenesis is not yet understood and the roles of the UmuD and UmuC proteins have not been elucidated. The umuDC operon is induced by UV irradiation and regulated as part of the SOS response. Genetic evidence now indicates that RecA-mediated cleavage activates UmuD for its role in mutagenesis. The COOH-terminal fragment of UmuD is both necessary and sufficient for this role. The RecA protein appears to have third role in UV mutagenesis besides mediating the cleavage of LexA and UmuD at the time of SOS induction. In addition, we have obtained evidence which indicates that the GroEL and GroES proteins also play a role in UV mutagenesis. Similarities of the amino acid sequence of UmuD to the sequence of gene 45 protein of bacteriophage T4 and of the sequence of UmuC to those of the gene 44 and gene 62 proteins suggest possible roles for UmuD and UmuC in mutagenesis that are supported by preliminary evidence.

Acetyl-CoA: N-hydroxyarylamine O-acetyltransferase is an enzyme involved in the intracellular metabolic activation of arylhydroxylamines derived from mutagenic nitroarenes and aromatic amines. The acetyltransferase gene of Salmonella typhimurium TA1538 was cloned into pBR322 and the plasmids harboring the gene were introduced into TA98 and TA100. The resulting strains (YG1024 and YG1029) had about 100 times higher 2-hydroxyamino-6-methyldipyrido[1,2-a: 3′, 2′-d]-imidazole (N-hydroxy-Glu-P-1) O-acetyltransferase activity than TA1538 containing pBR322, and were extremely sensitive to the mutagenic actions of 2-nitrofluorene, 1-nitropyrene, 1,8-dinitropyrene, 2-amino-6-methyldipyrido[1,2-a: 3′, 2-d]-imidazole (Glu-P-1), 2-aminofluorene and 2-aminoanthracene. These results indicate that the new strains permit the efficient detection of the mutagenicity of environmental nitroarenes and aromatic amines.

Salmonella typhimurium YG1024, an O-acetyltransferase-overproducing derivative of TA98, was found to be more sensitive in detecting mutagenicity in human urine caused by cigarette smoking, than the conventional strain TA98, in Ames test in the presence of S9 mix. YG1021, another derivative of TA98 with elevated nitroreductase levels, did not show increased sensitivity. These results suggest that the mutagenic compounds concentrated by Blue Rayon from smokers' urine are aromatic amino compounds.

The products of the SOS-regulated umuDC operon are required for most UV and chemical mutagenesis in Escherichia coli. The UmuD protein shares homology with a family of proteins that includes LexA and several bacteriophage repressors. UmuD is posttranslationally activated for its role in mutagenesis by a RecA-mediated proteolytic cleavage that yields UmuD'. A set of missense mutants of umuD was isolated and shown to encode mutant UmuD proteins that are deficient in RecA-mediated cleavage in vivo. Most of these mutations are dominant to umuD+ with respect to UV mutagenesis yet do not interfere with SOS induction. Although both UmuD and UmuD' form homodimers, we provide evidence that they preferentially form heterodimers. The relationship of UmuD to LexA, lambda repressor, and other members of the family of proteins is discussed and possible roles of intact UmuD in modulating SOS mutagenesis are discussed.

'Classical nitroreductase' is an enzyme involved in theintracellular metabolic activation ofmutagenic nitroarenes (1).Wehave already cloned the nitroreductase gene ofSalmonellatyphimuriumTA1538(2) anddeterminedthenucleotide sequenceby dideoxy sequence analysis ofboth strands. Below is shownthe nucleotide sequence of 1690base fragment which containsanopenreadingframeof651 nucleotides withpotential to encodethe nitroreductase. Themaxicell technique wasusedto identifythe nitroreductase and its molecular weight was estimated as28KDa, which is close to the calculated molecular weight of23,955. Possible sequence of -35, -10, S.D. and rho-independent transcriptional termination signal are indicated.

umuD and umuC are essential genes for UV and most chemical mutagenesis in Escherichia coli (4, 15, 18). Both umuD and umuC mutants are virtually nonmutable with ultraviolet (UV) and a variety of chemicals (6, 17). The umuD and umuC genes are organized as an operon (4, 15) and encode proteins of 15.0 and 47.7 kilodaltons (kDa), respectively (7, 12). The umuDC operon is repressed by the LexA protein (1, 4) and regulated as a part of SOS response of E. coli (10, 18, 19). SOS response occurs by activated RecA mediating the proteolytic cleavage of a bond between Ala84 and Gly85 of LexA (9), apparently facilitating a conditional autodigestion of LexA (16).

In Escherichia coli, most UV and chemical mutagenesis is not a passive process and requires the participation of the umuD and umuC gene products. However, the molecular mechanism of UV mutagenesis is not yet understood and the roles of the UmuD and UmuC proteins have not been elucidated. The umuDC operon is induced by UV irradiation and regulated as part of the SOS response. Genetic evidence now indicates that RecA-mediated cleavage activates UmuD for its role in mutagenesis. The COOH-terminal fragment of UmuD is both necessary and sufficient for this role. The RecA protein appears to have a third role in UV mutagenesis besides mediating the cleavage of LexA and UmuD at the time of SOS induction. In addition, we have obtained evidence which indicates that the GroEL and GroES proteins also play a role in UV mutagenesis. Similarities of the amino acid sequence of UmuD to the sequence of gene 45 protein of bacteriophage T4 and of the sequence of UmuC to those of the gene 44 and gene 62 proteins suggest possible roles for UmuD and UmuC in mutagenesis that are supported by preliminary evidence.

Nitroreductase and acetyltransferase genes of S. typhimurium TA1538 are cloned into pBR322 (see Watanabe et al., Biochem. Biophys. Res. Commun., 147 974–979 (1987)). The plasmids (pYG216, which contains a nitroreductase gene, and pYG219, which contains an acetyltransferase gene) were introduced into TA98 and TA100. TA98(pYG216), TA98(pYG219), TA100(pYG216) and TA100(pYG219) were named as YG1021, YG1024, YG1026 and YG1029, respectively. YG1021 and YG1026 had nitrofurazone-reductase activity about 50 times higher than the original strain, TA1538(pBR322), and were highly sensitive to 2-nitrofluorene (2-NF), 1-nitropyrene (1-NP) and 2-nitronaphthalene (2-NN)(Mutat. Res., in press (1989)). YG1024 and YG1029 had isoniazid-, 2-aminofluorene-N-acetyltransferase and N-hydroxy-Glu-P-1-0-acetyltransferase activities 100 times higher than TA1538(pBR322), and were highly sensitive to 2-NF, 1-NP, 1,8-dinitropyrene, 2-NN, Glu-P-1(+S9) and 2-aminoanthracene(+S9). These newly established strains are recommended to use for the detection of a small amount of nitroarenes and aromatic amines in the environment, and also for the estimation of metabolic pathway of chemical mutagens.

A co-expression plasmid (p1A2OR) carrying human CYP1A2 and NADPH-P450 reductase cDNAs and an expression plasmid (pOAT) carrying Salmonella TA1538/ARO cells. The CYP and OAT expressed in the Salmonella cells showed catalytic activity. The TA1538/ARO strain exhibited high sensitivity to heterocyclic amines (HCAs) such as 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,8-dimenthylimidazo[4,5-f]quinoline (MeIQx), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) as compared with the parent Ames tester strain TA1538. It is suggested that the intracellular expression of drug-metabolising enzymes makes the Salmonella cells highly sensitive to the HCAs.

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.

Summary The nitroreductase and acetyltransferase genes of Salmonella typhimurium TA1538 have been cloned into pBR322. When transformed into TA1538 derivatives, these plasmids provided the basis for an extremely sensitive assay for the detection of mutagenic nitroarenes.

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.

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.