Zebrafish irritant responses to wildland fire-related biomass smoke are influenced by fuel type, combustion phase, and byproduct chemistry.

Human exposure to wildfire-derived particulate matter (PM) is linked to adverse health outcomes; however, little is known regarding the influence of biomass fuel type and burn conditions on toxicity. The aim of this study was to assess the irritant potential of extractable organic material (EOM) of biomass smoke condensates from five fuels (eucalyptus, pine, pine needle, peat, or red oak), representing various fire-prone regions of the USA, burned at two temperatures each [flaming (approximately 640°C) or (smoldering approximately 500°C)] using a locomotor assay in zebrafish (Danio rerio) larvae. It was postulated that locomotor responses, as measures of irritant effects, might be dependent upon fuel type and burn conditions and that these differences relate to combustion byproduct chemistry. To test this, locomotor activity was tracked for 60 min in 6-day-old zebrafish larvae (25-32/group) immediately after exposure to 0.4% dimethyl sulfoxide (DMSO) vehicle or EOM from the biomass smoke condensates (0.3-30 µg EOM/ml; half-log intervals). All EOM samples produced concentration-dependent irritant responses. Linear regression analysis to derive rank-order potency indicated that on a µg PM basis, flaming pine and eucalyptus were the most irritating. In contrast, on an emission-factor basis, which normalizes responses to the amount of PM produced/kg of fuel burned, smoldering smoke condensates induced greater irritant responses (>100-fold) than flaming smoke condensates, with smoldering pine being the most potent. Importantly, irritant responses significantly correlated with polycyclic aromatic hydrocarbon (PAH) content, but not with organic carbon or methoxyphenols. Data indicate that fuel type and burn condition influence the quantity and chemical composition of PM as well as toxicity.

Predictive models for carcinogenicity and mutagenicity: frameworks, state-of-the-art, and perspectives.

Mutagenicity and carcinogenicity are endpoints of major environmental and regulatory concern. These endpoints are also important targets for development of alternative methods for screening and prediction due to the large number of chemicals of potential concern and the tremendous cost (in time, money, animals) of rodent carcinogenicity bioassays. Both mutagenicity and carcinogenicity involve complex, cellular processes that are only partially understood. Advances in technologies and generation of new data will permit a much deeper understanding. In silico methods for predicting mutagenicity and rodent carcinogenicity based on chemical structural features, along with current mutagenicity and carcinogenicity data sets, have performed well for local prediction (i.e., within specific chemical classes), but are less successful for global prediction (i.e., for a broad range of chemicals). The predictivity of in silico methods can be improved by improving the quality of the data base and endpoints used for modelling. In particular, in vitro assays for clastogenicity need to be improved to reduce false positives (relative to rodent carcinogenicity) and to detect compounds that do not interact directly with DNA or have epigenetic activities. New assays emerging to complement or replace some of the standard assays include Vitotox, GreenScreenGC, and RadarScreen. The needs of industry and regulators to assess thousands of compounds necessitate the development of high-throughput assays combined with innovative data-mining and in silico methods. Various initiatives in this regard have begun, including CAESAR, OSIRIS, CHEMOMENTUM, CHEMPREDICT, OpenTox, EPAA, and ToxCast. In silico methods can be used for priority setting, mechanistic studies, and to estimate potency. Ultimately, such efforts should lead to improvements in application of in silico methods for predicting carcinogenicity to assist industry and regulators and to enhance protection of public health.

Mutagenicity and cytotoxicity of five antitumor ellipticines in mammalian cells and their structure-activity relationships in Salmonella.

The mutagenicity and cytotoxicity of five antitumor compounds (ellipticines) were investigated in the Chinese hamster ovary cell hypoxanthine-guanine phosphoribosyltransferase assay and in six strains of Salmonella. All five compounds (ellipticine, 9-methoxyellipticine, 9-hydroxyellipticine, 9-aminoellipticine, and 2-methyl-9-hydroxyellipticinium) were cytotoxic and mutagenic in the Chinese hamster ovary cell hypoxanthine-guanine phosphoribosyltransferase assay in the presence or absence of Aroclor 1254-induced rat liver S9, and all except the last compound were mutagenic in Salmonella. Based on the reversion pattern obtained in various frame-shift and DNA repair-proficient (uvrB+) or -deficient (uvrB) strains of Salmonella in the presence or absence of S9, the first three compounds appear to cause frame-shift mutations by both intercalation and covalent bonding with DNA; thus, these are classified as reactive intercalators. However, 9-aminoellipticine intercalates only weakly and may instead exert its mutagenic activity primarily (or exclusively) by forming a covalent adduct with DNA. Compared to the published antitumor data obtained in mice, the results in Salmonella and Chinese hamster ovary cells suggest that the ability of ellipticine, 9-methoxyellipticine, and 9-hydroxyellipticine to intercalate with DNA, induce frame-shift mutations, and cause cell killing is associated with and may be the basis for their antitumor activity. The observation that the ellipticines are mutagenic in mammalian cells suggests that these antitumor agents may be carcinogenic.

Lung tumor KRAS and TP53 mutations in nonsmokers reflect exposure to PAH-rich coal combustion emissions.

We determined the TP53 and codon 12 KRAS mutations in lung tumors from 24 nonsmokers whose tumors were associated with exposure to smoky coal. Among any tumors studied previously, these showed the highest percentage of mutations that (a) were G --> T transversions at either KRAS (86%) or TP53 (76%), (b) clustered at the G-rich codons 153-158 of TP53 (33%), and (c) had 100% of the guanines of the G --> T transversions on the nontranscribed strand. This mutation spectrum is consistent with an exposure to polycyclic aromatic hydrocarbons, which are the primary component of the smoky coal emissions. These results show that mutations in the TP53 and KRAS genes can reflect a specific environmental exposure.

Hotspot sites for acridine-induced frameshift mutations in bacteriophage T4 correspond to sites of action of the T4 type II topoisomerase.

The type II topoisomerase of bacteriophage T4 is a central determinant of the frequency and specificity of acridine-induced frameshift mutations. Acridine-induced frameshift mutagenesis is specifically reduced in a mutant defective in topoisomerase activity. The ability of an acridine to promote topoisomerase-dependent cleavage at specific DNA sites in vitro is correlated to its ability to produce frameshift mutations at those sites in vivo. The specific phosphodiester bonds cleaved in vitro are precisely those at which frameshifts are most strongly promoted by acridines in vivo. The cospecificity of in vitro cleavage and in vivo mutation implicate acridine-induced, topoisomerase-mediated DNA cleavages as intermediates of acridine-induced mutagenesis in T4.

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 approximately 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 (delta uvrB). In strain TA98 (delta uvrB, pKM101), which contained the SOS DNA repair system provided by the pKM101 plasmid, approximately 85% of the revertants also contained the hotspot deletion; the remaining approximately 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.

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.

Spontaneous and MNNG-induced reversion of an EGFP construct in HeLa cells: an assay for observing mutations in living cells by fluorescent microscopy.

A HeLa cell line stably expressing the enhanced green fluorescence protein (EGFP) gene, interrupted by the HBB IVS2-654 intron, was studied without treatment and after treatment with a single standard dose of 15 microM of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). This assay was done in order to prove that such a construct can revert by a variety of mechanisms and that it produces a visible phenotype, i.e., green fluorescence. The system permits visual detection of living mutant cells among a background of non-mutant cells and does not require a selective medium. The results show that the construct reverts by large deletions (-62, -100, and -162 bp), small insertions (+4 bp), small rearrangements (19 bp duplication), base substitutions at purines (G652, G653, A655, G579), and a pyrimidine (T654) between nucleotide positions 579 and 837. Splice-site mutations were recovered, and some of the mechanisms underlying these mutations are discussed. Because of the ease of detection of revertant cells under fluorescent light and the wide variety of mutations that can be recovered, further development of this system could make it a useful new mammalian cell mutagenicity assay.

The glutathione S-transferase M1 (GSTM1) null genotype and benzidine-associated bladder cancer, urine mutagenicity, and exfoliated urothelial cell DNA adducts.

Multiple studies in the general population have suggested that subjects with the glutathione S-transferase M1 (GSTM1)-null genotype, who lack functional GSTM1, are at higher risk for bladder cancer. To evaluate the impact of the GSTM1-null genotype on bladder cancer caused by occupational exposure to benzidine and to determine its influence on benzidine metabolism, we carried out three complementary investigations: a case-control study of bladder cancer among workers previously exposed to benzidine in China, a cross-sectional study of urothelial cell DNA adducts and urinary mutagenicity in workers currently exposed to benzidine in India, and a laboratory study of the ability of human GSTM1 to conjugate benzidine and its known metabolites in vitro. There was no overall increase in bladder cancer risk for the GSTM1-null genotype among 38 bladder cancer cases and 43 controls (odds ratio, 1.0; 95% confidence interval, 0.4-2.7), although there was some indication that highly exposed workers with the GSTM1-null genotype were at greater risk of bladder cancer compared to similarly exposed workers without this allele. However, the GSTM1 genotype had no impact on urothelial cell DNA adduct and urinary mutagenicity levels in workers currently exposed to benzidine. Furthermore, human GSTM1 did not conjugate benzidine or its metabolites. These results led us to conclude that the GSTM1-null genotype does not have an impact on bladder cancer caused by benzidine, providing a contrast to its association with elevated bladder cancer risk in the general population.

Induction of mutation spectra by complex mixtures: approaches, problems, and possibilities.

More complex environmental mixtures have been evaluated for mutagenic activity at the hisD3052 allele of Salmonella, primarily in strain TA98, than in any other target or mutation assay. Using colony probe hybridization to detect a common hot spot deletion, followed by polymerase chain reaction and DNA sequencing, we have generated 10 mutation spectra from three classes of mixtures (i.e., urban air, cigarette smoke condensate, and municipal waste incinerator emissions). The mutation spectra are distinctly different among the three classes of mixtures; however, the spectra for samples within the same class of mixture are similar. In addition to the hot spot mutation, the mixtures induce complex mutations, which consist of a small deletion and a base substitution. These mutations suggest a mechanism involving misinsertion of a base opposite a DNA adduct followed by a slippage and mismatch. A role for DNA secondary structure also may be the basis for the mutational site specificity exhibited by the various mixtures. The results suggest that unique mutation spectra can be generated by different classes of complex mixtures and that such spectra are a consequence of the dominance of a particular chemical class or classes within the mixture. The problems associated with this type of research are discussed along with the potential value of mutation spectra as a tool for exposure and risk assessment.

Molecular analysis of mutations induced at the hisD3052 allele of Salmonella by single chemicals and complex mixtures.

More single chemicals and complex environmental mixtures have been evaluated for mutagenicity at the hisD3052 allele of Salmonella, primarily in strain TA98, than in any other mutation assay. The development of colony probe hybridization procedures and the application of the polymerase chain reaction and direct DNA sequencing has permitted rapid molecular access to this allele. We discuss these techniques and the resulting mutation spectra that have been induced by a variety of environmental mutagens and complex mixtures. A common GC or CG deletion within a hot-spot region of the sequence dominates most of the spectra. In addition to this two-base deletion, we have recovered about 200 other types of mutations within the 72-base target for reversion of the hisD3052 allele. These include a variety of deletions (as large as 35 bases), duplications (as large as 46 bases), and complex mutations involving base substitutions. The quasipalindromic nature of the target sequence and its potential to form DNA secondary structures and slippage mismatches appear to be an important basis for the mutability of this allele.

Development of source testing, analytical, and mutagenicity bioassay procedures for evaluating emissions from municipal and hospital waste combustors.

Incineration is currently being used for disposal of about 10% of the solid waste generated in the United States, and this percentage will likely increase as land disposal declines. Siting new incinerators, however, is often controversial because of concerns related to the possibility of adverse health effects and environmental contamination from long-term exposure to stack emissions. Specific concerns relate to the adequacies of a) stack emission testing protocols, b) existing regulations, and c) compliance monitoring and enforcement of regulations. U.S. Environmental Protection Agency laboratories are cooperatively conducting research aimed at developing new testing equipment and procedures that will allow a more comprehensive assessment of the complex mixture of organics that is present in stack emissions. These efforts are directed specifically toward developing source testing equipment and procedures, analytical procedures, and bioassay procedures. The objectives of this study were to field test two types of high-volume source dilution samplers, collect stack samples for use in developing analytical and mutagenicity bioassay procedures, and determine mutagenicity of organics associated with emission particles from two municipal waste combustors and a hospital waste combustor. Data are presented for particle concentrations and emission rates, extractable organic concentrations and emission rates, and Salmonella (Ames) mutagenic potency and emission rates. The mutagenic emission rates and emission factors are compared to other incinerators and combustion sources.

Multiple-site carcinogenicity of benzene in Fischer 344 rats and B6C3F1 mice.

Toxicology and carcinogenesis studies of benzene (CAS No. 71-43-2; greater than 99.7% pure) were conducted in groups of 60 F344/N rats and 60 B6C3F1 mice of each sex for each of three exposure doses and vehicle controls. These composite studies on benzene were designed and conducted because of large production volume and widespread human exposure, because of the epidemiologic association with leukemia, and because previous experiments were considered inadequate or inconclusive for determining carcinogenicity in laboratory animals. Using the results from 17-week studies, doses for the 2-year studies were selected based on clinical observations (tremors in higher dosed mice), on clinical pathologic findings (lymphoid depletion in rats and leukopenia in mice), and on body weight effects. Doses of 0, 50, 100, or 200 mg/kg body weight benzene in corn oil were administered by gavage to male rats, 5 days per week, for 103 weeks. Doses of 0, 25, 50, or 100 mg/kg benzene in corn oil were administered by gavage to female rats and to male and female mice for 103 weeks. Ten animals in each of the 16 groups were killed at 12 months, and necropsies were performed. Hematologic profiles were performed at 3-month intervals. For the 2-year studies, mean body weights of the top dose groups of male rats and of both sexes of mice were lower than those of the controls. Survivals of the top dose group of rats and mice of each sex were reduced; however, at week 92 for rats and week 91 for mice, survival was greater than 60% in all groups; most of the dosed animals that died before week 103 had neoplasia. Compound-related nonneoplastic or neoplastic effects on the hematopoietic system, Zymbal gland, forestomach, and adrenal gland were found both for rats and mice. Further, the oral cavity was affected in rats, and the lung, liver, Harderian gland, preputial gland, ovary, and mammary gland were affected in mice. Under the conditions of these 2-year gavage studies, there was clear evidence of carcinogenicity of benzene in male F344/N rats, female F344/N rats, male B6C3F1 mice, and female B6C3F1 mice. In male rats, benzene caused increased incidences of Zymbal gland carcinomas, squamous cell papillomas and squamous cell carcinomas of the oral cavity, and squamous cell papillomas and squamous cell carcinomas of the skin. In female rats, benzene caused increased incidences of Zymbal gland carcinomas and squamous cell papillomas and squamous cell carcinomas of the oral cavity.(ABSTRACT TRUNCATED AT 400 WORDS)

Induction of prophage lambda by chlorinated organics: detection of some single-species/single-site carcinogens.

Twenty-eight chlorinated organic compounds were evaluated for their ability to induce DNA damage using the Microscreen prophage-induction assay in Escherichia coli. Comparison of the performance characteristics of the prophage-induction and Salmonella assays to rodent carcinogenicity assays showed that the prophage-induction assay had a somewhat higher specificity than did the Salmonella assay (70% vs. 50%); sensitivity, concordance, and positive and negative predictivity were similar for the two microbial assays. The Microscreen prophage-induction assay failed to detect eight carcinogens, perhaps due to toxicity or other unknown factors; five of these eight carcinogens were detected by the Salmonella assay. However, the prophage-induction assay did detect six carcinogens that were not detected by the Salmonella assay, and five of these were single-species, single-site carcinogens, mostly mouse liver carcinogens. Some of these carcinogens, such as the chloroethanes, produce free radicals, which may be the basis for their carcinogenicity and ability to induce prophage. The prophage-induction (or other SOS) assay may be useful in identifying some genotoxic chlorinated carcinogens that induce DNA damage that does not revert the standard Salmonella tester strains.

Use of the microscreen phage-induction assay to assess the genotoxicity of 14 hazardous industrial wastes.

The Microscreen phage-induction assay, which quantitatively measures the induction of prophage lambda in Escherichia coli WP2s(lambda), was used to test 14 crude (unfractionated) hazardous industrial waste samples for genotoxic activity in the presence and absence of metabolic activation. Eleven of the 14 wastes induced prophage, and induction was observed at concentrations as low as 0.4 pg per ml. Comparisons between the ability of these waste samples to induce prophage and their mutagenicity in the Salmonella reverse mutation assay indicate that the phage-induction assay detected genotoxic activity in all but one of the wastes that were mutagenic in Salmonella. Moreover, the Microscreen assay detected as genotoxic five additional wastes that were not detected in the Salmonella assay. The applicability of the Microscreen phage-induction assay for screening hazardous wastes for genotoxic activity is discussed, as are some of the problems associated with screening highly toxic wastes containing toxic volatile compounds.

Workshop overview: new molecular techniques in genome analysis.

An overview of the application of various molecular techniques to the analysis of genomic DNA is presented. For the analysis of small-scale changes, the polymerase chain reaction (PCR), colony probe hybridization, mismatch hybridization, and denaturing gradient gel electrophoresis (DGGE) are providing information on mutations within prokaryotic and eukaryotic genes. For large-scale changes, fluorescence in situ hybridization (FISH), pulsed-field gel electrophoresis (PFGE), Southern blotting, multiplex PCR, hybridization of linked probes, and restriction enzyme mapping are permitting analysis of genomic alterations that are larger than point mutations but below the resolution of standard cytogenetic analysis. Many of these techniques, either alone or in combination, produce DNA that can be subjected to DNA sequence analysis, which provides the most detailed information regarding genomic changes.

Mutation spectra in Salmonella of complex mixtures: comparison of urban air to benzo[a]pyrene.

We used an ion-exchange procedure coupled to the Salmonella assay to fractionate the dichloromethane-extractable particulate organics from an urban air sample collected in Boise, Idaho. A resulting base/neutral fraction contained 81% of the mutagenic activity but only 36% of the mass of the unfractionated sample. Chemical analysis showed that polycyclic aromatic hydrocarbons (PAHs) accounted for much of the mutagenic activity of the air sample. Colony probe hybridization, PCR, and DNA sequence analysis were then used to determine the mutations induced by the complex mixtures and a model PAH, benzo[a]pyrene (BAP) in approximately 900 revertants of the frameshift hisD3052 allele and approximately 400 revertants of the base-substitution hisG46 allele. The majority (93-94%) of the mutations induced at the frameshift allele in strain TA98 by the whole or base/neutral fraction of the urban air sample was a hotspot 2-base deletion of a CG or GC within the sequence CGCGCGCG. The remaining mutations were complex frameshifts that consisted of -2 or +1 frameshifts associated with a flanking base substitution. BAP induced a somewhat similar pattern of mutations, with 70% being the hotspot mutation, 23% being complex frameshifts, and the remaining being deletions. The inferred base-substitution specificity associated with the complex frameshifts at the hisD3052 allele (primarily G.C-->T.A transversions) was consistent with the observation that this same transversion was the primary mutation induced by the whole urban air sample and BAP at the base-substitution allele in strain TA100. At the frameshift allele, adducts that promote correct incorporation/slippage could account for hotspot mutations, whereas those that promote misincorporation/slippage could account for complex frameshifts. At the base-substitution allele, a mixture of adducts or of adducts with multiple conformations could account for the observed proportion of transitions and transversions. Combined with the bioassay-directed chemical analysis, these results from the first mutation spectra of a complex mixture suggest that such spectra reflect the dominance of particular classes of chemical mutagens within the mixture.

Mutation spectra of Glu-P-1 in Salmonella: induction of hotspot frameshifts and site-specific base substitutions.

We used colony probe hybridization and PCR/DNA sequence analysis to determine the mutations in approximately 1,640 revertants of the -1 frameshift allele hisD3052 and approximately 260 revertants of the base substitution allele hisG46 of Salmonella typhimurium induced by the heterocyclic amine cooked food mutagen 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1). All of the mutations were at sites containing guanine, which is the base at which Glu-P-1 forms DNA adducts. A hotspot mutation involving the deletion of a CG or GC within the sequence CGCGCGCG accounted for 100% of the Glu-P-1-induced mutations at the frameshift allele in strains TA1978 (uvr+) and TA1538 (delta uvrB) and 99% in TA98 (delta uvrB, pKM101). To explain the induction of these hotspot mutations by Glu-P-1, we describe here a more detailed version of our recently proposed correct incorporation/slippage model [Genetics:136:731, 1994]. We propose that after cytosine is incorporated correctly opposite a Glu-P-1-adducted guanine, various slipped intermediates may form (a total of 18), depending on which guanine is adducted and whether it remains within the helix or becomes extrahelical. This variety of mutational pathways may account for the high mutability of the hotspot sequence by Glu-P-1. Although the pKM101 plasmid does not influence the mutagenic potency or mutational spectrum of Glu-P-1 at the frameshift allele, it is required by Glu-P-1 to revert the base substitution allele, where Glu-P-1 induces G-C --> T-A transversions (75%) and G-C --> tA-T transitions (25%) exclusively at a single site (the second position of the CCC codon of the hisG46 allele). The limited (20-30 times less) base substitution mutagenic potency of Glu-P-1 relative to its frameshift mutagenic potency as well as the extreme site specificity exhibited by Glu-P-1 for base substitutions may have bearing on the lack of base substitutions identified in ras genes in Glu-P-1-induced rat colon tumors.

Induction of prophage lambda by chlorophenols.

Chlorinated phenols, which are used primarily as wood preservatives and fungicides, are present in most air, water, and soil samples in industrialized areas as well as in the urine of most people. We have examined the ability of phenol and the 19 isomers of chlorophenol to induce DNA damage using the Microscreen prophage-induction assay in Escherichia coli. Seven of the isomers (2,3,4,-tri, 2,4,5-tri, 3,4,5-tri, 2,3,4,5-tetra, 2,3,6-tri, 2,4,6-tri, and pentachlorophenol) induced prophage lambda in the presence of S9, with the first three being approximately 10 times more potent than the last three. The more potent isomers have either one or no chlorine atom ortho to the OH group; whereas the less potent isomers have two chlorine atoms ortho to the OH group. Although none of the 20 compounds is mutagenic in Salmonella, the prophage-induction results agree with findings by others that most of these seven isomers are clastogenic, are associated with cancer and chromosomal aberrations in humans (pentachlorophenol), and are carcinogenic in rodents (2,4,6-tri and pentachlorophenol). A likely basis for the genotoxicity of the seven isomers involves the metabolism of the parent isomer to a chlorohydroquinone, which can form a chlorobenzosemiquinone in the presence of oxygen. These two metabolites can produce free radicals that can cause DNA strand breaks, resulting in prophage induction in E. coli or, possibly, the chromosomal aberrations/cancer associated with human exposure to chlorophenols.

Molecular analysis of mutations induced by the intercalating agent ellipticine at the hisD3052 allele of Salmonella typhimurium TA98.

We have used DNA colony hybridization, the polymerase chain reaction (PCR), and direct DNA sequencing to determine the mutations induced by the intercalating agent ellipticine in Salmonella typhimurium TA98 in the presence of S9. Of 400 ellipticine-induced revertants that were selected at a mutant yield that was ninefold over the background, 85.5% contained a GC or CG deletion within a common CGCGCGCG hotspot; this deletion occurred among 47% of the spontaneous revertants. In addition to this hotspot, the ellipticine spectrum contained two deletion warmspots that reside opposite each other in looped-out regions of a possible DNA secondary structure. Ellipticine and its metabolites likely revert Salmonella strain TA98 by forming DNA adducts that promote slippage-mismatches and by stabilizing these slipped mismatched sequences via intercalation. The involvement of these mechanisms, along with a likely role for DNA secondary structures and a possible role for DNA gyrase, may account for the site specificity exhibited by ellipticine in strain TA98.