Genotoxic Polycyclic Aromatic Hydrocarbons Research Articles

Evidence of chromosomal damage in common eiders ( Somateria mollissima) from the Baltic Sea

Common eiders nesting in the Baltic Sea are exposed to generally high levels of contaminants including potentially genotoxic polycyclic aromatic hydrocarbons and organochlorines. Blood samples were collected from eiders at eight sites in the Baltic Sea and two sites in the Beaufort Sea. DNA content variation was estimated using the flow cytometric method, and subsequently utilized as a biomarker of genetic damage. We observed no significant differences in genetic damage among populations within either the Baltic or Beaufort Seas. However, eider populations from the Baltic Sea had significantly elevated estimates of genetic damage compared to populations from the Beaufort Sea.

Nickel (II) enhances benzo[a]pyrene diol epoxide-induced mutagenesis through inhibition of nucleotide excision repair in human cells: a possible mechanism for nickel (II)-induced carcinogenesis.

Nickel (II), a ubiquitous environmental and industrial contaminant, is a well-known human carcinogen, particularly in human lung cancer. Although by itself it is a weak mutagen, nickel (II) is able to significantly enhance the genotoxicity of other mutagens and carcinogens, such as polycyclic aromatic hydrocarbons (PAHs) and ultraviolet light. Certain human populations, especially cigarette smokers, are frequently exposed to both nickel (II) and PAHs. To understand the interplay of nickel (II) and PAHs in mutagenesis and human carcinogenesis, we used a shuttle vector mutagenicity assay to examine the effect of nickel (II) on (+/-) anti-7beta, 8alpha-dihydroxy-9alpha, 10alpha-epoxy-7,8,9,10-tetrahydroxybenzo[a]pyrene (BPDE)-induced mutagenesis in human cells. BPDE is an activated metabolite of benzo[a]pyrene (BP), a major carcinogen in cigarette smoke. The shuttle vector pSP189 modified with BPDE was transfected into human cells with and without nickel (II) exposure. We found that nickel (II) exposure significantly enhanced BPDE-induced mutation frequency, but did not change BPDE-induced mutational spectrum in the supF gene of pSP189 plasmids replicated in nucleotide excision repair (NER)-proficient human cells. However, the enhancing effect of nickel (II) on BPDE-induced mutation frequency was not observed in NER-deficient human XPA cells. We also found that nickel (II) exposure of human cells did not change the spontaneous mutation frequency of the supF gene in NER-proficient or NER-deficient human cells, indicating that nickel (II) did not affect the replication fidelity in human cells. Using a plasmid containing a luciferase reporter gene and a host cell reactivation assay, we have found that nickel (II) exposure greatly inhibited the repair of BPDE-DNA adducts in NER-proficient but not in NER-deficient cells. Together these results strongly suggest that nickel (II) can greatly enhance the mutagenicity and genotoxicity of PAHs by inhibiting the NER pathway in human cells, and this may constitute an important mechanism for nickel (II)-induced human carcinogenesis.

Emission characterization of particulate/gaseous phases and size association for polycyclic aromatic hydrocarbons from residential coal combustion

The emission characterization of polycyclic aromatic hydrocarbons (PAHs) emitted from the commonly used residential coal-stove in China was studied, including the partition between the particulate and gaseous phases and the distribution as a function of particle size. Smoke samples were taken by a PUF sampler and a size-segregated sampler through a dilution equipment and analyzed for PAHs using GC/MS. The contribution of vapor phase to the total PAHs emitted was very important. The emission factors of 17 PAHs and 10 genotoxic PAHs on a coal-weight basis were 116.65 and 18.41 μg/kg, respectively, greatly lower than the extraction rates of the raw anthracite coal (2514.50 and 1343.56 μg/kg, respectively). The residential coal-stove burning honeycomb coal briquettes was identified as a clean combustion system. Submicron particles dominated the size distribution of PAHs mass, and higher molecular weight PAHs tend to distribute in finer particles.

Screening of Chemopreventive Tea Polyphenols Against PAH Genotoxicity in Breast Cancer Cells by a XRE-Luciferase ReporterConstruct

Polycyclic aromatic hydrocarbons (PAHs) are established cancer initiators that can be found in our food and environment. Some dietary plant polyphenols are strong inhibitors to PAH-induced mutagenesis, whereas others may not be as effective. To identify the chemopreventive compounds from a huge volume of dietary components, the development of an efficient screening method is required. In this study, a xenobiotic response element (XRE)-luciferase reporter plasmid was constructed to screen for some potential chemopreventive agents in tea against PAH-induced DNA damage. Tea is one of the most consumed beverages worldwide, and its beneficial effects on health have been documented. Previous studies have claimed that tea polyphenols could be protective against various cancers, and the rich database can be a source for comparison. Among the green and black tea polyphenols, the XRE-luciferase reporter assays suggested that only epigallocatechin gallate (EGCG) was effective in reducing XRE-driven luciferase assay in MCF-7 cells at the concentrations tested. Further study indicated EGCG could reduce CYP1A1 and CYP1B1 mRNA abundances and decrease the DMBA-DNA lesions. The results of DNA covalent binding of all tea polyphenols tested were consistent with the XRE-reporter assays. This study illustrated that the XRE-reporter assay was a viable screening test for dietary chemopreventive agents against PAH-initiated breast mutagenesis. It has the advantages of shorter sample processing time and producing no radioactive waste over directly measuring the CYP1A1/1B1 expressions, DNA lesion, or gel mobility shift assay.

A Comparison of Two Methods for Fractionating Complex Mixtures in Preparation for Toxicity Analysis

Chemical fractionation is a widely used tool for the chemical and toxicological characterization of complex mixtures. The objective of this research was to compare two frequently employed methods for fractionating a wood preserving waste (WPW) containing polycyclic aromatic hydrocarbons (PAHs) and pentachlorophenol (PCP). The first method involved fractionation of the WPW into acid, base, and neutral fractions using a liquid-liquid acid/base/neutral (A/B/N) technique. The second method utilized alumina column chromatography to produce two fractions, A1 and A2. Gas chromatography and mass spectrometry were used to quantify the chemical components in all fractions. The alumina method recovered 473,338 mg of total PAHs (tPAHs) per kilogram crude, while the A/B/N method yielded only 193,379 mg tPAHs/kg crude. In contrast, the A/B/N method recovered 13.7 mg PCP/kg crude while the alumina method yielded only 0.5 mg PCP/kg crude. Three bioassays were used to determine the toxicity of the crude extract and fractions. The neutral and A1 fractions contained the highest levels of tPAHs and benzo[a]pyrene (BaP) but failed to induce a positive response in the Salmonella/microsome assay with concentrations containing as much as 1800 and 2500 ng BaP/plate, respectively. In the Escherichia coli prophage induction assay, the acid fraction, which contained 472 mg PCP/kg fraction, induced a positive response, as did the base fraction, which did not contain detectable PCP. Significant reduction of gap junctional intercellular communication in hepatic cells occurred with the crude extract and acid, base, and neutral fractions. Overall, the results of these bioassays suggest that PCP genotoxicity was expressed in the acid fraction, whereas the cumulative genotoxicity of genotoxic PAHs appeared to be masked in the isolates from either fractionation method. The optimal fractionation method for a mixture of chlorophenols and PAHs may involve a refined hybrid method.

Exposure to genotoxins present in ambient air in Bangkok, Thailand--particle associated polycyclic aromatic hydrocarbons and biomarkers.

Exposure to genotoxic compounds in ambient air has been studied in Bangkok, Thailand, by analysis of polycyclic aromatic hydrocarbons (PAHs) associated with particles and using different biomarkers of exposure. Eighty-nine male, non-smoking Royal Thai police officers were investigated. The police officers were divided into a high exposure group (traffic police) and low exposure (office duty). Particulate matter was collected using personal pumps (2 l/min) and the eight carcinogenic PAHs were analysed by standard procedures. The traffic police was exposed to a 20-fold higher level of total PAHs than office police (74.25 ng/m3 vs. 3.11; P= 0.001). A two-fold variation was observed between the different police stations. The major PAHs in all groups was benzo[g,h,l]pyrelene. Large inter-individual differences in biomarker levels were observed, but the level of all markers was statistically significantly higher in the traffic police group than in the office group. The level of 1-hydroxypyrene (1-HOP) was 0.181+/-0.078 (range 0.071-0.393) micromol/mol creatinine in the traffic group and 0.173+/-0.151 (P = 0.044) in the office group. The bulky carcinogen DNA-adduct level, determined by P32-post-labelling, was 1.6+/-0.9 (range 0.4-4.3) adducts/10(8) nucleotides in the traffic group and 1.2+/-1.0 (0.2-4.9) in the office group (P = 0.029; Mann-Whitney U-test). The serum PAH-albumin adduct level was 1.76 (0.51-3.07) fmol adducts/microg albumin in the traffic group and 1.35+/-0.77 (0.11-3.45; P = 0.001) in the office group. Lower biomarker levels were observed during the period when the traffic police officers were wearing a simple facemask, indicating that these masks protect against particle-associated PAHs. No statistically significant correlations were observed between biomarker levels and the level of individual PAHs or total PAH. Our data show, that people in Bangkok, who spend most of the day outside air-conditioned offices, are exposed to high levels of genotoxic PAHs. However, for people who spend their working day in offices, the exposure is similar to people living in other metropolitan areas.

In Vivo Assessment of the Genotoxicity of PAH-Contaminated Soils in Rats Exposed by Natural Routes

In order to assess the genotoxicity of polycyclic aromatic hydrocarbons (PAHs) in vivo, male laboratory rats were exposed during 4 days to a soil highly polluted with PAHs (1200 mg.kg−1) obtained from a former coke-plant. A range of dilution, from 1 to 1/8 was realized with sand. Lymphocytes were sampled every day during the exposure. Injured nuclei were quantified by the technique of Comet assay after inclusion in an agarose gel, cells lysis and electrophoresis migration (modified from [1]). This study showed an optimum response after three days in fact whatever the dilution degree 95% of the nuclei were injured. The dilution range showed a dose-response relationship. Moreover, the quantity of injured nuclei depended on the duration of exposure. In control animals, less than 5% of the nuclei were damaged at the end of the exposure period. So the mode of exposure has shown in vivo both the bioavailability and the clastogenicity of PAHs from polluted soils. Therefore, Comet assay can be utilized as a biomarker to evaluate the genotoxic effects of a polluted soil.

Evaluation of benzo(a)pyrene-induced DNA damage in human endothelial cells using alkaline single cell gel electrophoresis

The alkaline version of the ‘comet assay’ was used to evaluate DNA damage in human umbilical vein endothelial cells (HUVEC) exposed to 0.1, 1.0, or 10 μM benzo(a)pyrene for 90 min. The genotoxicity was monitored in HUVEC pretreated with the Ah-receptor agonist β-naphthoflavone (BNF), previously shown to induce cytochrome P4501A1 (CYP1A1) activity in these cells, and in vehicle-treated HUVEC with only constitutive levels of this enzyme. Increased DNA damage was observed only in cells that had been exposed to 10 μM benzo(a)pyrene, cells exposed to BNF being subjected to the most extensive damage. The CYP1A/B-inhibitor α-naphthoflavone (ANF) reduced the benzo(a)pyrene-induced DNA-damage in the BNF-treated HUVEC to the same level as in the uninduced cells. The fact that benzo(a)pyrene induced DNA damage in vehicle-treated HUVEC suggests that there may be at least one alternative route of bioactivation for benzo(a)pyrene in these cells. Consequently, judging from the present results it seems as if tobacco-related polycyclic aromatic hydrocarbons (PAHs) may disrupt the function of the endothelial lining in blood vessels with low monooxygenase activity. It is proposed that exposure to Ah receptor agonists via, for example, tobacco smoke, may enhance the DNA-damaging effects of smoke-related genotoxic PAHs in human endothelial cells. The role of PAHs in endothelial dysfunction of tobacco smokers should therefore be further studied.

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.

Effects of solvent on DNA adduct formation in skin and lung of CD1 mice exposed cutaneously to benzo(a)pyrene.

The effect of solvent polarity and lipophilicity on DNA adduct formation by polycyclic aromatic hydrocarbons in skin and lung has been studied in CD1 mice exposed cutaneously in vivo to benzo(a)pyrene ( approximately 0.01-7.0 microg/animal) in either tetrahydrofuran or n-dodecane. The nature and amounts of DNA adducts, measured as 7R,8S, 9R-trihydroxy-10S-(N(2)-deoxyguanosyl-3'-phosphate)-7,8,9, 10-tetrahydrobenzo(a)pyrene, in relation to exposure dose and treatment regime was determined by (32)P-postlabelling. In skin DNA there was a linear relationship between exposure dose and adduct formation with both solvents, though the amount of adduct formed was significantly lower from treatment with benzo(a)pyrene in n-dodecane than in tetrahydrofuran. The amounts of adducts measured in skin DNA ranged from 67 amol adducts/microg DNA at the lowest exposure dose of benzo(a)pyrene in n-dodecane to 3.5 fmol adducts/microg DNA (1 adduct in 5 x 10(7) nucleotides to 1 adduct in 9 x 10(5) nucleotides) at the highest dose. In tetrahydrofuran the corresponding levels were 89 amol adducts/microg DNA (1 adduct in 3 x 10(7) nucleotides) to 16.9 fmol adducts/microg DNA (1 adduct in 2 x 10(5) nucleotides). DNA adducts could not be detected in lung tissue following cutaneous treatment of animals with benzo(a)pyrene in n-dodecane. Cutaneous treatment of animals with benzo(a)pyrene in tetrahydrofuran, however, resulted in adducts in lung DNA at a level of 88 amol/microg DNA from exposures only at the highest dose (6.72 microg/animal). The difference in octanol-water partition coefficient, log P(ow) between n-dodecane compared to tetrahydrofuran is considered to be the most likely reason for the reduction in the bioavailability of benzo(a)pyrene and/or its metabolites and hence the degree of genotoxicity in tissues. The results suggest that other paraffinic hydrocarbon solvents may moderate the genotoxicity of polycyclic aromatic hydrocarbons in vivo. The assessment of the genotoxicity in vivo of mixtures of compounds should be carried out on complete mixtures of substances of interest in order to take account of these possible antagonistic or synergistic effects.

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- a]pyrene and naphtho[2,3- a]pyrene. The results of this study underscore the significant contribution that molecular mass 302 PAH make to the biological activity of complex environmental mixtures.

DNA adducts in perch ( Perca fluviatilis) from a creosote contaminated site in the River Ångermanälven, Sweden

Levels of hepatic DNA adducts, analysed by 32P-postlabelling, histopathological lesions and organosomatic indices, were measured in perch ( Perca fluviatilis). The fish were caught at five sites in the river Ångermanälven, Sweden: one site known to be contaminated with creosote, sites downstream from the contaminated site and a reference site upstream. Perch were also caught at a long-distance reference site. The level of DNA adducts in fish from the creosote-contaminated site was 6.8±4.1 nmol mol −1 nucleotides compared to 0.21±0.21 nmol mol −1 nucleotides in fish from the long-distance reference site. The adduct level was also significantly increased compared to adduct levels in fish from the local reference site. Hepatocellular degeneration and macrophage aggregates were observed but did not correlate with a specific site in the river. No effects on organosomatic indices that correlated with distance from the contaminated site were observed. In the laboratory, perch were exposed to an organic solvent extract prepared from sediment collected at the creosote-contaminated site or to benzo(a)pyrene (BaP) by oral administration. Perch treated with the extract had adduct patterns very similar to those observed in perch from the contaminated field site. Two diagonal zones including 10–12 adduct spots were observed on the autoradiograms. One adduct was tentatively identified by co-chromatography with a 32P-labelled standard of 7 R,8 S,9 S-trihydroxy, 10 R-( N 2-deoxyguanosyl 3′-phosphate)-7,8,9,10-tetrahydrobenzo(a)pyrene (BaPDE-dG-3′p) in two different solvent systems. Autoradiograms derived from the BaP-exposed perch showed one major adduct and several less intense spots. The major BaP adduct had the same chromatographic properties as the standard BaPDE-dG-3′p adduct. This study shows that 32P-postlabelling analysis of DNA adducts in perch can be used as a sensitive indicator of exposure to genotoxic polycyclic aromatic hydrocarbons, and that comparison of DNA adduct patterns of fish exposed in the field and laboratory can be used to determine the responsible source of pollutants.

Genotoxic substances in the St. Lawrence system II: Extracts of fish and macroinvertebrates from the St. Lawrence and Saguenay rivers, Canada

AbstractAquatic biota frequently accumulate organic contaminants and maintain steady state tissue concentrations that are as much as 105 times higher than those in the surrounding water. Although many researchers have studied the accumulation of genotoxic polycyclic aromatic hydrocarbons (PAHs) by aquatic biota, few researchers have used bioassays to investigate the accumulation of genotoxins. In several previous studies we used the SOS Chromotest to investigate the genotoxicity of industrial effluent extracts, sediment extracts, and bivalve tissue extracts. In this study we use the SOS Chromotest to investigate the accumulation of organic genotoxins by macroinvertebrates and fish in the St. Lawrence and Saguenay rivers (Quebec, Canada). Tissue concentrations of genotoxins (expressed as μg benzo[a]pyrene genotoxic equivalents) reveal bioconcentration factors in the 102 to 103 range. Concentrations are partially determined by lipid content (r2 = 0.22). Lipid‐normalized values indicate that genotoxin concentrations in invertebrate tissues are significantly higher than those in fish. Fish values indicate that tissue concentrations are biodiminished, with fish at higher trophic levels having lower tissue burdens of genotoxins. The biodiminution pattern observed corresponds exceptionally well with trophic position assignments made by other authors. More contaminated sites yielded less contaminated specimens. This may be due to the induction of phase I and phase II detoxification enzymes that is likely to occur at high levels of exposure. Although the results do not support PAHs as the putative genotoxins, the results do indicate that the accumulated genotoxins have similar properties. Tissue to sediment ratios of genotoxins are similar to those observed for genotoxic PAHs, and far lower than those of more persistent organochlorines. Although we did not investigate genotoxic effects, we might expect the most dramatic effects in fish that consume contaminated macroinvertebrates.

Electrostatic effects on the rates of DNA-catalyzed reactions

Abstract Diol-epoxide metabolites of many genotoxic polycyclic aromatic hydrocarbons are hydrolyzed to tetraols in a detoxification reaction. The hydrolysis reaction has both spontaneous and acid-catalyzed components; moreover, the reaction rate increases in the presence of DNA. The best studied of these diol epoxide metabolites are the trans 7,8 diol-9,10 epoxides of benzo[a]pyrene: anti -BPDE, the proximate carcinogen, in which the oxirane ring is anti with respect to the 7-hydroxyl group, and its syn diastereomer, syn -BPDE. Jerina and coworkers have studied the kinetics of the hydrolysis of syn - and anti -BPDE as a function of pH and DNA concentration and have measured the equilibrium constant for the formation of a noncovalent complex with DNA. They constructed a two-state model in which the diol epoxide is either free or statically bound: the free fraction is hydrolyzed with the same kinetics as it exhibits in solution without DNA; the bound diol epoxide reacts at faster rates. In this model, the dependence of the observed hydrolysis rates on both DNA concentration and pH is explained by using the rate constants, k 0 and k H , for reactions of the free diol epoxide, the rate constants, k cat 0 and k cat H , for the bound molecule, and the binding constant, K eq . The present work uses an acidic-domain interpretation of the two-state model to explain the catalytic effect of DNA on the acid-catalyzed hydrolysis of syn - and anti -BPDE. Postulating that the rate enhancement is a result of acidic domains at the DNA surface, we assumed the relationship k cat H = k H [H + ] b , where [H + ] b is the hydrogen ion concentration near the bound molecule. Using numerical solutions to the Poisson-Boltzmann equation, the pH dependence of acidic domains at the surface of the polyelectrolyte, DNA, was calculated. Energy-minimization calculations were used to estimate the conformations of diol epoxide-DNA intercalation complexes. Poisson-Boltzmann (PB) calculations on these structures yielded hydrogen-ion concentrations near the epoxide group consistent with the k cat H /k H ratio over a range of added-salt concentrations. The results strongly suggest that DNA catalysis of diol-epoxide hydrolysis is a polyelectrolyte effect. The mechanisms and rate constants observed for the acid-catalyzed hydrolysis in the absence of DNA are consistent with the increase in the rate constant induced by DNA. It may be concluded that the catalysis is primarily an effect of the acidic domains in the surface grooves of the nucleic acid.

Tumor markers in serum, polyamines and modified nucleosides in urine, and cytogenetic aberrations in lymphocytes of workers exposed to polycyclic aromatic hydrocarbons

Some polycyclic aromatic hydrocarbons (PAHs) such as benzo(a)pyrene and benzo(a)anthracene are well-established genotoxic agents. Long-term exposure to PAHs may lead to proliferative cell disorders in humans, predominantly in the skin, lung, and bladder. The concentration of several tumor markers in serum, of polyamines and modified nucleosides in urine, and of cytogenetic endpoints in peripheral lymphocytes (sister-chromatid exchanges, high frequency cells [HFC], and micronuclei) were measured in 149 male workers exposed to PAHs in two coke oven and one graphite electrode plants, and in 137 controls. We have assessed whether these biomarkers were related to several parameters reflecting exposure to PAHs, i.e., the sum of the airborne concentration of 13 PAHs, 1-hydroxypyrene (1-OHP) concentration in postshift urine, benzo(a)pyrene-diolepoxide adducts to hemoglobin (BPDE-Hb adducts), and duration of exposure, taking also into account several possible confounding factors. HFC was the biomarker most consistently associated with the intensity of current exposure to PAHs. Smoking exerts an independent effect on the same parameter. On the basis of the logistic regression between the prevalence of abnormal HFC values and PAHs in air and 1-OHP in postshift urine found in nonsmokers, it is suggested that the latter should be kept below 6.4 micrograms/m3 and 2.7 micrograms/g creatinine, respectively. No relationship was found between the cytogenetic effects and BPDE-Hb adducts although both parameters are statistically correlated with the airborne PAH level. Some tumor markers in serum (carcinoembryonic antigen, tissue polypeptide antigen, sialic acid) and the urinary concentration of some polyamines were correlated with either PAHs in air or 1-OHP in urine. The associations, however, were very weak which suggests that these biomarkers have limited practical value for the health surveillance of groups of workers exposed to genotoxic PAHs.

Influences of chronic cholangiohepatitis and cholestasis on hepatic metabolism of benzo[a]pyrene in white suckers (Catostomus commersoni) from industrially polluted areas of Lake Ontario.

White suckers from polluted regions of western Lake Ontario have an increased prevalence of cholangiocellular and hepatocellular and hepatocellular neoplasms associated with an idiopathic chronic cholangiohepatitis. We examined the hypothesis that bile duct obstructions and cholestasis in these fish might increase the susceptibility of liver to administered benzo[a]pyrene (B[a]P). Cytosolic glutathione S-transferase (GST) activity (CDNB) was reduced in obstructed liver to 45% of activity in adjacent unobstructed liver. At micromolar concentrations, chenodeoxycholic acid, deoxycholic acid, bilirubin and haematin each inhibited GST activity of hepatic cytosolic and S-hexylglutatione-affinity-purified GST preparations from unobstructed liver. Liver cytosol and affinity-purified hepatic GSTs from normal white sucker liver reduced DNA binding of 3H-benzo[a]-pyrene-7,8-diol-9,10-epoxide (3H-BPDE) after preincubation in vitro in the presence of 5 mM GSH. Under these conditions, cytosol from adjacent unobstructed liver had a moderately stronger protective activity against DNA binding by BPDE (16.4 +/- 1.3 pmol BPDE/mg DNA) than did cytosol from obstructed liver (20.6 +/- 1.6 pmol BPDE/mg DNA). Suckers with obstructed livers identified by laparotomy were orally administered 3H-benzo[a]pyrene (3H-B[a]P) (0.2 mmol/kg) or unlabelled B[a]P (2.0 mg/kg) and the level of B[a]P macromolecular binding was analyzed in liver tissue by liquid scintillation counting and by immunohistochemistry with antibodies to BPDE-DNA adducts. Covalent binding of 3H-B[a]P to hepatic protein was 30% less in adjacent unobstructed liver compared to obstructed liver; however, there was no significant difference in the levels of 3H-B[a]P bound to DNA in the obstructed lobes compared with non-obstructed adjacent liver. These studies demonstrate that some endogenous non-substrate ligands that accumulate during cholestasis can reduce hepatic GST activity in white suckers. While these changes are insufficient to influence total 3H-B[a]P-DNA adducts in obstructed liver, the preferential localization of BPDE-DNA adducts in GST-deficient hyperplastic biliary tracts suggests that cholangiohepatitis might increase susceptibility to cholangiolar neoplasia in fish exposed to genotoxic polycyclic aromatic hydrocarbons.

Determinants of urinary thioethers, D-glucaric acid and mutagenicity after exposure to polycyclic aromatic hydrocarbons assessed by air monitoring and measurement of 1-hydroxypyrene in urine: a cross-sectional study in workers of coke and graphite-electrode-producing plants.

A cross-sectional epidemiological study was performed on 286 workers from two coke oven and one graphite electrode plants. The aim was to evaluate the usefulness of monitoring 1-hydroxypyrene (1-HOP) in urine for assessing exposure to polycyclic aromatic hydrocarbons (PAHs), and that of the urinary excretion of thioethers and D-glucaric acid, and the mutagenic activity of urine as indicators or biological effects of PAHs. The results confirm that 1-HOP determination in urine probably reflects exposure to PAHs by all routes and is not significantly influenced by the smoking habit. In comparison with the total PAHs in the air and 1-hydroxypyrene in urine, taken as reference exposure parameters, the results indicate that urinary D-glucaric acid excretion is not positively influenced by PAHs exposure; thioethers determination in urine is of poor value, since the smoking habit is a strong confounding factor. The determination of urinary mutagenicity might contribute to the detection of groups of workers exposed to potentially genotoxic PAHs.

Development of chiral stationary phases for the enantiomeric resolution of dihydrodiols of polycyclic aromatic hydrocarbons by π-donor-acceptor interactions

Chiral stationary phases (CSPs) derived from ( R)-(−)-2-(2,4,5,7-tetranitrofluoren-9-ylideneaminooxy) propionic acid (TAPA) covalently bound to silica gel have been developed by altering the alkyl group at the chiral centre or the number of aromatic rings and their degree of nitration. The chromatographic properties of the CSPs were characterized by use of a racemic model solute. Depending on the solvent strength of the mobile phase, the CSPs exhibit the quality of a normal or a reversed phase. The chromatographic behaviour of 30 racemic hydroxylated derivatives of polycyclic aromatic hydrocarbons (PAHs) on ( R)-(−)-TAPA CSP revealed the structural requirements for chiral recognition. The applicability of the CSPs for the enantiomeric separation of trans-dihydrodiols of PAHs on an analytical as well as preparative scale and for investigating the enantioselectivity of the biotransformation and genotoxicity of PAHs is demonstrated.

Determination and Genotoxicity of Polycyclic Aromatic Hydrocarbons Isolated from Dreissina polymorpha (Zebra Mussels) Sampled from Hamilton Harbour

Polycyclic aromatic hydrocarbons (PAH) were determined in extracts of zebra mussels sampled from sites in Hamilton Harbour. The mussels were extracted using a tissue homogenizer method and the resulting solvent extracts were subjected to an alumina column and a Sephadex LH20 column clean-up procedure. Total PAH concentrations in Zebra mussel extracts ranged from 0.35 μg/g to 10.0 μg/g of mussel homogenate. The extracts were also found to exhibit significant mutagenic responses in the Ames Salmonella/microtome assay. The results indicate that zebra mussels might be used as biological indicators of PAH contamination in freshwater environments such as Hamilton Harbour.

Effect of caloric restriction on the metabolism of 7-bromobenz[a]anthracene and 7-fluorobenz[a]anthracene by male B6C3F1 mouse liver microsomes: Reduction of metabolic activation pathway

The effect of caloric restriction (CR) on the in vitro metabolism of 7-bromobenz[a]anthracene (7-Br-BA) and 7-fluorobenz[a]anthracene (7-F-BA) by liver microsomes isolated from 5-and 12-month-old male B6C3F1 mice was studied. Mice were fed ad libitum (AL), or starting at 14 weeks of age, received 60% of the calories consumed by control mice. After microsomal incubation, metabolites were separated by HPLC and their structures identified by comparison of their spectra with known standards. The metabolites formed were: trans-3,4-dihydrodiol, trans-5,6-dihydrodiol, trans-8,9-dihydrodiol and trans-10,11-dihydrodiol, 5,6-epoxide, and 4-, 5-, 6-, 8-, and 9-phenolic derivatives. The formation of the 7-Br-BA trans-3,4-dihydrodiol and 7-F-BA trans-3,4-dihydrodiol, the proximate mutagens of 7-Br-BA and 7-F-BA, respectively, was lower in microsomal incubation mixtures derived from the 5-month-old CR mice as compared to those from the AL. These results suggest that CR can diminish the genotoxicity of polycyclic aromatic hydrocarbons by directly altering metabolic activation.