Chemico/biological investigation of contaminated sediment from the Hamilton Harbour area of western Lake Ontario.

Highly contaminated sediment from the Hamilton Harbour area of western Lake Ontario was examined using a bioassay-directed fractionation methodology. A sediment sample was extracted using a Soxhlet apparatus and the resulting extract was fractionated into compound classes using an alumina clean-up step and high performance liquid chromatographic techniques. The resulting fractions were subjected to bioassays using TA98- and TA100-like strains modified by the inclusion of genes for the activating enzymes nitroreductase and O-acetyl-transferase. The majority of the mutagenic activity displayed by the sample extract was found to be present in the fraction containing the polycyclic aromatic hydrocarbons (PAH). Extracts of the PAH-containing fraction displayed dramatically higher responses with the TA100 type strains with metabolic activation. Further separation of the PAH-containing fraction showed the majority of the biological activity coeluted with PAH having molecular masses of 276, 278, and 302 amu.

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.

Analysis of high-molecular-mass polycyclic aromatic hydrocarbons in environmental samples using liquid chromatography-atmospheric pressure chemical ionization mass spectrometry.

Polycyclic aromatic hydrocarbons (PAHs) with molecular masses higher than 300 u were analysed using LC-atmospheric pressure chemical ionization (APCI) MS in extracts of environmental samples from Hamilton, Canada including zebra mussels from Hamilton Harbour, air particulate and coal tar. The LC-APCI-MS profiles of three molecular mass classes of PAHs (326 u, 350 u and 374 u) were compared to identify potential sources of PAH contamination in Hamilton Harbour. The Hamilton air particulate profile was also compared with an urban air reference standard (NIST SRM 1649) from Washington, DC, USA. Profiles of all extracts were similar and suggested an environmental predominance of PAHs within the three isomeric molecular mass classes studied. However, PAHs of molecular mass 326 u and 350 u were detected in extracts of coal tar and zebra mussels from Hamilton Harbour but were not detected in Hamilton air. These results indicated that some high-molecular-mass PAHs may be characteristic of contamination by coal tar.

Nitrofuran induced mutagenesis and error prone repair in Escherichia coli.

Nitrofuran derivatives are a class of compounds which exhibit mutagenic and cytotoxic effects in bacteria and mammalian cells in tissue culture after metabolic activation by endogenous nitroreductases. The relationship between mutation and induction of pleiotropic error-prone repair functions (the 'SOS' system) in bacteria following exposure to nitrofurans was examined. A variety of nitrofurans were found to induce protein X, the recA+ protein, which is characteristic of error-prone repair. Furthermore, induction of the 'SOS' system depended on reductive activation of the mutagen. The use of a mutant thermally inducible for error-prone repair functions (tif-1) provided direct examination of bacteria exposed to non-lethal doses of nitrofuran. These results distinguish between mutants which arose from directly induced base mispairing and those which occur only after induction of error-prone repair functions. The mutational activity of AF2 (furylfuramide) was almost entirely dependent on the induction of error-prone repair since very few tryptophan revertants were detected in conditions which did not induced the 'SOS' repair system. We present a model for mutation induction by nitrofurans in bacteria.

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.

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.

Action of nitrofurans on E. coli: mutation and induction and repair of daughter-strand gaps in DNA.

The antibacterial and mutagenic potency of 9 nitrofurans in "treat and plate" experiments varied over almost 5 orders of magnitude. The relative toxicities were as follows: FANFT greater than AF2 greater than ANFT greather than furazolidone greater than furagin greater than nitrofurantoin greater than nitrofurazone greater than methylnitrofuroate greater than nitrofuroic acid. In general, mutagenic activity paralleled toxicity. The compounds at concentrations corresponding to their LD50's, induced mutations at frequencies which ranged from 2.5/10(6) survivors for FANFT to 130/10(6) survivors for furagin (NF416). The observed differences in antibacterial and mutagenic activity are unlikely to be due to lack of activation of the weaker agents since the two most potent agents were reduced somewhat more slowly than many of the less active agents. The relative sensitivities to the antibacterial effects of AF2 of strains WP2, WP2 uvrA, CM561 (lexA) and CM571 (recA) were 1 : 1.6 : 3 : 7 and to nitrofurazone 1 : 1 : 25 : 50. The wvrA strain was 6--7-fold more mutable with both these agents than was WP2. No increase over the spontaneous mutation frequency was observed when recA or lexA strains were exposed to either AF2 or nitrofurazone in these experiments. When wild-type of wvrA bacteria containing nitrofuran-induced lesions replicated their DNA in drug-free medium in the presence of [3H]thymidine for 5 min, the label was found in low molecular weight DNA indicating that daughter-strand gaps were formed. During subsequent incubation in nonradioactive medium the molecular weight of the DNA increased to the control value. A recA strain (which was very sensitive to the lethal effects of AF2 and nitrofurazone) lacked the ability to repair daughter-strand gaps caused by nitrofuran-induced lesions.

Results of the IPCS collaborative study on complex mixtures.

The International Programme on Chemical Safety (IPCS) sponsored a collaborative study to examine the intra- and inter-laboratory variation associated with the preparation and bioassay of complex chemical mixtures. The mixtures selected were National Institute of Standards and Technology (NIST) Standard Reference Materials (SRMs). 20 laboratories worldwide participated in the collaborative trial. The participating laboratories extracted the organic portion of two particulate samples--an air-particulate sample and a diesel-particulate sample--and bioassayed the extracts. The laboratories simultaneously bioassayed a NIST-prepared extract of coal tar and two control compounds (benzo[a]pyrene, and 1-nitropyrene). The bioassay method used was the Salmonella/mammalian microsome plate-incorporation test using strains TA98 and TA100. Study design also allowed for a comparison of sonication and Soxhlet extraction techniques. The mean extractable masses for the air particles and diesel particles were approximately 5% and 17.5%, respectively. The particulate samples were mutagenic in both strains with and without activation in all 20 laboratories. For TA100 the with and without activation slope values for the air particulate were 162 and 137 revertants per mg particles, respectively. For TA98 the respective diesel slope values were 268 and 269. The mutagenicity slope values for the diesel particles ranged from 3090 (TA98, +S9) to 6697 (TA100, +S9) revertants per mg particles. The coal tar solution was negative for both strains when exogenous activation was not used but was mutagenic in both strains with exogenous activation. The benzo[a]pyrene and 1-nitropyrene were used as positive controls and gave results consistent with the literature. This paper provides a complete summary of the data collected during the collaborative study. Companion papers provide further analysis and interpretation of the results.

Bioassay-directed fractionation of PAH of molecular mass 302 in coal tar-contaminated sediment.

Bioassay-directed fractionation was used to characterise genotoxic polycyclic aromatic hydrocarbons (PAH) of molecular mass 302 amu in organic solvent extracts of coal tar-contaminated sediment from Sydney Harbour, Nova Scotia. A normal phase HPLC technique was employed to separate PAH-rich solvent extracts into fractions containing PAH of single molecular mass classes. The 302 amu molecular mass fraction was isolated and further separated using reversed phase HPLC; subfractions were collected every 30 s and subjected to bioassay analyses with Salmonella typhimurium strain YG1025 with the addition of oxidative metabolism (4% S9). Compounds eluting in the most active subfractions included naphtho[2,1-alpha]pyrene and naphtho[2,3-alpha]pyrene. The results of this study underscore the significant contribution that molecular mass 302 PAH make to the biological activity of complex environmental mixtures.

Chemical and biological profiles of sediments as indicators of sources of genotoxic contamination in Hamilton Harbour. Part I: analysis of polycyclic aromatic hydrocarbons and thia-arene compounds.

Bottom sediment and suspended sediment samples from Hamilton Harbour (western Lake Ontario) and from a major tributary were profiled using polycyclic aromatic hydrocarbons (PAH) and thia-arenes as source apportionment tracers. Ratios of selected PAH and ratios of monomethyl and dimethyl/ethyl dibenzothiophenes to the parent dibenzothiophenes were calculated. Thia-arene and PAH profiles of Standard Reference Material SRM 1649 (urban dust/organics), SRM 1650 (diesel), SRM 1597 (coal tar), Hamilton coal tar and a composite Hamilton air particulate sample provided source sample data. The gas chromatography-mass spectrometry (GC-MS) chromatograms of all sample extracts were dominated by homocyclic PAH but interpretation of PAH profiles with respect to source was difficult. In contrast, thia-arene analyses revealed more distinct differences in profiles of samples collected in different areas of the harbour, including the tributary. These results indicated that areas of coal tar-contaminated sediment are potential contributors to the overall contaminant burden of sediments and suspended sediments in Hamilton Harbour. These data also indicated that contaminants related to mobile combustion sources were entering the harbour via a major tributary.

Chemical and biological profiles of sediments as indicators of sources of contamination in Hamilton Harbour. Part II: bioassay-directed fractionation using the Ames Salmonella/microsome assay.

Bottom sediment and suspended sediment samples from Hamilton Harbour (western Lake Ontario) and from a major tributary were profiled using a bioassay-directed fractionation approach. Sample extracts were fractionated using an alumina/Sephadex gel clean-up procedure to afford non-polar aromatic fractions which were characterized using chemical analyses and the Ames/microsome bacterial assay in Salmonella typhimurium strains YG1025 with the addition of oxidative metabolism (S9), and YG1024 without S9. Non-polar aromatic fractions of selected samples were separated by normal phase HPLC into 1-min fractions which were subjected to bioassay analyses. The bioassays using strain YG1025+S9, a TA100-type strain, were performed to assess genotoxicity arising from the presence of polycyclic aromatic hydrocarbons (PAH). Fractions which exhibited mutagenic activity contained PAH with molecular masses of 252, 276 and 278 amu; these fractions contained over 80% of the genotoxicity attributable to PAH. Individual compounds identified using Gas Chromatography-Mass Spectrometry analyses in these active fractions included benzo[a]pyrene, indeno[cd]pyrene and dibenz[a,h]anthracene. The YG1025+S9 mutagenic activity profiles were similar for all samples. Mutagenic activity profiles generated using strain YG1024-S9, a TA98-type strain sensitive to compounds characteristic of mobile source emissions, were very different. The mutagenic activities in strain YG1024-S9 were greatest for harbour-suspended sediment samples collected from sites impacted by a major tributary. Suspended sediments collected near areas known to contain high levels of coal tar-contamination in the bottom sediments contained higher levels of genotoxic PAH than suspended sediments collected from other areas of the harbour.

Sarcocystosis in cattle in Kentucky.

Sarcocystosis was diagnosed in 41 eighteen-month-old heifers and steers. Clinical signs included anorexia, severe weight loss, nervousness, hypersalivation, lameness, and hair loss on the extremities. Hair loss was noticed especially at the end of the tail, where there was complete loss of the switch, giving the animals a "rat-tail" appearance. Consistent gross changes observed at necropsy of four affected animals included generalized lymphadenopathy, erosions and ulcerations in the oral cavity and esophagus, and severe laminitis. Microscopically, young cysts of Sarcocystis sp were disseminated in the heart, skeletal muscle, and brain. Ultrastructural examination indicated that the cysts were young because they contained metrocytes. Affected animals had moderate to severe nonsuppurative myocarditis and myositis, with focal degeneration of myofibers and infiltration by macrophages, lymphocytes, and plasma cells. Indirect hemagglutination of sera from 19 animals revealed a mean antibody titer of 1:24,000 against Sarcocystis bovicanis antigen. Epidemiologic investigation incriminated resident farm dogs that had been housed in a farm hayloft as the source of infection. Hay contaminated with sporocysts in dog feces was thought to have been fed to the heifers and steers.

Endonuclease alpha from Saccharomyces cerevisiae shows increased activity on ultraviolet irradiated native DNA.

Endonuclease alpha isolated from the nucleus of the yeast Saccharomyces cerevisiae is a DNA endonuclease which has been shown to act preferentially on denatured T7 DNA. The purified enzyme is more active with UV-irradiated native T7 DNA than with unirradiated substrate. The relation between damage, measured by pyrimidine dimer concentration, and excess endonuclease activity is most readily explained by local denaturation caused by presence of pyrimidine dimers. When three radiation sensitive mutants of yeast were tested for the level of endonuclease alpha present, none were found lacking the enzyme. However, nuclei of strain rad 1-1, a mutant that may be defective in heteroduplex repair as well as excision repair, were found to contain reduced levels of the endonuclease. The enzyme isolated from this strain had less than one half the specific activity of similar preparations from wild type yeast.

A DNA endonuclease isolated from yeast nuclear extract.

We have isolated and partially purified a DNA endonuclease from nuclei of the yeast Saccharomyces cerevisiae. Although purified on the basis of its ability to degrade denatured DNA, the enzyme can also attack native DNA. Denatured oligonucleotide products of the enzyme are sensitive to venom phosphodiesterase (EC3.1.4.1.) but not to bovine spleen phosphodiesterase (EC3.1.4.18). The enzyme has an estimated molecular weight of 6.6--7.5 X 10(4), more than twice as large as the endonucleases involved in DNA repair in Escherichia coli. When analyzed on glycerol gradients, the endonuclease sedimented as a single activity against both denatured DNA and closed circular DNA duplexes. The enzyme showed a 10-fold preference for denatured over native T7 DNA substrate, and appears to produce random nicks in a supercoiled replicative form of phiX174 DNA (RFI) with no discernable preference for the unpaired bases in the supercoiled duplex. The endonuclease appears to be distinct from the yeast endonucleases previously described.

Toxicological significance of DNA adducts: summary of discussions with an expert panel.

A workshop was held to discuss the uses of data on DNA adduct measurement in humans and in experimental systems in vitro and in vivo. The discussions focused principally on the understanding of the toxicological significance of DNA adducts as provided by information from animal models. An Expert Panel concluded that human DNA adduct data have utility in several aspects of risk assessment. The presence and amount of specific adducts that can be correlated with a chemical exposure are relevant for hazard identification and risk evaluation. Data from experimental systems have established dose-response relationships between the level of adducts and exposure, but these remain complex and depend on metabolic fate. Although structure-activity relationships have been useful retrospectively to explain the DNA-reactive nature of some chemicals or classes of chemicals, there are currently no means outside the laboratory to specifically predict the adduct-producing potency of a compound. Analysis of DNA adducts in tissues of laboratory animals and humans has revealed sensitive subpopulations, a finding that has important relevance for human risk assessment. Adduct analysis may be one of the best tools available to characterize exposures to DNA from complex mixtures for purposes of epidemiological investigation. Consensus statements were developed based on presentations by R. Gupta, W. Lutz, R. Nath, and B. Singer [see Regul. Toxicol. Pharmacol. 23(1), 1996] and subsequent discussions. First, rigorous scientific criteria should be met for the detection and characterization of specific DNA adducts in vitro and in target tissues in vivo. Second, the use of adduct data in risk extrapolation has the greatest value when there is characterization of adduct structure, an understanding of the role of repair in DNA adduct removal, and demonstration of biological relevance for each adduct. Third, the detection of DNA adducts in a tissue does not necessarily indicate a specific tumorigenic risk for that tissue. Fourth, the mutagenic potency for specific adducts varies by several orders of magnitude. Fifth, the role of DNA adducts induced by exogenous agents must be placed in perspective of endogenously produced adducts. The biological significance of a type of DNA adduct is related to several factors, including the efficiency of conversion to mutation, the amounts of similar endogenous adducts, and the variety of exogenous DNA adducts found in DNA from humans. The biological relevance of DNA adducts may be deduced from the dose-response relationships for adducts and tumors at physiologically relevant doses as well as from data showing mutations in targets such as oncogenes or tumor suppressor genes. There is convincing evidence in the literature for an association between some specific DNA adducts, mutation, and the carcinogenic process. As a general conclusion, the Panel suggested that the current technological capabilities for detection of DNA adducts exceed our ability to define the biological significance of adducts as it relates to toxicity or health outcome. DNA adducts are likely to play an important role in human risk for cancer induction and progression, but the quantitative aspects of this relationship remain to be determined.

Type I nitroreductases of Escherichia coli.

Analysis of partially purified crude extract of Escherichia coli K12 by chromatography and gel electrophoresis has resulted in the separation of three distinct activities which catalyse the reduction of nitrofurazone (semicarbazone of 5-nitro-2-furaldehyde) in the presence of oxygen (type I nitroreductases). The major enzymatic activity (type IA), which was dependent solely on NADPH as a cofactor, was absent from nitrofurazone-resistant strains NFR 402 and NFR 502, but present in SIL 41, a strain which is only marginally resistant to the nitrofuran. The remaining nitroreductase activities (IB1 and IB2) utilize either NADH or NADPH as a cofactor. These activities coelute from DEAE-cellulose at pH 7.2, but may be differentiated by their behaviour on CM-cellulose at pH 5.8. The reductase activity missing in SIL 41 was observed in extracts of strain NFR 402 but not NFR 502. This enzyme (IB1) though retained by DEAE-cellulose had no affinity for CM-cellulose. The only reductase present in extracts of NFR 502 (a nitrofuran-resistant strain selected after two mutational events) was type IB2. This activity, also detectable in SIL 41 and NFR 402, has not been mapped genetically. An interesting feature of the type IB2 enzyme is its apparent inactivation by MnCl2 which has been routinely used as a partial purification step in the past.

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.

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.

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)