Melissa L. Shelton

Glutathione S-transferase-mediated induction of GC AT transitions by halomethanes in salmonella

Halomethanes are among the most common mutagenic and carcinogenic disinfection by‐products present in the volatile/semivolatile fraction of chlorinated drinking water. Recent studies have demonstrated that the mutagenicity of dichloromethane (CH2Cl2) and bromodichloromethane (BrCHCl2) can be mediated by a theta‐class glutathione S‐transferase (GSTT1‐1). These studies used strain RSJ100 of Salmonella, which is a derivative of the base‐substitution strain TA1535 (hisG46, rfa, δuvrB), into which has been cloned the GSTT1‐1gene from rat. In the present report, we have ex tended these studies by demonstrating that the mutagenicity of two additional brominated trihalomethanes, bromoform (CHBr3) and chlorodibromomethane (ClCHBr2), are also mediated by GSTT1‐1 in RSJ100. Using a Tedlar bag vaporization technique, the mutagenic potencies (revertants/ppm) for these two compounds as well as the compounds tested previously rank as follows: CHBr3 ≈ ClCHBr2 > BrCHCl2 ≈ CH2Cl2. To explore the mutational mechanism, we determined the mutation spectra of all four halomethanes at the hisG46 allele by per forming colony probe hybridizations of ∼100 revertants induced by each compound. The majority (96–100%) of the mutations were GC → AT transitions, and 87–100% of these were at the second position of the CCC/GGG target. In contrast, only15% of mutants induced by CH2Cl2 were GC → AT transitions in the absence of the GSTT1‐1 gene in strain TA100 (a homologue of TA1535 containing the plasmid pKM101). The ability of GSTT1‐1 to mediate the mutagenicity of these di‐ and trihalomethanes and the induction of almost exclusively GC → AT transitions by these compounds suggest that these halomethanes are activated by similar pathways in RSJ100, possibly through similar reactive intermediates. The implications of these findings are discussed in relation to previous experimental work on the GST‐mediated bioactivation of dihalomethanes, which includes the possible formation of GSH intermediates and/or GSH‐DNAadducts. Environ. Mol. Mutagen. 30:440–447, 1997 Published 1997 Wiley‐Liss, Inc. This article is a US Government work and as such, is in the public domain in the United States of America.

Mutation spectra of chemical fractions of a complex mixture : role of nitroarenes in the mutagenic specificity of municipal waste incinerator emissions

Using an ion-exchange procedure coupled to a microsuspension Salmonella assay, we fractionated the dichloromethane-extractable particulate organics emitted by a municipal waste incinerator. Most (80-95%) of the mutagenic activity resided in the neutral/base fraction; however, the polar neutral fraction accounted for 12% of the direct-acting mutagenic activity. The mutagenic potencies of the whole extract and the various fractions were 4-15 times greater in the absence than in the presence of S9. Results with strains deficient in classical nitroreductase (TA98NR) and transacetylase (TA98/1,8-DNP6) indicated that a majority of the direct-acting mutagenicity was due to nitroarenes. This was confirmed by bioassay-directed subfractionation of the neutral/base faction by a cyanopropyl/HPLC method. The mutations in -3,000 revertants (approximately 400 each induced in TA98 by the whole extract, the neutral/base and polar neutral fractions from the ion-exchange column and 3 of the neural/base subfractions from the HPLC column; along with 200 revertants each induced by the model nitroarene 1-nitropyrene (1NP) in strains TA98, TA1538 and TA100) were analyzed by probe hybridization and PCR/DNA sequence analysis. The results indicated that nitroarenes such as 1NP that eluted in the neutral/base fraction accounted for at least 50% of the direct-acting mutagenicity and induced only a hotspot 2-base deletion in the sequence (CG)4 in TA98. In contrast, most of the complex frameshifts (a frameshift with a flanking base substitution) induced by the whole extract were induced by nitroarenes other than 1NP that were activated by transacetylation and that eluted in the polar neutral fraction. This study (1) identifies nitroarenes as an important contributor to the mutagenic activity of the emissions from municipal waste incinerators; (2) confirms our previous conclusion that the mutation spectrum of a complex mixture reflects the dominance of particular classes of chemical mutagens within the mixture; and (3) demonstrates the possibility of isolating certain chemical fractions of a complex mixture that induce certain classes of mutations produced by the whole, unfractionated mixture.

Mutation spectra in Salmonella of sunlight, white fluorescent light, and light from tanning salon beds: induction of tandem mutations and role of DNA repair.

We evaluated the mutagenicity of sunlight (SUN), uncovered coolwhite fluorescent light (FLR), and light from a tanning salon bed (TAN) at the base-substitution allele hisG46 of Salmonella in four DNA repair backgrounds (wild type, uvrB, pKM101, and uvrB + pKM101). Approximately 80% of the radiation emitted by TAN was within the ultraviolet (UV) range, whereas only approximately 10% of the SUN and approximately 1% of the FLR radiation was UV. TAN emitted similar amounts of UVA and UVB, whereas SUN emitted 50-60x and FLR emitted 5-10x more UVA relative to UVB. Based on total dose (UV + visible), the mutagenic potency ranking was TAN > FLR > SUN. Using colony probe hybridization and PCR/DNA sequence analysis, approximately 3000 revertants were analyzed to determine the mutational specificity of the three light sources. The mutation spectra and those induced by 254-nm UV had common features. The uvrB mutation enhanced the mutagenicity of the environmental UV sources more (20-216x) than did the pKM101 plasmid (approximately 20x) relative to wild type DNA repair. All light sources induced equal proportions of transitions and transversions in excision repair-proficient strains, but they induced more transitions relative to transversions in uvrB-containing strains. The majority of the mutations were G.C-->A.T transitions that were induced equally frequently at the first or second position of the CCC codon of the hisG46 allele in all strains except TA1535 (uvrB), where SUN and FLR induced transitions preferentially at the first position, and TAN induced them preferentially at the second position. Identified or presumptive multiple mutations, which constituted the only mutational class enhanced by all three light sources in the presence of uvrB and pKM101 either alone or together, accounted for 3-5% of the induced mutations in the plasmid-containing strains, and their increases (38-82-fold) in TA100 (uvrB, pKM101) were the highest of any mutational class. Of the TAN-induced multiple mutations, 83% (19/23) were CC-->TT tandem transitions. These results show that exposures to the nonsolar environmental UV sources FLR and TAN produce mutations similar to those produced by SUN, a known carcinogen.

Mutagenicity and mutation spectra of 2-acetylaminofluorene at frameshift and base-substitution alleles in four DNA repair backgrounds of Salmonella.

We used colony probe hybridization procedures to determine the mutations in approximately 600 revertants of the -1 frameshift allele hisD3052 and approximately 200 revertants of the base-substitution allele hisG46 of Salmonella typhimurium induced by 2-acetylaminofluorene (2-AAF) in the presence of Aroclor-induced rat liver S9. 2-AAF was primarily a frameshift mutagen, exhibiting 5 times more frameshift than base-substitution activity. The only frameshift mutation 2-AAF induced at the hisD3052 allele was a hotspot (-2) deletion within the sequence CGCGCGCG. The addition of the pKM101 plasmid had a small effect on the mutagenic potency of 2-AAF at this allele in a uvr+ background and no effect on the mutation spectra in either a uvr+ or uvr- background. The small amount of base-substitution activity exhibited by 2-AAF at the hisG46 allele required the presence of both the pKM101 plasmid and the uvrB mutation. The base substitutions were G.C-->T.A transversions (86%) and G.C-->A.T transitions (14%), and 85% of the substitutions were at the second position of the CCC target of the hisG46 allele; the remainder were at the first position. We propose that the hotspot frameshift may be initiated by N-acetyl-2-aminofluorene adducts located at the C(8) position of any of the guanines except the first one in the CGCGCGCG hotspot sequence. The mutation might then result from correct incorporation of cytosine opposite the adducted guanine, followed by a 2-base slippage according to our recently proposed correct-incorporation/slippage model. The hotspot mutation may also result from a 2-AAF-induced B- to Z-DNA transition at the repeating GpC site as well as by the action of enzymes involved in DNA metabolism, such as DNA resolvases or topoisomerases, on DNA structures that have been distorted by 2-AAF adducts. The small amount of 2-AAF-induced base-substitution activity may be due to mispairing of adenine opposite the minor aminofluorene adduct at the C(8) position of guanine.

Mutagenicity in lung of Big Blue® mice and induction of tandem-base substitutions in Salmonella by the air pollutant peroxyacetyl nitrate (PAN): predicted formation of intrastrand cross-links

Peroxyacetyl nitrate (PAN) is a ubiquitous air pollutant formed from NO(2) reacting with acetoxy radicals generated from ambient aldehydes in the presence of sunlight and ozone. It contributes to eye irritation associated with photochemical smog and is present in most urban air. PAN was generated in a chamber containing open petri dishes of Salmonella TA100 (gas-phase exposure). After subtraction of the background mutation spectrum, the spectrum of PAN-induced mutants selected at 3.1-fold above the background mutant yield was 59% GC-->TA, 29% GC-->AT, 2% GC-->CG, and 10% multiple mutations - primarily GG-->TT tandem-base substitutions. Using computational molecular modeling methods, a mechanism was developed for producing this unusual tandem-base substitution. The mechanism depends on the protonation of PAN near the polyanionic DNA to release NO(2)(+) resulting in intrastrand dimer formation. Insertion of AA opposite the dimerized GG would account for the tandem GG-->TT transversions. Nose-only exposure of Big Blue((R)) mice to PAN at 78ppm (near the MTD) was mutagenic at the lacI gene in the lung (mutant frequency +/-S.E. of 6.16+/-0.58/10(5) for controls versus 8.24+/-0.30/10(5) for PAN, P=0.016). No tandem-base mutations were detected among the 40 lacI mutants sequenced. Dosimetry with 3H-PAN showed that 24h after exposure, 3.9% of the radiolabel was in the nasal tissue, and only 0.3% was in the lung. However, based on the molecular modeling considerations, the labeled portion of the molecule would not have been expected to have been bound covalently to DNA. Our results indicate that PAN is weakly mutagenic in the lungs of mice and in Salmonella and that PAN produces a unique signature mutation (a tandem GG-->TT transversion) in Salmonella that is likely due to a GG intrastrand cross-link. Thus, PAN may pose a mutagenic and possible carcinogenic risk to humans, especially at the high concentrations at which it is present in some urban environments.

Mutation spectrum of a binary mixture of mutagens (methapyrilene and sodium azide) in strain TA1535 of Salmonella

Methapyrilene (MP) is a rat-liver carcinogen and cocarcinogen that exhibits a narrow spectrum of mutagenic activity in Salmonella typhimurium, inducing only a 2-fold increase in revertants only in the base-substitution strain TA1535; it also enhances the mutagenic activity of sodium azide (NaN3) in the same strain. To examine the effects of MP at the molecular level, we used the colony probe hybridization procedure developed by Cebula and Koch (Mutation Res., 229 (1990) 79-87) to identify the base substitutions in approximately 800 background, MP-, NaN3-, and MP + NaN3-induced revertants of the hisG46 allele of strain TA1535. The predominant mutation in all 4 mutation spectra was a CCC-->CTC transition. The results suggest a mechanism by which MP enhances the infidelity of the DNA replication complex or inhibits a DNA repair or proofreading function, resulting in the production of more of the same error that occurs normally and that is also induced by NaN3. Such a mechanism might be the basis for the carcinogenic and cocarcinogenic activities of MP. To our knowledge, this is the first report of the molecular analysis of mutants produced by exposure of cells to a binary mixture of mutagens.