Radiation induced COX-2 expression and mutagenesis at non-targeted lung tissues of gpt delta transgenic mice.

BACKGROUND: Although radiation-induced bystander effects have been confirmed using a variety of endpoints, the mechanism(s) underlying these effects are not well understood, especially for in vivo study. METHODS: A 1-cm(2) area (1 cm × 1 cm) in the lower abdominal region of gpt delta transgenic mice was irradiated with 5 Gy of 300 keV X-rays, and changes in out-of-field lung and liver were observed. RESULTS: Compared with sham-treated controls, the Spi(-) mutation frequency increased 2.4-fold in non-targeted lung tissues at 24 h after partial body irradiation (PBIR). Consistent with dramatic Cyclooxygenase 2 (COX-2) induction in the non-targeted bronchial epithelial cells, increasing levels of prostaglandin, together with 8-hydroxydeoxyguanosine, in the out-of-field lung tissues were observed after PBIR. In addition, DNA double-strand breaks and apoptosis were induced in bystander lung tissues after PBIR. CONCLUSION: The PBIR induces DNA damage and mutagenesis in non-targeted lung tissues, especially in bronchial epithelial cells, and COX-2 has an essential role in bystander mutagenesis.

Report of the IWGT working group on strategies and interpretation of regulatory in vivo tests I. Increases in micronucleated bone marrow cells in rodents that do not indicate genotoxic hazards.

In vivo genotoxicity tests play a pivotal role in genotoxicity testing batteries. They are used both to determine if potential genotoxicity observed in vitro is realised in vivo and to detect any genotoxic carcinogens that are poorly detected in vitro. It is recognised that individual in vivo genotoxicity tests have limited sensitivity but good specificity. Thus, a positive result from the established in vivo assays is taken as strong evidence for genotoxic carcinogenicity of the compound tested. However, there is a growing body of evidence that compound-related disturbances in the physiology of the rodents used in these assays can result in increases in micronucleated cells in the bone marrow that are not related to the intrinsic genotoxicity of the compound under test. For rodent bone marrow or peripheral blood micronucleus tests, these disturbances include changes in core body temperature (hypothermia and hyperthermia) and increases in erythropoiesis following prior toxicity to erythroblasts or by direct stimulation of cell division in these cells. This paper reviews relevant data from the literature and also previously unpublished data obtained from a questionnaire devised by the IWGT working group. Regulatory implications of these findings are discussed and flow diagrams have been provided to aid in interpretation and decision-making when such changes in physiology are suspected.

Report of the IWGT working group on strategy/interpretation for regulatory in vivo tests II. Identification of in vivo-only positive compounds in the bone marrow micronucleus test.

A survey conducted as part of an International Workshop on Genotoxicity Testing (IWGT) has identified a number of compounds that appear to be more readily detected in vivo than in vitro. The reasons for this property varies from compound to compound and includes metabolic differences; the influence of gut flora; higher exposures in vivo compared to in vitro; effects on pharmacology, in particular folate depletion or receptor kinase inhibition. It is possible that at least some of these compounds are detectable in vitro if a specific in vitro test is chosen as part of the test battery, but the 'correct' choice of test may not always be obvious when testing a compound of unknown genotoxicity. It is noted that many of the compounds identified in this study interfere with cell cycle kinetics and this can result in either aneugenicity or chromosome breakage. A decision tree is outlined as a guide for the evaluation of compounds that appear to be genotoxic agents in vivo but not in vitro. The regulatory implications of these findings are discussed.

Adenine excisional repair function of MYH protein on the adenine:8-hydroxyguanine base pair in double-stranded DNA.

Adenine paired with 8-hydroxyguanine (oh(8)G), a major component of oxidative DNA damage, is excised by MYH base excision repair protein in human cells. Since repair activity of MYH protein on an A:G mismatch has also been reported, we compared the repair activity of His(6)-tagged MYH proteins, expressed in Spodoptera frugiperda Sf21 cells, on A:oh(8)G and A:G mismatches by DNA cleavage assay and gel mobility shift assay. We also compared the repair ability of type 1 mitochondrial protein with type 2 nuclear protein, as well as of polymorphic type 1-Q(324) and 2-Q(310) proteins with type 1-H(324) and 2-H(310) proteins by DNA cleavage assay and complementation assay of an Escherichia coli mutM mutY strain. In a reaction buffer with a low salt (0-50 mM) concentration, adenine DNA glycosylase activity of type 2 protein was detected on both A:oh(8)G and A:G substrates. However, in a reaction buffer with a 150 mM salt concentration, similar to physiological conditions, the glycosylase activity on A:G, but not on A:oh(8)G, was extremely reduced and the binding activity of type 2 protein for A:G, but not for A:oh(8)G, was proportionally reduced. The glycosylase activity on A:oh(8)G and the ability to suppress spontaneous mutagenesis were greater for type 2 than type 1 enzyme. There was apparently no difference in the repair activities between the two types of polymorphic MYH proteins. These results indicate that human MYH protein specifically catalyzes the glycosylase reaction on A:oh(8)G under physiological salt concentrations.

Establishment of a Salmonella tester strain highly sensitive to mutagenic heterocyclic amines.

Heterocyclic amines (HCAs) that are present in cooked foods require metabolic activation to exert their genotoxicity. They undergo activation via N-hydroxylation by cytochrome P450 1A2 (CYP1A2), followed by O-esterification by O-acetyltransferase (OAT). To develop a Salmonella tester strain that is highly sensitive to mutagenic HCAs, we introduced a coexpression plasmid (p1A2OR) carrying human CYP1A2 and NADPH-CYP reductase cDNAs and an expression plasmid (pOAT) carrying Salmonella OAT to Salmonella typhimurium TA1538 to yield a TA1538/ARO strain. The TA1538/ARO strain was proven to express the enzymes, as indicated by high activities of 7-ethoxyresorufin O-deethylase and isoniazid N-acetylase. The TA1538/ARO strain exhibited very high sensitivity to mutagenic HCAs 2-amino-3,4-dimethylimidazo[4,5-f]quinoline, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline and a somewhat higher sensitivity to 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine compared with the parent Ames tester strain TA1538. The minimum concentrations of 2-amino-3,4-dimethylimidazo[4,5-f]quinoline, IQ, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline, and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine giving positive results were defined by evidence that the number of colonies increased in a dose-dependent manner and reached a number two times higher than that obtained by vehicle alone as a control in the TA1538/ARO strain at concentrations of 0.3, 3, 30, and 1000 pM, respectively. When the membrane and cytosol fractions prepared from TA1538/ARO were added to a mixture containing the parental TA1538, the sensitivity of TA1538 to IQ was much lower than that seen with TA1538/ARO. These results indicate that the intracellular expression of drug-metabolizing enzymes makes the established strain of Salmonella highly sensitive to mutagenic HCAs.

Molecular nature of ultraviolet B light-induced deletions in the murine epidermis.

Depletion of the stratospheric ozone layer leads to an increase in ambient UV loads, which are expected to raise skin cancer incidences. Tumor development in the skin could be a multistep process in which various genetic alterations, such as point mutations and deletions, occur successively. Here, we demonstrate that UVB irradiation efficiently induces deletions in the epidermis using a novel transgenic mouse, gpt delta. In this mouse model, deletions in lambda DNA integrated in the chromosome are preferentially selected as Spi(-) (sensitive to P2 interference) phages, which can then be subjected to molecular analysis. The mice were exposed to UVB at single doses of 0.3, 0.5, 1.0, 1.5, and 2.0 kJ/m(2). After 4 weeks, lambda phage was rescued from the genomic DNA of the epidermis by in vitro packaging reactions. The mutant frequencies of Spi(-) with large deletions in the epidermis increased >15-fold at a UVB dose of 0.5 kJ/m(2) over the control. Molecular sizes of most of the large deletions were >1000 bp. More than one-half of the large deletions occurred between short direct-repeat sequences from 1 to 6 bp, and the remainder had flush ends. In the unirradiated mouse, almost all of the Spi(-) mutants were 1-bp frameshifts in runs of identical bases. These results suggest that UVB irradiation induces deletions in the murine epidermis, and most of the deletions are generated through end-joining of double strand breaks in DNA.

New sublines of Chinese hamster CHL stably expressing human NAT1 or NAT2 N-acetyltransferases or Salmonella typhimurium O-acetyltransferase: comparison of the sensitivities to nitroarenes and aromatic amines using the in vitro micronucleus test.

New sublines of Chinese hamster CHL cells stably expressing human NAT1 or NAT2 N-acetyltransferases or O-acetyltransferase of Salmonella typhimurium were established, and their sensitivities to carcinogenic nitroarenes and aromatic amines were compared using the in vitro micronucleus test. The subline expressing human NAT2 N-acetyltransferase exhibited the highest sensitivity to the clastogenicities of 1,8-dinitropyrene and 2-nitrofluorene. These results raise the possibility that human NAT2 N-acetyltransferase is involved in the metabolic activation of 1,8-dinitro-pyrene and 2-nitrofluorene. Since human NAT2 N-acetyltransferase exhibits a marked genetic polymorphism, the polymorphic status of human N-acetyltransferase could be a genetic predisposing factor to cancers caused by the nitroarenes. In contrast, the subline expressing O-acetyltransferase of S. typhimurium exhibited the highest sensitivity to the clastogenicity of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) when the microsomes prepared from rat liver were present. This suggests that O-acetyltransferase of S. typhimurium has a higher ability to activate IQ than do the human acetyltransferases. Acetyltransferase enzymes of human enteric bacteria might contribute to the metabolic activation of IQ. The sublines could provide a new tool for investigation of the mechanism of metabolic activation and for assessment of cancer risk of nitroarenes and aromatic amines to humans.

Cloning of a human homolog of the yeast OGG1 gene that is involved in the repair of oxidative DNA damage.

We report the cloning of a human homolog of the yeast OGGC1 gene, which encodes a DNA glycosylase that excises an oxidatively damaged form of guanine, 8-hydroxyguanine (also known as 7,8-dihydro-8-oxoguanine). Since the deduced amino acid sequence (68 amino acids) of a human expressed sequence tag, N55394, matched a short stretch of yeast OGG1 protein with greater than 40% amino acid identity, a full length cDNA clone was isolated from a HeLa cell cDNA library with the N55394 clone as a probe. The cDNA clone encodes a predicted protein of 345 amino acids which is homologous to yeast OGG1 protein throughout the entire polypeptide sequence and shares 38% amino acid identity with yeast OGG1 protein. Moreover, we found that both a human homolog and yeast OGG1 protein possess two distinct DNA binding motifs, a helix-hairpin-helix (HhH) motif and a C2H2 zinc finger like motif, and a domain homologous to human and E. coli MutY proteins. Expression of a human homolog suppressed spontaneous mutagenesis of an E. coli (mutM mutY) mutant as in the case of yeast OGG1 protein. The gene was ubiquitously expressed in a variety of human organs and mapped to chromosome 3p26.2. These results strongly suggest that the gene isolated here is a human counterpart of the yeast OGGI gene and is involved in the repair of oxidative DNA damage in human cells.

Genetic polymorphisms and alternative splicing of the hOGG1 gene, that is involved in the repair of 8-hydroxyguanine in damaged DNA.

The hOGG1 gene encodes a DNA glycosylase that excises 8-hydroxyguanine (oh8Gua) from damaged DNA. Structural analyses of the hOGG1 gene and its transcripts were performed in normal and lung cancer cells. Due to a genetic polymorphism at codon 326, hOGG1-Ser326 and hOGG1-Cys326 proteins were produced in human cells. Activity in the repair of oh8Gua was greater in hOGG1-Ser326 protein than in hOGG1-Cys326 protein in the complementation assay of an E. coli mutant defective in the repair of oh8Gua. Two isoforms of hOGG1 transcripts produced by alternative splicing encoded distinct hOGG1 proteins: one with and the other without a putative nuclear localization signal. Loss of heterozygosity at the hOGG1 locus was frequently (15/ 23, 62.2%) detected in lung cancer cells, and a cell line NCI-H526 had a mutation leading to the formation of the transcripts encoding a truncated hOGG1 protein. However, the oh8Gua levels in nuclear DNA were similar among lung cancer cells and leukocytes irrespective of the type of hOGG1 proteins expressed. These results suggest that the oh8Gua levels are maintained at a steady level, even though multiple hOGG1 proteins are produced due to genetic polymorphisms, mutations and alternative splicing of the hOGG1 gene.

Establishment of a Salmonella tester strain highly sensitive to mutagenic heterocylic amines.

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 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP) in the new gpt delta transgenic mouse.

Gender differences and organ specificity of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-induced mutagenesis were examined with the new gptdelta transgenic mouse (T. Nohmi, M. Katoh, H. Suzuki, M. Matsui, M. Yamada, M. Watanabe, M. Suzuki, N. Horiya, O. Ueda, T. Shibuya, H. Ikeda, T. Sofuni, A new transgenic mouse mutagenesis test system using Spi-and 6-thioguanine selections (Environ. Mol. Mutagen. 28 (1996) 465-470). In this mouse model, two distinct selections are employed to efficiently detect different types of mutations, i.e 6-thioguanine (6-TG) selection for point mutations and Spi-selection for deletions, respectively. In both selections, the highest mutant frequencies were observed in colon, followed by in spleen and liver. No increases in mutations were observed in testis, brain and bone marrow in PhIP-treated male mice. No significant differences in 6-TG and Spi- mutant frequencies were observed in colon and liver between male and female treated mice. The correlation between PhIP-induced mutagenesis and carcinogenesis in colon is discussed.

N-hydroxyarylamine O-acetyltransferase of Salmonella typhimurium: proposal for a common catalytic mechanism of arylamine acetyltransferase enzymes.

Acetyl-CoA:N-hydroxyarylamine O-acetyltransferase is an enzyme involved in the metabolic activation of N-hydroxyarylamines derived from mutagenic and carcinogenic aromatic amines and nitroarenes. The O-acetyltransferase gene of Salmonella typhimurium has been cloned, and new Ames tester substrains highly sensitive to mutagenic aromatic amines and nitroarenes have been established in our laboratory. The nucleotide sequence of the O-acetyltransferase gene was determined. There was an open reading frame of 843 nucleotides coding for a protein with a calculated molecular weight of 32,177, which was close to the molecular weight of the O-acetyltransferase protein determined by using the maxicell technique. Only the residue of Cys69 in O-acetyltransferase of S. typhimurium and its corresponding residue (Cys68) in N-acetyltransferase of higher organisms were conserved in all acetyltransferase enzymes sequenced so far. The amino acid sequence Arg-Gly-Gly-X-Cys, including the Cys69, was highly conserved. A mutant O-acetyltransferase of S. typhimurium, which contained Ala69 instead of Cys69, no longer showed the activities of O- and N-acetyltransferase. These results suggest that the Cys69 of S. typhimurium and the corresponding cysteine residues of the higher organisms are essential for the enzyme activities as an acetyl-CoA binding site. We propose a new catalytic model of acetyltransferase for S. typhimurium and the higher organisms.

Characterization of three forms of cytochrome P-450 inducible by 3-methylcholanthrene in Golden hamster livers with special reference to aflatoxin B1 activation.

Three forms of cytochrome P-450 of liver microsomes of 3-methylcholanthrene-treated Golden hamsters were purified and characterized as regards their catalytic activity toward aflatoxin B1-related hepatocarcinogenic mycotoxins. These include two major forms, designated as cytochrome P-450-AFB (P-450-I) and P-450-II, and one minor form, P-450-III. Cytochromes P-450-AFB, P-450-II, and P-450-III have their absorption maximum in the carbon monoxide-complex of the reduced form at 448.5, 447.0, and 448.0 nm, have apparent molecular weights of 56,000, 58,000, and 59,500, and are in the low spin, high spin, and low spin state, respectively. Of these, cytochrome P-450-AFB was shown to be highly active in the mutagenic activation of aflatoxin B1-related hepatocarcinogens such as sterigmatocystin and O-methylsterigmatocystin. Activation of aflatoxin B1 by hepatic microsomes of 3-methylcholanthrene-treated hamsters was inhibited almost completely by the antibody against P-450-AFB but not by the antibody against P-450-II, indicating that P-450-AFB is the major component responsible for the activation of aflatoxin B1 by hamster liver. Western blot analysis demonstrated that no protein cross-reacted with the antibody to P-450-AFB in the liver microsomes from guinea pig, rat, mouse, and house musk shrew (Suncus murinus) treated with 3-methylcholanthrene, while one or two proteins cross-reacted with the antibody to P-450-II in the liver microsomes of these animals.

A glucocorticoid antagonist, mifepristone affects anti-Candida activity of murine neutrophils in the presence of prednisolone in vitro and experimental candidiasis of prednisolone-treated mice in vivo.

The effects of a glucocorticoid-antagonist, mifepristone on the suppressive action of prednisolone for anti-Candida activity of murine neutrophils were examined. Prednisolone suppressed inhibitory activity of neutrophils to mycelial growth of Candida albicans. This suppression was cancelled in the presence of 10(-7)-10(-6) M of mifepristone in vitro. Corresponding to this in vitro action, mifepristone protected prednisolone-treated mice from lethal C. albicans infection in vivo. These results suggest that glucocorticoid-induced vulnerability to Candida infection may be recovered or normalized by application of mifepristone.

Efficient detection of deletions induced by a single treatment of mitomycin C in transgenic mouse gpt delta using the Spi(-) selection.

Transgenic rodent mutation assays permit the detection and molecular analysis of various types of gene mutations, such as base changes and frameshifts, in a number of tissues. It is reported, however, that deletion mutations are not efficiently detected by the assays, in particular those using lambda phage shuttle vectors. Recently, a new transgenic mouse model, i.e., gpt delta, has been developed to selectively detect some types of deletions by Spi(-) selection. Spi(-) selection has an advantage over the other selections to preferentially identify deletions because only lambda phages deficient in both the red and gam gene functions are allowed to form phage plaques. In this study, we examined whether in vivo deletions induced by the treatment of mitomycin C (MMC) are detectable by the Spi(-) assay in the mouse model. The mice were treated with MMC (0.5, 1.0, 2.0, and 4.0 mg/kg, single intraperitoneal injection) and sacrificed 14 days after the dosing. The treatment at 4.0 mg/kg approximately doubled the mutant frequency of Spi(-) in the bone marrow, i.e., 2.52 x 10(-6) vs. 1.31 x 10(-6). The molecular analyses using polymerase chain reaction (PCR) and DNA sequencing indicated that seven Spi(-) mutants at 4.0 mg/kg group had deletions with molecular sizes from 0.8 kilo basepairs (kb) to 8.5 kb, whereas no such deletions were observed in the Spi(-) mutants in the control group. The results suggest that deletions induced by MMC in the bone marrow are efficiently detectable by Spi(-) selection and also that the molecular analyses are useful to evaluate the significance of a marginal increase in mutant frequency in the transgenic rodent mutation assays.

Highly sensitive umu test system for the detection of mutagenic nitroarenes in Salmonella typhimurium NM3009 having high O-acetyltransferase and nitroreductase activities.

A highly sensitive umu test system for the detection of genotoxic activities of a variety of mutagenic nitroarenes has been developed using a new tester strain, Salmonella typhimurium NM3009 having high O-acetyltransferase (O-AT) and nitroreductase (NR) activities. The NM3009 was constructed by subcloning both the O-AT and NR genes into plasmid vector pACYC184, and the resulting plasmid was introduced into the parent tester strain S. typhimurium TA1535/pSK1002 harboring an umuC'-'lacZ fusion gene. The induction of umuC gene expression could be monitored by measuring the cellular beta-galactosidase activity produced by fusion gene. The purpose of the study was to evaluate whether the newly developed strain NM3009 is highly sensitive toward nitroarene compounds. The sensitivity of the strain NM3009 was compared with those of the parent TA1535/pSK1002 strain, the NR-overexpressing strain NM1011, the NR-deficient strain NM1000, the O-AT-overexpression strain NM2009, and the O-AT-defective strain NM2000. The newly developed NM3009 strain had about 13-fold and 3-fold higher activities for N-AT and NR, respectively, than the original S. typhimurium TA1535/pSK1002 strain. Among six strains tested, NM3009 showed the highest sensitivity toward such chemicals as 1-nitronaphthalene, 2-nitrofluorene, 3,7-dinitrofluoranthene, 3-nitrofluoranthene, 5-nitroacenaphthene, 2-nitronaphthalene, 1-nitropyrene, 1,6-dinitropyrene, 3,9-dinitrofluoranthene, 4,4'-dinitrobiphenyl, 1,8-dinitropyrene, m-dinitrobenzene, 2,4-dinitrotoluene, and 1,3-dinitropyrene. We have also found that the order of sensitivities to induce umuC gene expression toward a variety of nitroarenes was NM3009 > NM2009 > NM1011 > TA1535/pSK1002 > NM2000 > NM1000. These results suggest that the newly developed tester strain NM3009 is of great use for the detection of genotoxic activities of numerous carcinogenic and mutagenic chemicals including nitroarenes, which require NR and/or O-AT for the activation.

Further characterization and validation of gpt delta transgenic mice for quantifying somatic mutations in vivo.

The utility of any mutation assay depends on its characteristics, which are best discovered using model mutagens. To this end, we report further on the characteristics of the lambda-based gpt delta transgenic assay first described by Nohmi et al. ([1996]: Environ Mol Mutagen 28:465-470). Our studies show that the gpt transgene responds similarly to other transgenic loci, specifically lacZ and cII, after treatment with acute doses of N-ethyl-N-nitrosourea (ENU). Because genetic neutrality is an important factor in the design of treatment protocols for mutagenicity testing, as well as for valid comparisons between different tissues and treatments, a time-course study was conducted. The results indicate that the gpt transgene, like cII and lacZ, is genetically neutral in vivo. The sensitivities of the loci are also equivalent, as evidenced by spontaneous mutant frequency data and dose- response curves after acute treatment with 50, 150, or 250 mg/kg ENU. The results are interesting in light of transgenic target size and location and of host genetic background differences. Based on these studies, protocols developed for other transgenic assays should be suitable for the gpt delta. Additionally, a comparison of the gpt and an endogenous locus, Dlb-1, within the small intestine of chronically treated animals (94 microg/mL ENU in drinking water daily) shows differential accumulation of mutations at the loci during chronic exposure. The results further support the existence of preferential repair at endogenous, expressed genes relative to transgenes.

Construction of Salmonella typhimurium YG7108 strains, each coexpressing a form of human cytochrome P450 with NADPH-cytochrome P450 reductase.

A series of Salmonella typhimurium (S. typhimurium) YG7108 strains, each coexpressing a form of human cytochrome P450 (CYP) (CYP1A1, CYP1A2, CYP1B1, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4, or CYP3A5) together with human NADPH-cytochrome P450 reductase (OR), was established. The parental S. typhimurium YG7108, derived from TA1535, lacks two O(6)-methylguanine-DNA methyltransferase genes, ada and ogt, and is highly sensitive to the mutagenicity of alkylating agents. The expression levels of CYP holo-protein in the genetically engineered S. typhimurium YG7108 cells, determined by carbon monoxide (CO) difference spectra, ranged from 62 nmol/L culture for CYP2C19 to 169 nmol/L culture for CYP3A4. The expression level of the OR varied, depending on the form of CYP coexpressed, and ranged from 214 to 1029 units/L culture. Each form of CYP expressed in the S. typhimurium YG7108 cells catalyzed the oxidation of a representative substrate at an efficient rate. The rates appeared comparable to the reported activities of CYP expressed in human liver microsomes or CYP in other heterologous systems, indicating that the OR was sufficiently expressed to support the catalytic activity of CYP. These S. typhimurium strains may be useful not only for predicting the metabolic activation of promutagens catalyzed by human CYP but also for identifying the CYP form involved.

UVB-induced gpt mutations in the skin of gpt delta transgenic mice.

Ultraviolet light B (UVB)-induced mutagenesis was studied in gpt delta transgenic mice, which contain the lambdaEG10 shuttle vector as a transgene. The mice were exposed to UVB at single doses of 0.3, 0.5, 1.0, 1.5, and 2.0 kJ/m(2). At 4 weeks after irradiation, the mutant frequencies (MF) of the gpt gene were determined in the epidermis and the dermis, and the gpt mutations in the epidermis were identified by DNA sequencing. The epidermis exhibited a higher sensitivity to UVB than the dermis at doses of 0.3 and 0.5 kJ/m(2) UVB: the MF of the epidermis were more than nine times higher than those of the nonirradiated mice, whereas the MF of the dermis were only two to three times higher than the nonirradiated level at the doses used. The UVB-induced mutation spectrum in the epidermis was dominated by G:C to A:T transitions at dipyrimidine sites, such as 5'-TC-3', 5'-CC-3', and 5'-T/C-CG-3'. Tandem transitions such as CC to TT were also observed. Interestingly, a remarkable bias towards the template strand of the gpt gene was observed in the single transitions at 5'-TC-3' and 5'-CC-3' sites, but not at 5'-T/C-CG-3' site. In contrast, G:C to A:T transitions at CpG sites and deletions were observed in nonirradiated mice. Hot spots of transitions were observed at different sites in UVB-irradiated and nonirradiated mice. These results indicate that gpt delta transgenic mouse is a suitable model for studying in vivo UVB-induced mutations at the molecular level.

Effects of O (6)-alkylguanine-DNA alkyltransferase deficiency in Escherichia coli as the host for the detection of mutations in lacI transgenic mice.

Transgenic mice are widely used to detect gene mutations in vivo induced by a variety of chemicals. It is known, however, that no mutagenicity of methyl methanesulfonate (MMS) is detected in epididymal sperm in various transgenic mice assays, although MMS induces the dominant lethal and specific locus mutations in male mice. To investigate the issue of whether unrepaired lesions in DNA of mature sperm can be transformed into mutations during replication of the lambda phage in Escherichia coli cells, we developed an E. coli strain YG5152, which is a derivative of strain SCS-8 but is deficient in the genes encoding O (6)-alkylguanine-DNA alkyltransferases. When lambda LIZalpha phages were treated with MMS or N-ethyl-N-nitrosourea (ENU) in vitro and infected to the E. coli strains, the mutant frequencies of lacI were markedly higher in strain YG5152 than in strain SCS-8. When Big Blue(trade mark) mice were treated with MMS (160 mg/kg) or ENU (125 or 250 mg/kg) and the phages rescued from mature sperm were infected to the strains, the mutation frequency (MF) of phages from ENU-treated mice at a dose of 250 mg/kg in strain YG5152 was about two times higher than that in strain SCS-8. However, no increase in the MF was observed in the MMS-treated mice even in strain YG5152. These results suggest that, although strain YG5152 efficiently detects ex vivo mutations caused by mutagenic alkyl adducts formed by MMS in lambda phage DNA, no detectable levels of mutagenic methyl adducts are present in mature sperm of MMS-treated mice. Possible reasons for this lack of mutagenicity of MMS in mature sperm using transgenic mice assays are discussed.