Mutation spectra induced by 1-nitropyrene 4,5-oxide and 1-nitropyrene 9,10-oxide in the supF gene of human XP-A fibroblasts

1-Nitropyrene 4,5-oxide and 1-nitropyrene 9,10-oxide are oxidative metabolites that are responsible for the mutagenicity of 1-nitropyrene. In this study, the mutation spectra induced by oxidative metabolites in human cells were determined using a shuttle vector assay. The mutation frequencies induced by 1-nitropyrene 9,10-oxide were 2-3 times higher than those induced by 1-nitropyrene 4,5-oxide. The base substitutions induced by 1-nitropyrene 4,5-oxide were G --> A transitions, G --> C transversions, and G --> T transversions. In the case of 1-nitropyrene 9,10-oxide, G --> A transitions, G --> T transversions, A --> G transitions and G --> C transversions were observed. Most base substitution mutations induced by oxidative metabolites occurred at the guanine sites in the supF gene. These sequence-specific hot spots were commonly identified as 5'-GA sequences for both metabolites. On the other hand, the sequence-specific hot spots at the adenine sites were identified as 5'-CAC sequences for 1-nitropyrene 9,10-oxide. These results suggest that the oxidative metabolites of 1-nitropyrene induce sequence-specific DNA mutations at the guanine and adenine sites at high frequency.     

Bioactivation of cysteine conjugates of 1-nitropyrene oxides by cysteine conjugate beta-lyase purified from Peptostreptococcus magnus.

To determine the role of cysteine conjugate beta-lyase (beta-lyase) in the metabolism of mutagenic nitropolycyclic aromatic hydrocarbons, we determined the effect of beta-lyase on the mutagenicities and DNA binding of cysteine conjugates of 4,5-epoxy-4,5-dihydro-1-nitropyrene (1-NP 4,5-oxide) and 9,10-epoxy-9,10-dihydro-1-nitropyrene (1-NP 9,10-oxide), which are detoxified metabolites of the mutagenic compound 1-nitropyrene. We purified beta-lyase from Peptostreptococcus magnus GAI0663, since P. magnus is one of the constituents of the intestinal microflora and exhibits high levels of degrading activity with cysteine conjugates of 1-nitropyrene oxides (1-NP oxide-Cys). The activity of purified beta-lyase was optimal at pH 7.5 to 8.0, was completely inhibited by aminooxyacetic acid and hydroxylamine, and was eliminated by heating the enzyme at 55 degrees C for 5 min. The molecular weight of beta-lyase was 150,000, as determined by fast protein liquid chromatography. S-Arylcysteine conjugates were good substrates for this enzyme. As determined by the Salmonella mutagenicity test, 5 ng of beta-lyase protein increased the mutagenicity of the cysteine conjugate of 1-NP 9,10-oxide (10 nmol per plate) 4.5-fold in Salmonella typhimurium TA98 and 4.1-fold in strain TA100. However, beta-lyase had little effect on the cysteine conjugate of 1-NP 4,5-oxide (10 nmol per plate). Both conjugates exhibited only low levels of mutagenicity with nitroreductase-deficient strain TA98NR. In vitro binding of 1-NP oxide-Cys to calf thymus DNA was increased by adding purified beta-lyase or xanthine oxidase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Formation of reactive 1-nitropyrene metabolites by lung microsomes and isolated lung cells

The metabolism and activation of 1-nitropyrene (1-NP) to reactive intermediates by lung microsomes and isolated lung cells was studied. Mutagenicity of 1-NP metabolites was assayed in Salmonella typhimurium TA98NR, a strain lacking a major component of nitroreductase activity. In the presence of NADPH, microsomes from rabbit, rat and hamster lung metabolized 1-NP to mutagenic products to a similar degree. Pretreatment with a mixture of polychlorinated biphenyls (PCB) decreased the formation of mutagenic metabolites by rabbit lung microsomes, but did not affect the production of mutagens by rat or hamster lung microsomes. ³H-1-NP was metabolized to covalently bound protein products at a rate of 82 and 10 pmol/mg by rabbit and hamster lung microsomes, respectively, whereas no binding was detected in rat lung microsomes. PCB-pretreatment increased covalent protein binding of ³ H-1-NP in lung microsomes from hamster and rat, but decreased the binding in rabbit lung microsomes. High performance liquid chromatography analysis indicated that ³H-1-NP was readily converted to ring-hydroxylated products by rabbit and hamster lung microsomes; the rate was much lower with rat lung microsomes. ³H-1-NP was activated to metabolites that covalently bound to protein in isolated rabbit lung cells, with the following rates being observed: Clara cells > lung digest > type II cells. In contrast, covalent protein binding in cells isolated from rat lung was very low. 1-NP was not activated to products mutagenic for S. typhimurium TA 98 N R when co-incubated with cells isolated either from rabbit or rat lung.Abbreviations 1-AP 1-aminopyrene - DMSO dimethyl sulfoxide - EGTA ethylene glycol-bis(ß-aminoethyl ether) - EM electron microscopy - HEPES N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid - HPBS HEPES-phosphate-buffered-saline - HPLC high performance liquid chromatography - NBT nitroblue tetrazolium - 1-NP 1-nitropyrene - 1-NP-4,5-diol trans-4,5-dihydro-4,5-dihydroxy-1-nitropyrene - 1-NP-9,10-diol trans-9,10-dihydro-9,10-dihydroxy-1-nitropyrene - 1-NP-4,5-oxide 1-nitropyrene-4,5-oxide - 1-NP-9,10-oxide 1-nitropyrene-9,10-oxide - 3-OH-1-NP 3-hydroxy-1-nitropyrene - 6-/8-OH-1-NP a mixture of 6- and 8-hydroxy-1-nitropyrene - PBS phosphate-buffered saline - PCB a mixture of polychlorinated biphenyls (Aroclor 1254) - TLC thin layer chromatography     

Genotoxicity of pyrene oxide and 1-nitropyrene oxides in hepatocyte primary culture/DNA repair test

The genotoxicity of a pyrene oxide, 1-nitropyrene (NP) oxides and other related compounds was examined in the hepatocyte primary culture (HPC)/DNA repair test. Pyrene 4,5-oxide and both 1-NP-4,5-oxide and 1-NP-9,10-oxide elicited clearly positive responses of DNA repair. In this assay, 1-NP itself was weakly positive. However, other related chemicals such as pyrene, 1-nitro-3-hydroxypyrene, 1-nitro-6-hydroxypyrene, and 1-nitro-8-hydroxypyrene did not generate positive responses.     

氟氏链霉菌离子束注入突变谱的分析

用低能N⁺离子束注入转谷氨酰胺酶产生菌氟氏链霉菌后,通过试验,初步确定了注入的效应曲线,获得了一系列突变菌株。提取原始菌株和突变菌株的DNA,采用PCR反应分段扩增出转谷氨酰胺酶基因进行单链构象多态性分析(SSCP),并将特异性条带克隆测序进行基因突变型的鉴定,分析离子束注入引起链霉菌基因的基因突变类型及特点。结果显示:碱基变异的类型包括转换、颠换和缺失。在检测到的24个碱基突变中,主要是碱基的置换(87.5%),碱基缺失的比例比较小(12.5%)。在碱基置换中,转换的频率(58.3%)高于颠换的频率(29.2%)。转换主要以C→T,A→G为主,颠换以G→T,C→G为主。此外构成DNA的4种碱基均可以被离子束辐照诱发变异,其中胞嘧啶发生突变的频率较高。     

Mutagenicity of K-region derivatives of 1-nitropyrene; remarkable activity of 1- and 3-nitro-5H-phenanthro[4,5-bcd]pyran-5-one

The mutagenic activities toward S. typhimurium strains TA98 and TA100 of K-region derivatives of 1-nitropyrene and pyrene were determined. The compounds tested were trans-4,5-dihydro-4,5-dihydroxy-1-nitropyrene (Compound 3), trans-4,5-dihydro-4,5-dihydroxypyrene (Compound 4), 1-nitropyrene-4,5-quinone (Compound 5), 1-nitropyrene-9,10-quinone (Compound 6), pyrene-4,5-quinone (Compound 7), and the lactones, 1-nitro-5H-phenanthro[4,5-bcd]pyran-5-one (Compound 8), 3-nitro-5H-phenanthro[4,5-bcd]pyran-5-one (Compound 9), and 5H-phenanthro[4,5-bcd]pyran-5-one (Compound 10). Neither pyrene nor any of its K-region derivatives was mutagenic, either in the absence or presence of S9 mix at the doses tested. Of the K-region derivatives of 1-nitropyrene, the lactones (Compounds 8 and 9) were generally the most active; 0.25 microgram/plate induced 900-2200 revertants in TA98 or TA100 without activation. The 4,5-dihydrodiol (Compound 3), an established mammalian metabolite of 1-nitropyrene, was less mutagenic than was 1-nitropyrene in TA98, but was more mutagenic than was 1-nitropyrene in TA100, regardless of the presence of S9 mix. The quinones (Compounds 5 and 6) were less mutagenic than was 1-nitropyrene in the absence of S9 mix in both strains, but their activities were increased in the presence of S9 mix. The mutagenic activities of the lactones (Compounds 8 and 9) were lower in strains TA98NR and TA98/1,8-DNP6 than in TA98, indicating that nitro-reduction and esterification are involved in their activation. The results of this study indicate that K-region derivatives of 1-nitropyrene may be important in its metabolic activation.     

Molecular analysis of hprt mutations induced by chromium picolinate in CHO AA8 cells

Chromium picolinate (CrPic) is a popular dietary supplement, marketed to the public for weight loss, bodybuilding, and control of blood sugar. Recommendations for long-term use at high dosages have led to questions regarding its safety. Previous studies have reported that CrPic can cause chromosomal aberrations and mutations. The purpose of the current work was to compare the mutagenicity of CrPic as a suspension in acetone versus a solution in DMSO, and to characterize the hprt mutations induced by CrPic in CHO AA8 cells. Treatments of 2% acetone or 2% DMSO alone produced no significant increase in 6-thioguanine (6-TG)-resistant mutants after 48 h exposures. Mutants resistant to 6-TG were generated by exposing cells for 48 h to 80 microg/cm(2) CrPic in acetone or to 1.0mM CrPic in DMSO. CrPic in acetone produced an average induced mutation frequency (MF) of 56 per 10(6) surviving cells relative to acetone solvent. CrPic in acetone was 3.5-fold more mutagenic than CrPic in DMSO, which produced an MF of 16.2. Characterization of 61 total mutations in 48 mutants generated from exposure to CrPic in acetone showed that base substitutions comprised 33% of the mutations, with transversions being predominant; deletions made up 62% of the mutations, with one-exon deletions predominating; and 1-4 bp insertions made up 5% of the characterized mutations. CrPic induced a statistically greater number of deletions and a statistically smaller number of base substitutions than have been measured in spontaneously generated mutants. These data confirm previous studies showing that CrPic is mutagenic, and support the contention that further study is needed to verify the safety of CrPic for human consumption.     

SOS-dependent A-->G transitions induced by hydroxyl radical generating system hypoxanthine/xanthine oxidase/Fe3+/EDTA are accompanied by the increase of Fapy-adenine content in M13 mp18 phage DNA.

Gas chromatography/isotope dilution-mass spectrometry with selected ion monitoring (GC/IDMS-SIM) was used to measure oxidised bases in hypoxanthine/xanthine oxidase/Fe3+/EDTA modified ss M13 mp18 phage DNA. A dose-dependent increase of oxidised bases content in DNA was observed with the biggest augmentation of FapyGua, thymine glycol and FapyAde. The amount of 8-OH-Gua was relatively high both in non-oxidised and oxidised DNA, and increased to the same extent as FapyAde and ThyGly. DNA oxidation caused a dramatic decrease in phage survival after transfection to E. coli. Survival was improved 2.8-fold after induction of the SOS system by UV irradiation of bacteria and mutation frequency of the lacZ gene in SOS conditions increased 7-fold over that in non-irradiated bacteria. Spectrum of mutations was different from those reported previously and mutations were distributed rather randomly within M13 lacZ sequence, which was in contrast to previous findings, where with non-chelated metal ions other types of mutations were found in several clusters. Thus, conditions of DNA oxidation and accessibility of metal ions for DNA bases might be important factors for generating different DNA damages and mutations. Major base substitutions found both in SOS-induced and non-induced E. coli but with higher mutation frequency in SOS-induced cells were C-->A (approximately 20-fold increase in SOS-conditions), G-->A (9-fold increase) and G-->C (4.5-fold increase). Very few G-->T transitions were found. A particularly large group of A-->G transitions appeared only in SOS-induced bacteria and was accompanied by augmentation of FapyAde content in the phage DNA with undetectable 2-OH-Ade. It is then possible that imidazole ring-opened adenine mimics guanine during DNA replication and pairs with cytosine yielding A-->G transitions in SOS-induced bacteria.     

Reliable and high-throughput mutation screening for beta-thalassemia by a single-base extension/fluorescence polarization assay

beta-thalassemia is one of the most common inherited diseases with incidence varying between 3% and 10% in the high-prevalence regions of South China. The molecular defects are mostly due to single-nucleotide substitutions, minor insertions, and deletions in the beta-globin gene. Large-scale population genetic screening combined with prenatal diagnosis is necessary for the effective prevention of this disease. We present a single base extension (SBE) method based on homogenous fluorescence polarization (FP) for simultaneous detection of the eight most common causative mutations [CDs 41-42 (-TCTT), IVS-2-654 (C-->T), -28 (A-->G), CD17 (A-->T), CD 71/72 (+A), CD26 (G-->A), -29 (A-->G), and CD43 (G-->T)] in the beta-globin gene in a Chinese population. This assay has been validated by a blind experiment with 100 clinical samples previously characterized by reverse dot-blot and direct sequencing. The results demonstrate that this high-throughput method is simple, reliable, and cost effective. We expect this approach can be used in large-scale genetic screening for beta-thalassemia.     

Bacterial mutagenicity of new cyclopenta-fused cata-annelated polycyclic aromatic hydrocarbons, and identification of the major metabolites of benz[j]acephenanthrylene formed by Aroclor-treated rat liver microsomes

Three novel cyclopenta-fused polycyclic aromatic hydrocarbons were synthesized, benz[d]aceanthrylene, benz[k]aceanthrylene, and benz[j]acephenanthrylene, and evaluated for mutagenic activity in the Ames Salmonella typhimurium plate incorporation assay. The two benzaceanthrylene derivatives were active at low S9 concentrations in strain TA98 (4 and 27 rev/nmole respectively), as had been predicted from the calculated delta Edeloc/beta values of the carbocations derived from opening of the cyclopenta-fused epoxide rings, but the majority of this mutagenicity appeared to be due to free-radical decomposition products of spontaneous endo-peroxide formation. These compounds were therefore not further investigated. Benz[j]acephenanthrylene was also an indirect-acting frameshift mutagen (8-12 rev/nmole in strain TA98), but unlike most of the previously assayed cyclopenta-fused polycyclic aromatic hydrocarbons exhibited no peak of activity at low S9 protein concentration. The principal metabolites formed from this compound by microsomes from Aroclor-treated rat liver were benz[j]acephenanthrylene-4,5-dihydro-4,5-diol (necessarily derived from hydration of benz[j]acephenanthrylene 4,5-oxide) and benz[j]acephenanthrylene-9,10-dihydro-9,10-diol (precursor to benz[j]acephenanthrylene-9,10-dihydrodiol 7,8-oxide, the bay-region diol-epoxide). Consideration of the reduced activity of this compound compared to the related structure chrysene, the S9 dependence curves, and the predicted delta Edeloc/beta values of the postulate active species, suggests that in contrast to most other cyclopenta-fused polycyclic aromatic hydrocarbons, bay-region diol-epoxide formation plays a greater role than epoxidation of the cyclopenta-fused ring in the metabolic activation of benz[j]acephenanthrylene.     

Factors determining mutagenic potential for individual cis and trans opened benzo[c]phenanthrene diol epoxide-deoxyadenosine adducts

Four adducts that would result from trans opening at C-1 of benzo[c]phenanthrene 3,4-diol 1,2-epoxide (B[c]PhDE) isomers (i.e., DE-1 enantiomers, where the epoxide oxygen and benzylic hydroxyl group are cis, and DE-2 enantiomers, where they are trans) by the N(6)-amino group of dAdo, together with the two cis opened N(6)-dAdo adducts of B[c]PhDE-1, were incorporated into two oligonucleotides at the underlined site in 5'-TTTAGAGTCTGCTCCC [context I(A)] and 5'-CAGATTTAGAGTCTGC [context II(A)]. After ligation of these, and the corresponding unsubstituted oligonucleotides, into single-stranded M13mp7L2 bacteriophage and transfection into SOS-induced Escherichia coli SMH77, base substitution mutations induced by the different B[c]PhDE-dAdo adducts were determined. These findings were compared with data [Pontén et al. (1999) Biochemistry 38, 1144-1152] for cis opened B[c]PhDE-2-dAdo adducts in the same sequence contexts. In most cases, adducts with S absolute configuration at the site of attachment of the nucleoside to the hydrocarbon had higher mutation frequencies (1.9-56.5%) than the corresponding adducts with R configuration (0.05-5.6%). For adducts derived from B[c]PhDE-1, the predominant mutations were A-->T transversions in context I(A) and A-->G transitions for most of these adducts in context II(A). For adducts derived from B[c]PhDE-2, A-->T base substitutions predominated for most of the trans adducts, but A-->G mutations were favored by the cis adduct with S configuration in either context. Thus, the structural feature that most dramatically affected mutagenic activity was the configuration of the carbon at the attachment point, with S configuration mostly being associated with greater mutagenicity than the R configuration. However, other structural variations and sequence context also affected mutagenicity, indicating that a combination of structure and context effects define mutagenicity.     

Metabolic activation of 3-methylcholanthrene: mutagenic and transforming activities of the 9,10-dihydrodiol.

3-Methylcholanthrene and five related dihydrodiols have been tested for microsome-mediated mutagenicity towards $\text{Salmonella}\text{typhimurium}$ TA100 and for the induction of mutation to 8-azaguanine resistance in V79 Chinese hamster cells and malignant transformation in M2 mouse fibroblasts. In both mutagenicity test systems, the 9,10-diol was considerably more active than either the parent hydrocarbon, the related $\text{cis}$-2α,3-diol, the $\text{trans}$-4,5-, the $\text{trans}$-7,8- or the $\text{trans}$-11,12-dihydrodiols. At a non-toxic concentration (1μg/ml medium), the 9,10-diol induced the formation of more transformed malignant foci in cultures of M2 cells than 3-methylcholanthrene and the other diols were either inactive or only weakly active in this test system. The results obtained indicate that the 9,10-dihydrodiol derived from 3-methylcholanthrene is involved, presumably following conversion into the corresponding vicinal diol-epoxide, 9,10-dihydro-9,10-dihydroxy-3-methylcholanthrene 7,8-oxide, in the metabolic activation of this carcinogenic polycyclic hydrocarbon.     

Structure of the SLC7A7 gene and mutational analysis of patients affected by lysinuric protein intolerance

Lysinuric protein intolerance (LPI) is a rare autosomal recessive defect of cationic amino acid transport caused by mutations in the SLC7A7 gene. We report the genomic structure of the gene and the results of the mutational analysis in Italian, Tunisian, and Japanese patients. The SLC7A7 gene consists of 10 exons; sequences of all of the exon-intron boundaries are reported here. All of the mutant alleles were characterized and eight novel mutations were detected, including two missense mutations, 242A-->C (M1L) and 1399C-->A (S386R); a nonsense mutation 967G-->A (W242X); two splice mutations IVS3 +1G-->A and IVS6 +1G-->T; a single-base insertion, 786insT; and two 4-bp deletions, 455delCTCT and 1425delTTCT. In addition, a previously reported mutation, 1625insATCA, was found in one patient. It is noteworthy that 242A-->C causes the change of Met1 to Leu, a rare mutational event previously found in a few inherited conditions. We failed to establish a genotype/phenotype correlation. In fact, both intrafamilial and interfamilial phenotypic variability were observed in homozygotes for the same mutation. The DNA-based tests are now easily accessible for molecular diagnosis, genetic counseling, and prenatal diagnosis of LPI.     

Long-chain adducts of trans-4-hydroxy-2-nonenal to DNA bases cause recombination, base substitutions and frameshift mutations in M13 phage

Oxidative stress enhances lipid peroxidation (LPO) implicated in the promotion and progression of carcinogenesis. One of the major LPO products is trans-4-hydroxy-2-nonenal (HNE), which was shown to react with guanosine and under peroxidizing conditions also with adenosine. We show here that all four DNA bases are targets for HNE, although displaying different reactivity: dG > dC > dA approximately equal to dT. HPLC and mass spectrometry analyses of HNE reactions with deoxynucleosides showed in each case the formation of several products, with mass peaks corresponding to HNE-dN adducts at a 1:1 and also 2:1 and 3:1 ratios. In the dA, dC and dG reactions, mass peaks corresponding to heptyl-substituted etheno-adducts were also detected, indicating HNE oxidation to its epoxide by air oxygen. In DNA pretreated with HNE, DNA synthesis by T7 DNA polymerase was stopped in a sequence-dependent manner at G > or = C > A and T sites. HNE increased the mutation rates in the lac Z gene of M13 phage transfected into wild type Escherichia coli. The most frequent event was the recombination between lacZ gene sequences in M13 and the E. coli F' factor DNA. Base substitutions and frameshifts were also observed in approximately similar numbers. Over 50% of base substitutions were the C-->T transitions, followed by the G-->C and A-->C transversions. In the E. coli recA strain recombination was not observed, although one mutational G-->T hot-spot appeared within the DNA fragment undergoing recombination in the wild type E. coli. We conclude that long chain HNE adducts to DNA bases arrest DNA synthesis and cause recombination, base substitutions and frameshift mutations in ssDNA.     

Mutagenesis of benzo[a]pyrene diol epoxide in yeast: requirement for DNA polymerase zeta and involvement of DNA polymerase eta

Benzo[a]pyrene is a potent environmental carcinogen, which can be metabolized in cells to the DNA damaging agent anti-benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (anti-BPDE). We hypothesize that mutations induced by BPDE DNA adducts are mainly generated through an error-prone translesion synthesis that requires a specialized DNA polymerase (Pol). Using an in vivo mutagenesis assay in the yeast model system, we have examined the potential roles of Pol(zeta) and Pol(eta) in (+/-)-anti-BPDE-induced mutagenesis. In cells proficient in mutagenesis, (+/-)-anti-BPDE induced 85% base substitutions with predominant G --> C followed by G --> T transversions, 9% deletions of 1-3 nucleotides, and 6% insertions of 1-3 nucleotides. In rad30 mutant cells lacking Pol(eta), (+/-)-anti-BPDE-induced mutagenesis was reduced and accompanied by a moderate decrease in base substitutions and more significant decrease in deletions and insertions of 1-3 nucleotides. In rev3 mutant cells lacking Pol(zeta), (+/-)-anti-BPDE-induced mutagenesis was mostly abolished, leading to a great decrease in both base substitutions and deletions/insertions of 1-3 nucleotides. In contrast, large deletions/insertions were significantly increased in cells lacking Pol(zeta). Consistent with the in vivo results, purified yeast Pol(zeta) performed limited translesion synthesis opposite (+)- and (-)-trans-anti-BPDE-N(2)-dG DNA adducts with predominant G incorporation opposite the lesion. These results show that (+/-)-anti-BPDE-induced mutagenesis in yeast requires Pol(zeta) and partially involves Pol(eta) and suggest that Pol(zeta) directly participates in nucleotide insertions opposite the lesion, while Pol(eta) significantly contributes to deletions and insertions of 1-3 nucleotides.     

Mutagenicity of phenanthrene and phenanthrene K-region derivatives.

Phenanthrene and 9 K-region derivatives, most of them potential metabolites of phenanthrene, were tested for mutagenicity by the reversion of histidine-dependent Salmonella typhimurium TA1535, TA1537, TA1538, TA98 and TA100 and the rec assay with Bacillus subtilis H17 and M45. The strongest mutagenic effects in the reversion assay were observed with phenanthrene 9,10-oxide, 9-hydroxyphenanthrene and N-benzyl-phenanthrene-9,10-imine. Interestingly, the mutagenic potency of the arene imine was similar to that of the corresponding arene oxide. This is the first report on the mutagenicity of arene imine. The mutagenic effects of all these phenanthrene derivatives were much weaker than that of the positive control benzo[a]pyrene 4,5-oxide. Even weaker mutagenicty was found with cis-9,10-dihydroxy-9,10-dihydrophenanthrene and with trans-9,10-dihydroxy-9-10-dihydrophenanthrene. The other derivatives were inactive in this test. However, 9-10-dihydroxyphenanthrene and 9,10-phenanthrenequinone were more toxic to the rec- B. subtilis M45 strain than to the rec+ H17 strain. This was also true for phenanthrene 9,10-oxide and 9-hydroxyphenanthrene, but not with the other test compounds that reverted (9,10-dihydroxy-9,10-dihydrophenanthrenes; N-benzyl-phenanthrene 9,10-imine; benzo[a]pyrene 4,5-oxide) or did not revert (phenanthrene, 9,10-bis-(p-chlorophenyl)-phenanthrene 9,10-oxide, 9-10-diacetoxyphenanthrene) the Salmonella tester strains. Although the K region is a main site of metabolism and although all potential K-region metabolites were mutagenic, phenanthrene did not show a mutagenic effect in the presence of mouse-liver microsomes and an NADPH-generating system under standard conditions. However, uhen epoxide hydratase was inhibited, phenanthrene was activated to a mutagen that reverted his- S. typhimurium. This shows that demonstration of the mutagenic activity of metabolites together with the knowledge that a major metabolic route proceeds via these metabolites dose not automatically imply a mutagenic hazard of the mother compound, because the metabolites in question may not accumulate in sufficient quantities and therefore the presence and relative activities of enzymes that control the mutagenically active metabolites are crucial. N-Benzyl-phenanthrene 9.10-imine was mutagenic for the episome-containing S. typhimurium TA98 and TA100 but not for the precursor strains TA1538 and TA1535. This arene imine would therefore be useful as a positive control during routine testing to monitor in the former strains the presence of the episome which is rather easily lost.     

Alteration of cadmium-induced mutational spectrum by catalase depletion in Chinese hamster ovary-K1 cells.

Previously, we have demonstrated that cadmium acetate significantly induces hprt mutation frequency in Chinese hamster ovary (CHO)-K1 and that 3-amino-1,2,4-triazole (3AT), a catalase inhibitor, potentiates the mutagenicity of cadmium [Chem. Res. Toxicol. 9 (1996) 1360-1367]. In this study, we investigate the role of intracellular peroxide in the molecular nature of mutations induced by cadmium. Using 2',7'-dichlorofluorescin diacetate and fluorescence spectrophotometry, we have shown that cadmium dose-dependently increased the amounts of intracellular peroxide and the levels were significantly enhanced by 3AT. Furthermore, we have characterized and compared the hprt mutation spectra in 6-thioguanine-resistant mutants derived from CHO-K1 cells exposed to 4 microM of cadmium acetate for 4h in the absence and presence of 3AT. The mutation frequency induced by cadmium and cadmium plus 3AT was 11- and 16-fold higher than that observed in untreated populations (2.2 x 10(-6)), respectively. A total of 40 and 51 independent hprt mutants were isolated from cadmium and cadmium plus 3AT treatments for mRNA-polymerase chain reaction (PCR), genomic DNA-PCR and DNA sequencing analyses. 3AT co-administration significantly enhanced the frequency of deletions induced by cadmium. Cadmium induced more transversions than transitions. In contrast, 3AT co-administration increased the frequency of GC-->AT transitions and decreased the frequencies of TA-->AT and TA-->GC transversions. Together, the results suggest that intracellular catalase is important to prevent the formation of oxidative DNA damage as well as deletions and GC-->AT transitions upon cadmium exposure.     

The Fanconi anemia pathway limits the severity of mutagenesis

Fanconi anemia (FA) is a developmental and cancer predisposition disorder in which key, yet unknown, physiological events promoting chromosome stability are compromised. FA cells exhibit excess metaphase chromatid breaks and are universally hypersensitive to DNA interstrand crosslinking agents. Published mutagenesis data from single-gene mutation assays show both increased and decreased mutation frequencies in FA cells. In this review we discuss the data from the literature and from our isogenic fancg knockout hamster CHO cells, and interpret these data within the framework of a molecular model that accommodates these seemingly divergent observations. In FA cells, reduced rates of recovery of viable X-linked hypoxanthine phosphoribosyltransferase (hprt) mutants are characteristically observed for diverse mutagenic agents, but also in untreated cultures, indicating the relevance of the FA pathway for processing assorted DNA lesions. We ascribe these reductions to: (1) impaired mutagenic translesion synthesis within hprt during DNA replication and (2) lethality of mutant cells following replication fork breakage on the X chromosome, caused by unrepaired double-strand breaks or large deletions/translocations encompassing essential genes flanking hprt. These findings, along with studies showing increased spontaneous mutability of FA cells at two autosomal loci, support a model in which FA proteins promote both translesion synthesis at replication-blocking lesions and repair of broken replication forks by homologous recombination and DNA end joining. The essence of this model is that the FANC protein pathway serves to restrict the severity of mutational outcome by favoring base substitutions and small deletions over larger deletions and chromosomal rearrangements.