Involvement of umuDCST genes in nitropyrene-induced -CG frameshift mutagenesis at the repetitive CG sequence in the hisD3052 allele of Salmonella typhimurium

Expression of the umuDC operon is required for UV and most chemical mutagenesis in Escherichia coli. The closely related species Salmonella typhimurium has two sets of umuDC-like operons, umuDC _ST on the chromosome and samAB on a 60-MDa cryptic plasmid. The roles of theumuDC-like operons in chemically induced frameshift mutagenesis of the hisD3052 allele of S. typhimurium were investigated. Introduction of a pBR322-derived plasmid carrying umuDCST increased the rate of reversion of hisD3052, following treatment with 1-nitropyrene (1-NP) or 1,8-dinitropyrene (1,-8DNP) tenfold and fivefold, respectively, whereas it did not substantially increase the rate of reversion induced by other frameshift mutagens, i.e. 2-nitrofluorene (2NF) and 2-amino- 3-methyldipyrido[1,2-a:3 ,2-d]imi-dazole (Glu-P-1). Introduction of a pBR322-derived plasmid carrying samAB did not increase the incidence of reversion of hisD3052 observed with any of the mutagens examined. Deletion of umuDC _STSubstantially lowered the reversion rate induced by l-NP or 1,8-DNP, but it did not affect reversion induced by 2-NF, Glu-P-1 or N-hydroxyacetylaminofluorene (N-OH-AAF). Deletion of samAB had little impact on reversion incidence induced by any of the five frameshift mutagens. DNA amplification using the polymerase chain reaction technique followed by restriction enzyme analysis using BssHII, suggested that the mutations induced by the five frameshift mutagens were all CG deletions at the CGCGCGCG sequence in hisD3052. These results suggest that umuDCST, but not samAB, is involved in the -2 frameshift mutagenesis induced by l-NP and 1,8-DNP at the repetitive CG sequence, whereas neither operon participates in induction of the same type of mutations by 2-NF, Glu-P-1 or N-OH-AAF.     

Spontaneous and induced mutability or frameshift strains of Salmonella typhimurium carrying uvrB and polA mutations

Three strains Salmonella typhimurium carrying frameshift mutations affecting the histidine genes (hisC3076, hisD3052 and hisC207) showed increased sensitivity to mutagenesis by ICR-191 (as judged by measuring back mutation to prototrophy), if they were made deficient in excision repair by deleting the uvrB gene. One frameshift strain, hisC3076, also showed increased sensitivity to mutagenesis by ICR-191 when it carried either of two different polA alleles, whereas the hidD305 and hisD207 frameshifts reduced sensitivity to mutagenesis in the presence of these alleles. Studies of spontaneous back mutation to prototrophy revealed siginificant mutator effects of the polA1 mutation on reversion of the hisD3052 frameshift and of the polA3 mutation on reversion of the hisC3076 frameshift. Other smaller mutator effects of the polA alleles on reversion of the his mutations may also be present. In an attempt to explain the complex interactions between different polA alleles and different frameshift mutations, it is tentatively suggested that deletion frameshift may arise mainly during DNA replication, while addition frameshifts may arise mainly during post-replication repair.     

Influence of the processing of MucA protein on the reversion of the frameshift hisD3052 and the base substitution hisG46 mutations by chemical mutagens in Salmonella typhimurium

The correlation between the proficiency at promoting mutagenesis of MucA/B proteins and MucA processing has been considered to be very high (Hauser et al., 1992) on the basis of the results of UV mutagenicity (Shiba et al., 1990). Here we show that this correlation is only partial. We have assayed the mutagenicity of benzo[a]pyrene (B[a]P) and aflatoxin B1 (AFB1) in Salmonella typhimurium tester strains containing plasmids which encode MucA proteins with an altered cleavage site. Reversion of the frameshift hisD3052 mutation by B[a]P or AFB1 was observed in the presence of non-cleavable MucA protein although at a lower level than that found in cells containing wild-type MucA protein. Reversion of the base substitution hisG46 mutation by AFB1 requires a significant processing of MucA, while lower levels of this processing would be enough for the hisG46 reversion by B[a]P. These results suggest that the specificity of mutations induced by mutagens forming DNA adducts is influenced by the activity of MucA protein. They also show the relevance of mutagenicity assays in the mechanistic studies of mutagenesis.     

Base-change analysis of revertants of the hisD3052 allele in Salmonella typhimurium

This report is an investigation of the specific sequence changes in the DNA of Salmonella hisD3052 revertants induced by a set of specific frameshift mutagens found in our diet. They include B[a]P, aflatoxin B1, and the cooked-food mutagens, IQ, MeIQ, and PhIP. The Salmonella DNA was cleaved with restriction enzymes Sau3A, EcoR1, and Alu1 to give a 620-bp fragment containing the hisD3052 site. The size-fractionated fragments were ligated to the bacteriophage vector M13mp8. After transformation into E. coli, the recombinants were screened with a nick-translated hisD+ gene probe, and the isolated single-stranded DNA was sequenced. All IQ (13), MeIQ (3), PhIP (5), and aflatoxin B1 (3) induced revertants isolated had a 2-base (-CG- dinucleotide) deletion situated 10 bases upstream from the original hisD3052 -C- deletion. In contrast, 9 of 24 revertants induced by B[a]P had extensive deletions varying from 8 to 26 nucleotides in length and located at various sites along a 45-base-pair sequence beginning at nucleotide 2085 of the his operon. The other 15 B[a]P-induced revertants had a -CG- deletion at the same location as the revertants induced by the other food mutagens. 7 spontaneous revertants were also analyzed; they showed 3 -CG- deletions, 1 insertion and 3 distinct deletions (varying from 2 to 11 bases in size). In total, 13 distinct base changes are described which lead to reversion of the hisD3052 mutation.     

Induction of base-pair substitution and prameshift mutations in wild-type and repair-deficient strains of Salmonella typhimurium by the photodynamic action of methylene blue

Induction of back mutations to prototrophy by methylene blue (MB)-sensitized photodynamic (PD) treatment has been studied in wild-type and repair-deficient strains of Salmonella typhimurium carrying either the base-pair substitution mutation hisG46 or the frameshift mutation hisD3052. We found that reversion of the hisG46 mutation was increased in a strain carrying a uvrB deletion and decreased in a strain carrying a recA-type mutation. Reversion of the hisD3052 (frameshift) mutation, on the other hand, was decreased in both uvrB deletion and recA-type strains. The former results are consistent with the hypothesis that the majority of MB-sensitized PD-induced base-pair substitution mutations arise by a mechanism similar to that currently believed to be involved in UV mutagenesis. The latter results suggest that PD-induced frameshift mutations may arise in some other way, and two possible mechanisms involving sequential action of the excision repair and recombinational repair pathways are considered.     

New O-acetyltransferase-deficient Ames Salmonella strains generated by specific gene disruption

CoASAc-dependent N-hydroxyarylamine O-acetyltransferase (OAT) is an enzyme involved in the intracellular metabolic activation of N-hydroxyarylamines derived from mutagenic nitroarenes and aromatic amines. The oat gene encoding the enzyme of S. typhimurium TA98 and TA100 was specifically disrupted and the sensitivities of the resulting strains, i.e., YG7130 and YG7126, to mutagens were compared with those of the conventional oat-deficient strains, i.e., TA98/1,8DNP6 and TA100/1,8DNP, respectively. The new oat-deficient strains and the conventional strains exhibited similar sensitivity against most of the chemicals tested: both strains YG7130 and strain TA98/1,8-DNP6 were resistant to mutagenicity by 1,8-dinitropyrene (1, 8-DNP), 1-nitropyrene, 2-amino-6-methyldipyrido[1,2-alpha:3', 2'-d]imidazole (Glu-P-1) and 2-amino-3-methyl-3H-imidazo[4, 5-f]quinoline (IQ); neither strain YG7130 nor strain TA98/1,8-DNP6 was resistant to the mutagenicity of 3-amino-1-methyl-5H-pyrido[4, 3-b]indole (Trp-P-2); strain YG7126 and strain TA100/1,8-DNP were refractory to the mutagenicity of 1,8-DNP. However, the order of the sensitivity against 2-nitrofluorene (2-NF) was TA98>YG7130>TA98/1, 8-DNP6 and TA100>YG7126>TA100/1,8-DNP. Since the strains YG7130 and YG7126 have chloramphenicol resistance (Cmr) gene in place of the chromosomal oat gene for gene disruption, the possible involvement of chloramphenicol acetyltransferase (CAT) encoded by the Cmr gene in the activation of 2-NF was examined. Strikingly, introduction of plasmid pACYC184 carrying the Cmr gene alone substantially enhanced the sensitivity of the conventional oat-deficient strains to 2-NF. These results suggest that the new strains as well as the conventional strains are useful to assess the roles of OAT in the metabolic activation of nitroaromatics and aromatic amines in S. typhimurium, and also that CAT has the ability to activate N-hydroxy aromatic amines to mutagens.     

Mutagenicity of the malondialdehyde oligomerization products 2-(3'-oxo-1'-propenyl)-malondialdehyde and 2,4-dihydroxymethylene-3-(2,2-dimethoxyethyl)glutaraldehyde in Salmonella

Malondialdehyde (MDA), a byproduct of non-enzymatic lipid peroxidation and prostaglandin biosynthesis, has been shown to be a weak frameshift mutagen in Salmonella mutagenicity assays. Because it is a dialdehyde, MDA can undergo self condensation to form polymeric products. It is possible that these condensation products are highly mutagenic and have contributed to previously reported estimates of MDA mutagenicity. We synthesized two major MDA polymerization products, (1) 2-(3'-oxo-1'-propenyl)-malondialdehyde [(MDA)2] and (2) 2,4-dihydroxymethylene-3-(2,2-dimethoxyethyl)glutaraldehyde [(MDA)3Me2] and tested their mutagenicity in the Salmonella frameshift tester strains hisD3052 and TA94 (hisD3052/pKM101). Analysis of the reversion rates revealed both (MDA)2 and (MDA)3Me2 to be weak mutagens, approximately equipotent to MDA. Although both (MDA)2 and (MDA)3Me2 are mutagenic, the fact that their formation is thermodynamically unfavorable under physiological conditions suggests they do not contribute significantly to the mutagenicity of MDA solutions.     

Bioactivation of diesel exhaust particle extracts and their major nitrated polycyclic aromatic hydrocarbon components, 1-nitropyrene and dinitropyrenes, by human cytochromes P450 1A1, 1A2, and 1B1

The genotoxicities of four samples of diesel exhaust particle (DEP) extracts (DEPE) and nine nitroarenes found in DEPE were investigated after activation catalyzed by human cytochrome P450 (P450) family 1 enzymes co-expressed with NADPH-cytochrome P450 reductase (NPR) in Escherichia coli membranes. The DEPE samples induced umu gene expression in Salmonella typhimurium TA1535/pSK1002 without any P450 system and were further activated by human P450 1B1/NPR membranes. Moderate activation of the DEPE sample by P450 1A2/NPR membranes was also observed, but not by either P450 1A1/NPR or NPR membranes. 1-Nitropyrene (1-NP) was strongly activated by human P450 1B1/NPR membranes. 1,8-Dinitropyrene (1,8-DNP) was most highly activated by P450 1A1 and 1B1 systems for the three DNPs tested. In contrast, 1,3-DNP was inactivated by P450 1A1/NPR, 1A2/NPR, and 1B1/NPR systems and slightly activated by NPR membranes. 2-Nitrofluoranthene (2-NF) and 3-nitrofluoranthene (3-NF) showed activities similar to 1-NP after bioactivation by P450 1B1/NPR membranes. However, the genotoxicities of 6-nitrochrysene, 7-nitrobenz[a]anthracene, and 6-nitrobenzo[a]pyrene were all weak in the present assay system. Apparent genotoxic activities of DEPE were very low compared with standard nitroarenes in the presence of P450s, possibly because unknown component(s) of DEPE had inhibitory effects on the bioactivation of 1-NP and 1,8-DNP catalyzed by human P450 1B1. These results suggest that environmental chemicals existing in airborne DEP, in addition to 1-NP, 1,6-DNP, 1,8-DNP, 2-NF, and 3-NF, can be activated by human P450 1B1. Biological actions of air pollutants such as nitroarenes to human extrahepatic tissues may be of concern in tissues in which P450 1B1 is expressed.     

Pentachlorophenol-mediated mutagenic synergy with aromatic amines in Salmonella typhimurium

Pentachlorophenol (PCP), a widely used pesticide, enhanced the mutagenic potency of plant- or mammalian-activated 2-aminofluorene (2AF) as well as the direct-acting mutagen 2-acetoxyacetylaminofluorene (2AAAF) when assayed with specific Salmonella typhimurium strains. With 2AF the mutagenic synergy was observed in strains YG1024, TA1538, and MP153. With 2AAAF the PCP-mediated synergy was observed with these strains and with strain TA98/1,8-DNP₆. The synergy was dependent upon the presence of an activated N-acetoxy functional group and was only expressed at the hisD3052 allele and not at the hisG46 allele. Spectrophotometric analysis demonstrated that the rate of degradation of 2AAAF was reduced in the presence of PCP in phosphate buffer or with S. typhimurium cytosol and thus PCP may be affecting the stability of the N-acetoxy group of activated aromatic amines.     

Conditions affecting the mutagenicity of sodium bisulfite in Salmonella typhimurium

Sodium bisulfite is a weak mutagen at pH 5 and 6 in S. typhimurium strains carrying the hisG46 and hisD6610 mutations, but is not mutagenic in strains with the hisC3076 or hisD3052 mutations. The bisulfite-induced base-pair substitution mutations were slightly enhanced by the presence of the plasmid, pKM101, but inhibited by the presence of the uvrB and rfa mutations. The hisO1242 mutation which causes constitutive expression of the histidine operon, produced a slight enhancement of frameshift (hisD6610), but not base-pair substitution (hisG46) mutations. Bisulfite-induced mutations appear to be the result of two different mechanisms which may be a function of the repair capacity of the strains. The data suggest that the deamination of cytosine may not be responsible for frameshift mutations, but may be responsible for base-pair substitution mutagenesis. Because the rate of bisulfite autooxidation appears to play a role in the mutagenic process, we are suggesting that the deamination of cytosine may be the result of oxidative damage rather than through the direct formation of a cytosine-bisulfite adduct. This is further supported by the much lower concentrations of bisulfite needed to cause mutagenicity than the 1 M concentrations cited to produce cytosine-bisulfite adducts.     

Complex Frameshift Mutations Mediated by Plasmid Pkm101: Mutational Mechanisms Deduced from 4-Aminobiphenyl-Induced Mutation Spectra in Salmonella

We used colony probe hybridization and polymerase chain reaction/DNA sequence analysis to determine the mutations in ~2,400 4-aminobiphenyl (4-AB) +S9-induced revertants of the -1 frameshift allele hisD3052 and of the base-substitution allele hisG46 of Salmonella typhimurium. Most of the mutations occurred at sites containing guanine, which is the primary base at which 4-AB forms DNA adducts. A hotspot mutation involving the deletion of a CG or GC within the sequence CGCGCGCG accounted for 100 and 99.9%, respectively, of the reversion events at the hisD3052 allele in the pKM101 plasmid-minus strains TA1978 (uvr(+)) and TA1538 (δuvrB). In strain TA98 (δuvrB, pKM101), which contained the SOS DNA repair system provided by the pKM101 plasmid, ~85% of the revertants also contained the hotspot deletion; the remaining ~15% contained one of two types of mutations: (1) complex frameshifts that can be described as a -2 or + 1 frameshift and an associated base substitution and (2) deletions of the CC or GG sequences that flank the hotspot site (CCGCGCGCGG). We propose a misincorporation/slippage model to account for these mutations in which (1) pKM101-mediated misincorporation and translesion synthesis occurs across a 4-AB-adducted guanine; (2) the instability of such a mispairing and/or the presence of the adduct leads to strand slippage in a run of repeated bases adjacent to the adducted guanine; and (3) continued DNA synthesis from the slipped intermediate produces a frameshift associated with a base substitution. This model readily accounts for the deletion of the CC or GG sequences flanking the hotspot site, indicating that these mutations are, in fact, complex mutations in disguise (i.e., cryptic complex frameshifts). The inferred base-substitution specificity associated with the complex frameshifts at the hisD3052 allele (primarily G·C -> T·A transversions) is consistent with the finding that 4-AB induced primarily G·C -> T·A transversions at the hisG46 base-substitution allele. The model also provides a framework for understanding the different relative mutagenic potencies of 4-AB at the two alleles in the various DNA repair backgrounds of Salmonella.     

The role of the excision and error-prone repair systems in mutagenesis by fluorinated quinolones in Salmonella typhimurium

Patterns of reversion produced by ciprofioxacin, enoxacin and ofloxacin in Salmonella typhimurium strains carrying the hisG428 ochre mutation have been studied. These fluorinated quinolones produce a significant increase in reversion of this mutation, even when it is located on the chromosome. Nevertheless, reversion is higher when the hisG428 mutation is on the multicopy plasmid pAQ1 than when it is on the chromosome. Reversion of hisG428 induced by fluorinated quinolones is abolished both in a uvrB genetic background and in the absence of the plasmid pKM101. Therefore, mutagenesis produced by fluorinated quinolones in the Salmonella mutagenicity assay is significantly affected by both the excision repair and the error-prone repair systems. Furthermore, fluorinated quinolones are also detected as moderate mutagens with the base substitution hisG46 mutation when both repair systems are functional in the tester strain.     

Spectra of spontaneous frameshift mutations at the hisD3052 allele of Salmonella typhimurium in four DNA repair backgrounds.

To characterize the hisD3052 -1 frameshift allele of Salmonella typhimurium, we analyzed approximately 6000 spontaneous revertants (rev) for a 2-base deletion hotspot within the sequence (CG)4, and we sequenced approximately 500 nonhotspot rev. The reversion target is a minimum of 76 bases (nucleotides 843-918) that code for amino acids within a nonconserved region of the histidinol dehydrogenase protein. Only 0.4-3.9% were true rev. Of the following classes, 182 unique second-site mutations were identified: hotspot, complex frameshifts requiring DeltauvrB + pKM101 (TA98-specific) or not (concerted), 1-base insertions, duplications, and nonhotspot deletions. The percentages of hotspot mutations were 13.8% in TA1978 (wild type), 24.5% in UTH8413 (pKM101), 31.6% in TA1538 (DeltauvrB), and 41.0% in TA98 (DeltauvrB, pKM101). The DeltauvrB allele decreased by three times the mutant frequency (MF, rev/10(8) survivors) of duplications and increased by about two times the MF of deletions. Separately, the DeltauvrB allele or pKM101 plasmid increased by two to three times the MF of hotspot mutations; combined, they increased this MF by five times. The percentage of 1-base insertions was not influenced by either DeltauvrB or pKM101. Hotspot deletions and TA98-specific complex frameshifts are inducible by some mutagens; concerted complex frameshifts and 1-base insertions are not; and there is little evidence for mutagen-induced duplications and nonhotspot deletions. Except for the base substitutions in TA98-specific complex frameshifts, all spontaneous mutations of the hisD3052 allele are likely templated. The mechanisms may involve (1) the potential of direct and inverted repeats to undergo slippage and misalignment and to form quasi-palindromes and (2) the interaction of these sequences with DNA replication and repair proteins.     

Functional complementation between chromosomal and plasmid mutagenic DNA repair genes in bacteria

Summary The umuDC operons of Escherichia coli and Salmonella typhimurium and the analogous plasmid operons mucAB and impCAB have been previously characterized in terms of their roles in DNA repair and induced mutagenesis by radiation and many chemicals. The interrelationships of these mutagenic DNA repair operons were examined in vivo in functional tests of interchangeability of operon subunits in conferring UV resistance and UV mutability phenotypes to wild-type S. typhimurium and umu mutants of E. coli. This approach was combined with DNA and protein sequence comparisons between the four operons and a fifth operon, samAB, from the S. typhimurium LT2 cryptic plasmid. Components of the E. coli and S. typhimurium umu operons were reciprocally interchangeable whereas impCA and mucA could not function with umuC in either of these species. mucA and impB could also combine to give a mutagenic response to UV. These active combinations were associated with higher degrees of conservation of protein sequence than in other heterologous gene combinations and related to specific regions of sequence that may specify subunit interactions. The dominance of the E. coli umuD44 mutation over umuD was revealed in both wild-type E. coli and S. typhimurium and also demonstrated against impCAB. Finally interspecies transfer showed that the apparently poor activity of the S. typhimurium umuD gene in situ is not the result of an inherent defect in umuD but is due to the simultaneous presence of the S. typhimurium umuC sequence. It is suggested that the limitation of umuD activity by umuC in S. typhimurium is the basis of the poor induced mutability of this organism.     

Mutagenicity of coolwhite fluorescent light for Salmonella

The most common fluorescent lamps in use today in homes and businesses in the United States, 'coolwhite' fluorescent lamps, emit light that is mutagenic for Salmonella. Strains that carry both a uvrB mutation and plasmid pKM101 are extremely susceptible to this light-induced mutation. Both base substitution and frameshift mutations can be induced without substantial lethal effects on the bacteria. Induced mutations accumulate essentially as a linear function of the time bacteria are exposed to illumination. Of Salmonella histidine-requiring strains with known nucleotide target sequences (Hartman et al., 1986; Cebula and Koch, 1989, 1990), strains either carrying one of the base substitution mutations, hisG428 and hisG46, or one of the frameshifts, hisC3076 and hisD6610, are most highly mutagenized whereas frameshift strains with hisD6580 and hisD3052 exhibit lower rates of mutagenesis. Mutagenicity does not appear to require the presence of oxygen. A filter blocking wavelengths below 370 nm eliminates mutagenesis. Polystyrene, cellulose acetate and, especially, mylar and glass filters reduce mutagenesis, indicating that at least some of the mutagenic effects can be attributed to leakage of radiations below 290 nm (far-ultraviolet light) from 'coolwhite' lamps. The more recently introduced fluorescent 'softwhite' lamps are roughly 10-fold less mutagenic at approximately equal light intensity. Incandescent light bulbs are much less mutagenic than are these fluorescent lamps. Our mutational data correlate closely with previous results in eukaryotic cells (Jacobson and Krell, 1982). A uvrB recA Salmonella double mutant is hypersensitive to the lethal effects of coolwhite fluorescent light, even when illuminated through the lids of glass Petri dishes. Thus, appropriate Salmonella strains would appear to be simple and useful screens for both the mutagenic and the lethal activities of fluorescent lamps. These systems are amenable to classroom laboratory use as relatively safe and effective means of demonstrating environmental mutagenesis.     

Identification of dinitropyrenes in diesel-exhaust particles. Their probable presence as the major mutagens

The direct-acting mutagens in diesel particulate extracts were identified. It is concluded that the major mutagens are in all probability 1,6- and 1,8-dinitropyrene (DNP). 1-Nitropyrene (NP) and 3-nitrofluoranthene (NF) were also present. The DNP isomers contributed 43% of the total mutagenic activity of the crude extracts, whereas 1-NP (or 3-NF) was responsible for less than 10% of the activity. The quantities of 1,6- and 1,8-DNP were 1.2 and 3.4 ppm of the crude extracts, respectively, and the induction of both DNPs in the diesel particulate matter corresponded to about 1.7-4.8% by weight of the 1-NP content (70.5 ppm in the crude extracts).     

A umuDC-independent SOS pathway for frameshift mutagenesis.

Summary The chemical carcinogen N-acetoxy-N-2-acetylaminofluorene induces mainly frameshift mutations, which occur within two types of sequences (mutation hot spots): –1 frameshift mutations within contiguous guanine sequences and –2 frameshift mutations within alternating GC sequences such as the NarI and BssHII restriction site sequences. We have investigated the genetic control of mutagenesis at these sequences by means of a reversion assay using plasmids pW17 and pX2, which contain specific targets for contiguous guanine and alternating GC sequences, respectively. Our results suggest that mutations at these hot spot sequences are generated by two different genetic pathways, both involving induction of SOS functions. The two pathways differ both in their LexA-controlled gene and RecA protein requirements. In the mutation pathway that acts at contiguous guanine sequences, the RecA protein participates together with the umuDC gene products. In contrast, RecA is not essential for mutagenesis at alternating GC sequences, except to cleave the LexA repressor. The LexA-regulated gene product(s), which participate in this latter mutational pathway, do not involve umuDC but another as yet uncharacterized inducible function. We also show that wild-type RecA and RecA430 proteins exert an antagonistic effect on mutagenesis at alternating GC sequences, which is not observed either in the presence of activated RecA (RecA^*), RecA730 or RecA495 proteins, or in the complete absence of RecA as in recA99. It is concluded that the –1 mutation pathway presents the same genetic requirements as the pathway for UV light mutagenesis, while the –2 mutation pathway defines a distinct SOS pathway for frameshift mutagenesis.     

UV and chemical mutagenesis in rev7 mutants of yeast

Summary We have examined induced mutagenesis in rev7-1 mutants of Baker's yeast' Saccharomyces cerevisiae, using a variety of contrasting test systems and several different mutagens. UV-induced reversion frequencies of the ochre allele arg4-17, the putative missense allele ilv1-92 and the frameshift allele his4-38 were 10 to 200 fold lower in haploid and diploid rev7-1 mutants compared with wild type strains, but UV-induced reversion frequencies of the frameshift allele leu2-3 and the proline missense allele cyc1-115 were reduced only a few fold. Ilv1-92 reversion frequencies induced by methyl methane sulfonate or by N-methyl-N-nitro-N-nitrosoguanidine were 10 to 20 times lower in rev7-1 mutants, but normal frequencies of these revertants were induced with ethyl methane sulfonate, even though rev7-1 strains are slightly sensitive to this mutagen as well as to the others tested. We conclude that the rev7 mutants, like the rev3 mutants they closely resemble, have a substantial but not total deficiency concerning induced mutagenesis.     

Comparative mutagenesis of the C8-guanine adducts of 1-nitropyrene and 1,6- and 1,8-dinitropyrene in a CpG repeat sequence. A slipped frameshift intermediate model for dinucleotide deletion

In the Ames Salmonella typhimurium reversion assay 1,6- and 1,8-dinitropyrenes (1,6- and 1,8-DNPs) are much more potent mutagens than 1-nitropyrene (1-NP). Genetic experiments established that certain differences in the metabolism of the DNPs, which in turn result in increased DNA adduction, play a role. It remained unclear, however, if the DNP adducts, N-(guanin-8-yl)-1-amino-6 ()-nitropyrene (Gua-C8-1,6-ANP and Gua-C8-1,8-ANP), which contain a nitro group on the pyrene ring covalently linked to the guanine C8, are more mutagenic than the major 1-NP adduct, N-(guanin-8-yl)-1-aminopyrene (Gua-C8-AP). In order to address this, we have compared the mutation frequency of the three guanine C8 adducts, Gua-C8-AP, Gua-C8-1,6-ANP, and Gua-C8-1,8-ANP in a CGCG*CG sequence. Single-stranded M13mp7L2 vectors containing these adducts and a control were constructed and replicated in Escherichia coli. A remarkable difference in the induced CpG deletion frequency between these adducts was noted. In repair-competent cells the 1-NP adduct induced 1.7% CpG deletions without SOS, whereas the 1,6- and 1,8-DNP adducts induced 6.8 and 10.0% two-base deletions, respectively. With SOS, CpG deletions increased up to 1.9, 11.1, and 15.1% by 1-NP, 1,6-, and 1,8-DNP adducts, respectively. This result unequivocally established that DNP adducts are more mutagenic than the 1-NP adduct in the repetitive CpG sequence. In each case the mutation frequency was significantly increased in a mutS strain, which is impaired in methyl-directed mismatch repair, and a dnaQ strain, which carries a defect in proofreading activity of the DNA polymerase III. Modeling studies showed that the nitro group on the pyrene ring at the 8-position can provide additional stabilization to the two-nucleotide extrahelical loop in the promutagenic slipped frameshift intermediate through its added hydrogen-bonding capability. This could account for the increase in CpG deletions in the M13 vector with the nitro-containing adducts compared with the Gua-C8-AP adduct itself.     

Bioactivation of diesel exhaust particle extracts and their major nitrated polycyclic aromatic hydrocarbon components, 1-nitropyrene and dinitropyrenes, by human cytochromes P450 1A1, 1A2, and 1B1

The genotoxicities of four samples of diesel exhaust particle (DEP) extracts (DEPE) and nine nitroarenes found in DEPE were investigated after activation catalyzed by human cytochrome P450 (P450) family 1 enzymes co-expressed with NADPH-cytochrome P450 reductase (NPR) in Escherichia coli membranes. The DEPE samples induced umu gene expression in Salmonella typhimurium TA1535/pSK1002 without any P450 system and were further activated by human P450 1B1/NPR membranes. Moderate activation of the DEPE sample by P450 1A2/NPR membranes was also observed, but not by either P450 1A1/NPR or NPR membranes. 1-Nitropyrene (1-NP) was strongly activated by human P450 1B1/NPR membranes. 1,8-Dinitropyrene (1,8-DNP) was most highly activated by P450 1A1 and 1B1 systems for the three DNPs tested. In contrast, 1, 3-DNP was inactivated by P450 1A1/NPR, 1A2/NPR, and 1B1/NPR systems and slightly activated by NPR membranes. 2-Nitrofluoranthene (2-NF) and 3-nitrofluoranthene (3-NF) showed activities similar to 1-NP after bioactivation by P450 1B1/NPR membranes. However, the genotoxicities of 6-nitrochrysene, 7-nitrobenz[a]anthracene, and 6-nitrobenzo[a]pyrene were all weak in the present assay system. Apparent genotoxic activities of DEPE were very low compared with standard nitroarenes in the presence of P450s, possibly because unknown component(s) of DEPE had inhibitory effects on the bioactivation of 1-NP and 1,8-DNP catalyzed by human P450 1B1. These results suggest that environmental chemicals existing in airborne DEP, in addition to 1-NP, 1,6-DNP, 1,8-DNP, 2-NF, and 3-NF, can be activated by human P450 1B1. Biological actions of air pollutants such as nitroarenes to human extrahepatic tissues may be of concern in tissues in which P450 1B1 is expressed.