The influence of local DNA sequence and DNA repair background on the mutational specificity of 1-nitroso-8-nitropyrene in Escherichia coli: inferences for mutagenic mechanisms.

We have examined the mutational specificity of 1-nitroso-8-nitropyrene (1,8-NONP), an activated metabolite of the carcinogen 1,8-dinitropyrene, in the lacI gene of Escherichia coli strains which differ with respect to nucleotide excision repair (+/- delta uvrB) and MucA/B-mediated error-prone translesion synthesis (+/- pKM101). Several different classes of mutation were recovered, of which frameshifts, base substitutions, and deletions were clearly induced by 1,8-NONP treatment. The high proportion of point mutations (> 92%) which occurred at G.C sites correlates with the percentage of 1,8-NONP-DNA adducts which occur at the C(8) position of guanine. The most prominent frameshift mutations were -(G.C) events, which were induced by 1,8-NONP treatment in all strains, occurred preferentially in runs of guanine residues, and whose frequency increased markedly with the length of the reiterated sequence. Of the base substitution mutations G.C-->T.A transversions were induced to the greatest extent by 1,8-NONP. The distribution of the G.C-->T.A transversions was not influenced by the nature of flanking bases, nor was there a strand preference for these events. The presence of plasmid pKM101 specifically increased the frequency of G.C-->T.A transversions by a factor of 30-60. In contrast, the -(G.C) frameshift mutation frequency was increased only 2-4-fold in strains harboring pKM101 as compared to strains lacking this plasmid. There was, however, a marked influence of pKM101 on the strand specificity of frameshift mutation; a preference was observed for -G events on the transcribed strand. The ability of the bacteria to carry out nucleotide excision repair had a strong effect on the frequency of all classes of mutation but did not significantly influence either the overall distribution of mutational classes or the strand specificity of G.C-->T.A transversions and -(G.C) frameshifts. Deletion mutations were induced in the delta uvr, pKM101 strain. The endpoints of the majority of the deletion mutations were G.C rich and contained regions of considerable homology. The specificity of 1,8-NONP-induced mutation suggests that DNA containing 1,8-NONP adducts can be processed through different mutational pathways depending on the DNA sequence context of the adduct and the DNA repair background of the cell.

The action of 1-nitroso-8-nitropyrene in Escherichia coli: DNA adduct formation and mutational consequences in the absence of nucleotide excision-repair.

To study the mechanisms of mutagenesis by the carcinogen 1,8-dinitropyrene we have determined the DNA adducts formed and mutations induced by its partially activated metabolite 1-nitroso-8-nitropyrene (1,8-NONP) in an Escherichia coli strain deficient in nucleotide excision-repair. Using DNA sequence analysis we have characterized a collection of 159 lacI- mutations recovered following treatment with 1,8-NONP. The mutational spectrum was dominated by -1 frameshifts (110 events) in runs of contiguous G or C residues. Frameshift frequency was observed to increase with the length of the reiterated sequence. Two mutations involved the loss of GpC from alternating (GpC)n sequences. The ratio of -1:-2 events observed following 1,8-NONP treatment was markedly different from that induced by N-acetoxy-N-acetyl-2-aminofluorene in the same genetic target. Other mutational classes recovered included 'spontaneous' hotspot mutations (19 events), base substitutions (12 events), deletions (7 events), one duplication, one + (A:T) frameshift and one mutation containing closely juxtaposed -(G:C) events. Of the 125 point mutations characterized, 124 occurred at G:C sites. The site specificity of mutation was consistent with the 32P-postlabeling profile of 1,8-NONP-DNA adducts which showed that 95% of the adducts migrated to the same position on the TLC plates as the guanine C(8) adduct 1-N-(2'-deoxyguanosin-8-yl)-amino-8-nitropyrene. Two minor 1,8-NONP-DNA mutations were also detected, one at dG/dC sites, and the other at dA/dT sites.

The mutational specificity of 2-(2-furyl)-3-(5-nitro-2-furyl)-acrylamide (AF2) in the lacI gene of Escherichia coli.

We have determined the mutational specificity of the 5-nitrofuran derivative furylfuramide (AF2) in the lacI gene of Escherichia coli. Treatment of a delta uvrB, pKM101 strain with 1 M AF2 yielded a mutation frequency approximately 300 times greater than that of untreated controls. Of the 165 AF2-induced mutants analysed by DNA sequencing, 145 were base substitution mutations, 11 were frameshifts, and the remainder small deletions, tandem base substitutions and complex mutations. Base substitution occurred primarily (greater than 93%) at G:C base pairs. The proportions of the various mutations are very similar to those that have been reported for AP sites. We suggest that the principal mechanism for AF2 mutagenesis is the formation of an adduct which depurinates to yield AP sites that serve as a substrate for error-prone repair. Seventy-two of the mutations occurred at four 5'-TGC-3' sites. The majority (10/11) of the frameshift mutations occurred at one such hotspot and could have been templated by an inverted repeat less than 100 bp removed from the site of the mutation.

Formation and persistence of DNA adducts in rats following intraperitoneal administration of 1,8-dinitropyrene.

DNA adduct formation was examined in rat tissues following a single i.p. injection with 1,8-dinitropyrene (1,8-DNP). A single common adduct was observed in mammary, mesentery, bladder, lung, kidney and liver tissue using the 32P-postlabelling technique. Adduct levels were highest in mammary and mesentery tissue. The mammary gland and soft tissues of the peritoneal cavity are primary tumour sites in rats injected i.p. with 1,8-DNP. Adducts were not detected in the small intestine, heart or reproductive tissue. Pretreatment of rats with Aroclor 1254, an inducer of hepatic oxidative enzymes, did not alter qualitative or quantitative aspects of adduct formation. Over a 2 week period the relative adduct labelling values declined in all tissues. The loss of DNA adducts was biphasic, with an initial rapid decrease followed by a slower rate of adduct removal.

Comparison of three assays for genetic effects of antineoplastic drugs on cancer patients and their nurses.

Three assays have been compared for their ability to detect genetic damage caused by antineoplastic drugs in cancer patients and possible damage in the nurses who administered these drugs. The assays were sister chromatid exchanges (SCE) and chromosomal aberrations in peripheral blood lymphocytes, and the Salmonella/mammalian microsome assay on urine. Three comparisons were made: 1) patients before versus after treatment; 2) the administering nurses immediately after their work period versus after a few days off that followed (work and off-work); 3) the exposed nurses versus other nurses who did not administer antineoplastic drugs (controls). The SCE assay detected the treatments in all eight patients from whom complete data were obtained, and was positive in two nurses with a long history of smoking. The Salmonella/mammalian microsome assay detected eight of ten treatments in patients but failed to detect smokers. Four of nine patients receiving treatment were detected by the analysis of chromosomal aberrations. The SCE assay did not distinguish between the work and off-work samples in either the exposed or control nurses. The exposed nurses, as a group, had slightly fewer SCEs than the controls due to the two smokers detected in the latter group. Chromosomal aberration was the only assay which showed significant difference between the two samples of the exposed nurses and, consequently, between the exposed and control nurses. These differences, however, arose primarily from a higher frequency of aberrations found among the exposed nurses in samples taken after a few days away from work, rather than at the end of their work period. There is no evidence that the increase was connected to occupational exposure.

DNA adduct formation in primary rabbit tracheal epithelial cells following treatment with 1,8-dinitropyrene and its partially reduced derivative, 1-nitro-8-nitrosopyrene.

Formation of DNA adducts, following treatment of primary rabbit tracheal epithelial cells (RTEC) with 1,8-dinitropyrene (1,8-DNP) and its partially reduced derivative, 1-nitro-8-nitrosopyrene (1,8-NONO2), was examined using the 32P-post-labelling technique. Treatment of aerobic cells with 1,8-DNP or 1,8-NONO2 produced qualitatively similar results. Cochromatography showed that the major adduct observed corresponded to the major adduct seen following treatment of poly(dG.dC) with N-hydroxyl-1-amino-8-nitropyrene, generated from 1,8-NONO2. A minor adduct migrated to the same area on the TLC plate as the major compound observed following a similar treatment with poly(dA.dT). Relative adduct labelling (RAL) values were consistently an order of magnitude higher with 1,8-NONO2 than with 1,8-DNP, suggesting that reduction of a nitro group of 1,8-DNP to a nitroso group may be a rate-limiting step in the cells. In studies on the formation and persistence of the 1,8-NONO2 adduct in RTEC maximum binding was observed at 1 h. Fifteen hours later the RAL value was less than 15% of this maximum level.

Metabolic activation of 3-nitroperylene in the Salmonella/S9 assay.

The mutagenicity of 3-nitroperylene (nitroPer), a nitrated polycyclic aromatic hydrocarbon (PAH) has been extensively studied using the Ames Salmonella test. In accord with previous work, nitroPer proved to be a potent frameshift mutagen which requires activation by mixed function oxidases (MFOs) in the microsomes of rat liver S9 preparations. The concentration of S9 required for optimal activation of this mutagen is several times lower than that recommended for routine screening by the Ames test. Studies with the MFO inducers Aroclor 1254, phenobarbital, 3-methylcholanthrene and beta-naphthoflavone, as well as with some MFO inhibitors indicate that both P450b and P450c appear to be involved in the activation of nitroPer. Two non-mutagenic PAHs (perylene and benzo[e]pyrene) inhibited the mutagenicity of nitroPer in a competitive fashion. The mutagenic activity of nitroPer was greatly decreased in a strain of bacteria (TA98/1,8-DNP6) that lacks an acetyltransferase needed for the activation of many nitroarenes. Incubation of nitroPer with microsomes from Aroclor-treated rats plus appropriate cofactors led to the formation of several metabolites which could be separated from one another and from nitroPer by h.p.l.c. Three of these were direct-acting mutagens with Salmonella typhimurium strain TA98, while another required microsomal activation. We postulate that the metabolic activation of nitroPer requires three steps: (i) metabolism by MOF enzymes to yield a ring-oxidized compound which is absorbed by the bacteria; (ii) reduction of this compound to the hydroxylamine by a bacterial nitroreductase; and (iii) O-acetylation of the hydroxylamine to yield a reactive ultimate mutagen.

Etoposide (VP16) and teniposide (VM26): novel anticancer drugs, strongly mutagenic in mammalian but not prokaryotic test systems.

The mutagenic effect of the antineoplastic drugs VP16 (etoposide; 4'-demethylepipodophyllotoxin-ethylidene-beta-D-glucopyranoside) and VM26 (teniposide; 4'-demethylepipodophyllotoxin-thenylidene-beta-D-glucopyr ano side) in mammalian and prokaryotic test systems have been compared. Both VP16 and VM26 which interact with mammalian DNA topoisomerase II, are strongly mutagenic in Chinese hamster ovary cells as indicated by the induction of mutations at the hypoxanthine-guanine phosphoribosyl transferase and adenosine kinase loci, and production of DNA strand breaks and sister-chromatid exchanges. Mouse L cells treated with these drugs also show a large dose-dependent (0.05-0.2 microgram/ml for VM26 and 0.5-1.5 micrograms/ml for VP16) increase in the frequency of 6-thioguanine-resistant mutants and extensive fragmentation of cellular DNA. In contrast to the results obtained with mammalian cells, VP16, which was extensively investigated, showed no increase in revertant frequencies in the Salmonella typhimurium strains TA98 and TA100 at concentrations up to greater than 500 micrograms/plate, in either the absence or presence of an exogenous rat liver activation system. However, a very weak mutagenic response to VP16 and VM26 (less than 2-fold increase in revertant frequency) at very high drug concentrations was observed in the strain TA102. VP16 also failed to show any mutagenic response (up to greater than 500 micrograms/ml) in an excision repair-proficient Escherichia coli strain 113/143 employing two different forward mutation detection systems [viz. ability to grow in galactose (Gal+) or in presence of 5-methyltryptophan], which are capable of detecting various types of genetic lesions.(ABSTRACT TRUNCATED AT 250 WORDS)

The mutagenicity of emissions from eight binder systems used in steel foundries.

Emissions generated when molten steel was poured into sand molds fabricated with eight different binder systems were collected separately and tested for mutagenic activity using the Ames Salmonella assay. The sampling device consisted of an initial 8 X 10 in. glass fiber filter in a custom fabricated apparatus through which air was drawn using a Hi-Vol motor and fan. A portion of the airstream, which passed this filter, was in turn passed through a smaller glass fiber filter and then into two glass impingers filled with ice-cold methanol. All the binder systems generated material, which induced mutations in the absence of added S-9 extract, and in all emissions a higher level of activity was found when S-9 was added. In the presence of S-9, the shell-core system produced by far the most mutagenic activity followed by the oil-clay-cereal system. The green sand, furan and Kold Set systems produced much lower levels while the two reclaimed sand systems and sodium silicate system were intermediate.

Identification of the DNA adduct formed by metabolism of 1,8-dinitropyrene in Salmonella typhimurium.

The incubation of [3H]1,8-dinitropyrene with Salmonella typhimurium TA98NR followed by isolation of the DNA from these cells, hydrolysis of the DNA to nucleosides, butanol extraction of the hydrolysate and purification by reversed-phase liquid chromatography afforded a single product. Calf thymus DNA, after treatment with N-hydroxyl-1-amino-8-nitro-pyrene, was hydrolyzed, extracted and purified in a similar fashion to give a single compound which was shown to be the deoxyguanosine derivative 1-N-(2'-deoxyguanosin-8-yl)-amino-8-nitropyrene by a combination of proton n.m.r. and u.v.-vis. spectroscopy and fast atom bombardment mass spectrometry. The DNA adducts formed in vivo and in vitro exhibited identical chromatographic and chemical behavior. Under acidic or basic conditions in the vivo and in vitro adducts were converted to identical products. Reduction of the adduct gave a new, highly fluorescent product that had a fluorescence emission spectrum identical to that of 1,8-diaminopyrene.

Dinitropyrene-resistant Salmonella typhimurium are deficient in an acetyl-CoA acetyltransferase.

Salmonella typhimurium strain TA98NR which is sensitive to 1,8-dinitropyrene mutagenesis possesses acetyl-CoA dependent acetyltransferase activity, while a strain selected for resistance to 1,8-dinitropyrene (TA98/1, 8-DNP6) is deficient in this activity. Acetyltransferase competent strains can acetylate 1,8-diaminopyrene, forming 1-N-acetylamino-8-aminopyrene and 1,8-N,N'-diacetyldiaminopyrene. The coincidence of dinitropyrene resistance and acetyltransferase deficiency implicates acetylation as an important process in the metabolic activation of dinitropyrene to a mutagenic intermediate.

Identification of polycyclic aromatic compounds in mutagenic emissions from steel casting.

Workers in ferrous foundries show increased risk of lung cancer. In the steel casting process hot metal is poured into sand moulds solidified with organic binders, producing a plume of smoke containing a variety of organic compounds and showing strong mutagenicity in the Salmonella/S9 assay. We have collected the emissions produced when steel is poured into an experimental sand mould solidified with oil, clay and cereal, a widely used binder system. The organic constituents of these emissions have been fractionated by preparative reverse-phase high performance liquid chromatography (HPLC) and mutagenic fractions have been analysed by capillary column gas chromatography/mass spectrometry (GC/MS). Of the 65 compounds for which mass spectra are reported, 54 have been tentatively identified as alkyl derivatives of polycyclic aromatic compounds. Many compounds of this class are known to be carcinogenic and mutagenic. In addition, several unsubstituted polycyclic aromatic hydrocarbons, including the carcinogenic benz[a]anthracene and benzo[a]pyrene, were found to be present.

Metabolism of 1,8-dinitropyrene by Salmonella typhimurium.

Earlier work has shown that many nitroaromatic and nitroheterocyclic compounds are directly 'activated' to their ultimate mutagenic forms through the action of bacterial nitroreductase enzymes. However, in the case of 1,8-dinitropyrene (DNP) and certain other nitroarenes the pathway of activation is more complex and neither the identity of the ultimate mutagens nor the nature of the DNA adducts formed are known. We now show that Salmonella typhimurium strains TA98 and TA1538, which are sensitive to DNP and have wild type nitroreductase complements, do metabolize DNP to 1-amino-8-nitropyrene (ANP) and 1,8- diaminopyrene (DAP) but that these compounds are much weaker mutagens than DNP. These two strains (TA98 and TA1538) contain two separable components of nitroreductase activity as determined using nitrofurazone as the substrate. The major component, at least, is capable of reducing both 1-nitropyrene (NP) and DNP although the rates are much lower than with nitrofurazone. TA98NR , a mutant of TA98 that is resistant to nitrofurazone and NP but not to DNP, lacked the major nitroreductase but retained two minor components. In contrast, a mutant ( DNP6 ) which is resistant to DNP (but not to NP) contained a full complement of nitroreductases. When the metabolism of [3H]DNP by crude extracts of TA98 was re-examined, previously undetected metabolites were found. These were more polar than DAP and ANP and were also seen when TA98NR was used as the source of enzyme. These metabolites were not formed when enzymes from TA98DNP6 or TA98NR / DNP6 were used. This work supports the notion that some enzymic activity other than (or in addition to) nitroreductase is required for the activation of DNP and that the new polar metabolites may be related to this process.

Aerobic reduction of 5-nitro-2-furaldehyde semicarbazone by rat liver xanthine dehydrogenase.

Previous work in several laboratories has shown that enzymatic reduction of nitroheterocyclic compounds to reactive but uncharacterized metabolites that damage DNA constitutes an important "activation" step in both bacteria and hypoxic mammalian cells. However, since the known mammalian enzymes having nitroreductase activity are reported to be strongly inhibited by molecular oxygen, the relation of reductive activation to the toxic and mutagenic effects of nitroheterocyclic compounds in intact animals or aerobic cultured cells is unclear. We report here that the process of net nitroreduction of 5-nitro-2-furaldehyde semicarbazone (nitrofurazone) by rat liver xanthine dehydrogenase was considerably less sensitive to inhibition by oxygen than was nitroreduction catalyzed by rat liver or milk xanthine oxidase. The dehydrogenase is the native form of xanthine oxidoreductase and is known to change to the oxidase form as liver extracts are aged or treated with various agents. Incubation at 65 degrees rapidly converted the dehydrogenase form to the oxidase form with concomitant loss of aerobic nitroreductase activity. Similarly, much of the aerobic nitroreductase activity was lost when the preparation was treated with p-hydroxymercuribenzoate but was regained upon subsequent treatment with dithiothreitol. Intermediates generated in the aerobic nitroreduction process bound tightly and probably covalently to protein. Thus, it is possible that aerobic reduction of nitrofurans and other nitroheterocyclic and nitroaromatic components by xanthine dehydrogenase may constitute a significant "activation" process which contributes to the toxic action of such agents.

Lung cancer in a steel foundry: a search for causation.

Epidemiological studies suggest an association between excess lung cancer risk and work in ferrous foundries. No causative factors have been identified. The foundry environment is complex, and little is known about the health effects of the levels of known or suspected carcinogens present. As a part of an effort to identify causative factors and to assess current risk, an Ames assay of particulate mutagenicity was undertaken. Mutagenic levels were found to exceed those of urban air and distribution findings were consistent with those of a previous epidemiological study that identified high- and low-risk areas within the foundry. The biological significance of these mutagenic levels is unknown but is under study. A mechanism is suggested to explain current and historical findings.

The 1-nitropyrene reductase of Salmonella typhimurium.

We have devised a sensitive fluorimetric assay to monitor the conversion of 1-nitropyrene to 1-aminopyrene. Application of this assay to extracts of Salmonella typhimurium strains TA98 and TA100 (which are sensitive to the mutagenic and lethal effects of 1-nitropyrene) has shown that these bacteria contain 'nitropyrene reductase' activity at the low level of 10(-11) mol/min/mg protein. NADPH and NADH serve equally well as reducing agents. The nitropyrene reductase activity of strain TA100 F50 (a mutant resistant to 1-nitropyrene) was found to be considerably lower.