PAH Exposure Research Articles

Urinary 1-hydroxypyrene as a biomarker of internal dose of polycyclic aromatic hydrocarbons in carbon black workers.

In this study, a total of 30 workers were selected, including eight wet pelletizing workers and 22 packaging workers. For all selected workers, urine samples were collected on the first day pre-shift, first day post-shift and fifth day post-shift, and their urinary 1-hydroxylpyrene levels (1-OHP) were determined (denoted as BM1pre, BM1post and BM5post, respectively). Personal respiratory exposures, including both inhalable particle-bound PAHs (Cinh) and gaseous PAHs (Cgas), together with dermal exposure to particle-bound PAHs (Cskin) were measured. Personal background information, including age, sex and smoking habit, was carefully registered. Pyrene exposure was statistically significantly correlated with exposure to PAHs and carcinogenic PAHs. Multiple linear regression analysis results showed that the BM1post values could not be explained by workers' exposures. For BM5post in packaging workers, both the regression model (R2 = 0.73) and the regression coefficients for Cgas, Cinh and Cskin were statistically significant (P < 0.05). For pelletizing workers, the R2 value was higher but was not statistically significant because of the smaller number of workers. The resultant regression coefficients for 'sex', 'smoking habit' and 'age' were statistically insignificant (P >> 0.05), which could be because these variables made relatively small contributions to BM5post. In conclusion, this study suggests BM5post could be a suitable indicator for PAH exposures of carbon black workers, on the condition that both respiratory (including gaseous PAHs and particle-bound PAHs) and dermal exposures have been assessed.

In Vivo and in Vitro Inhibition of CYP1A-Dependent Activity in Fundulus heteroclitus by the Polynuclear Aromatic Hydrocarbon Fluoranthene

Certainpolynuclear aromatic hydrocarbons (PAHs) such as benzo[a]pyrene (BaP) induce CYP1A-dependent enzyme activities. Because PAHs are ubiquitous environmental contaminants, and some are aryl hydrocarbon agonists, CYP1A has been used as a biomarker for PAH exposure. However, PAHs exist in the environment in complex mixtures that may confound biomarker results. In in vitro studies, the PAH fluoranthene (FL) failed to increase or enhance CYP1A1-dependent ethoxyresorufin-O-deethylase (EROD) activity in cells, but rather inhibited activities induced by AhR agonists such as 2,3,7,8-tetrachlorodibenzo-p-dioxin and benzo(k)fluoranthene. In order to determine the in vivo effects of FL on CYP1A and DNA adduct levels, Fundulus heteroclitus were given single ip injections of either BaP (5 mg/kg), BaP + FL (5 mg/kg each), BaP + FL (5 and 50 mg/kg, respectively), FL (5 mg/kg), FL (50 mg/kg), or corn oil control. BaP-treated fish had liver microsome EROD activities significantly higher than controls, whereas FL-treated fish were not different from controls. EROD activities in BaP + FL cotreatments were significantly lower compared to fish treated with BaP alone. When FL was incubated with BaP-induced microsomes, the IC50 for inhibition of EROD activity was 1.4 × 10−5 M FL. Kinetic studies indicated a significant noncompetitive component to the FL inhibition. When fish were treated with [14C]FL, the concentration of radiolabel associated with microsomal preparations was four orders of magnitude lower than the IC50. Therefore, the presence of FL or a FL metabolite was insufficient to account for the inhibition by a kinetic mechanism. In contrast to cell studies, CYP1A immunoreactive protein was significantly decreased in vivo by FL cotreatment, indicating that FL may inhibit EROD activity by down-regulating the CYP1A protein. A covalent interaction of [14C]FL with CYP1A was not detected. Total 32P-postlabeled DNA adducts were not significantly changed by cotreatment of FL and BaP; however, cotreatment with 50 mg/kg FL decreased one adduct and increased another significantly. Because FL and perhaps other inhibitory PAHs, cooccur in the environment with CYP1A inducers, CYP1A-dependent bioassays may cause an underestimation of PAH exposures.

Bioavailability of PAHs in the Biobio river (Chile): MFO activity and biliary fluorescence in juvenile Oncorhynchus mykiss

Cytochrome P450-dependent monooxygenase (MFO) activity and levels of bile PAH metabolites were measured in fish ( Oncorhynchus mykiss) exposed to Biobio river (Chile) water. Experiments were performed simultaneously in both the field and laboratory to investigate whether the river water contained notable bioavailable PAH fractions. The field experiment was conducted using fish caged for 41 days at La Mochita (Biobio river mouth) whereas the laboratory experiment involved a 21-day exposure treatment with water collected in the same area. Induction of hepatic MFO, assayed by benzo(a)pyrene monooxygenase activity (BaPMO), and levels of PAH metabolites in the bile were measured in groups of eight (field experiment) and four specimens (laboratory experiment) sampled after 5, 10, 21 and 41 days of exposure. BaPMO was induced by a factor of 23 and 25 in fish caged for 21 and 41 days, respectively. Likewise, a significant BaPMO induction was found in the fish group experimentally exposed to river water for 21 days. Biliary fluorescence measurements, recorded by fixed wavelength fluorescence (FF) and synchronous fluorescence spectroscopy (SFS), revealed significantly high levels of PAH metabolites in bile samples of fish caged for 21 and 41 days. A positive relationship was found between BaPMO activity and biliary PAH metabolites. This study provides evidences that (i) Biobio river water contains a significant bioavailable PAH fraction in terms of marked effects on fish and (ii) the biological indicators MFO activity and bile PAH metabolites represent an excellent screening methodology to assess PAH exposure.

POLYCYCLIC AROMATIC HYDROCARBON EXPOSURE AND BURDEN OF OUTDOOR WORKERS IN BUDAPEST

Polycyclic aromatic hydrocarbons exposure (PAHs: (benz[a]anthracene, benzo[a]pyrene, dibenz[a,h]anthracene, benzo[b]fluoranthene, benzo[k]fluoranthene, indeno[1,2,3-cd]-pyrene, fluoranthene, chrysene, pyrene) of policemen on street duty in downtown Budapest and workers repairing the road (asphalting) at a traffic junction and their excretion of PAH metabolites (1-hydroxypyrene, 3-hydroxybenz[a]anthracene, and 3-hydroxybenzo[a]pyrene) were determined. As controls, health-care workers were investigated. In addition PAH pollution of the air of a factory processing asphalt was also measured. The measurements were performed on air samples gained using personal samplers and from urine of end-shift samples using a high-performance liquid chromatography method. It was found that PAH pollution of the most crowded and busy center of Budapest was similar to that of several other cities in the world. PAH exposure of road builders was actually not higher than that of policemen; the slight difference resulted from diverging life-styles. PAH metabolite excretion of smoking health-care workers, road builders, or policemen significantly exceeded that of the nonsmokers. The PAH metabolite values of the three groups engaged in various activities did not show any difference. It was concluded that cancer-related risk due to PAH compounds in the case of policemen on street duty and road builders (asphalting) does not exceed significantly that of workers not exposed occupationally to PAHs in the ambient air, but that smoking is a decisive factor.

Enzymatic biomarker measurement and study of DNA adduct formation in benzo[a]pyrene-contaminated mussels, Mytilus galloprovincialis

The aim of this study was to improve the knowledge on the metabolic pathways involved in benzo[a]pyrene (B[a]P) activation and on the relationship between adduct levels and enzymatic biomarker activities. With this purpose, a model to assess pollutant exposure via food supply has been developed for the sentinel organism, Mytilus galloprovincialis. Mussels were fed for 4 weeks with B[a]P-contaminated feed (50 mg/kg dry weight mussel). Bioaccumulation was studied by determination of B[a]P concentration in whole mussel by GC/MS analysis. Different biomarkers of pollutant exposure were measured to assess the metabolic state of the exposed organisms. CYP1A-like immunopositive protein titration and B[a]P hydroxylase (BPH) activity were assessed as indicators of phase I biotransformation. Glutathione- S-transferase (GST) activity was measured as an indicator of the conjugation activities. Catalase (CAT) and DT-diaphorase (DTD) activities were assessed as potential biomarkers of oxidative stress, whereas acetylthiocholine esterase (AChE) activity was measured as an indication of possible neurotoxicity of B[a]P exposure. DNA adduct levels were determined in digestive gland DNA by applying the 32P-postlabeling technique with nuclease P1 enhancement. For the developed conditions of exposure, B[a]P concentration reached in whole mussel tissues was very high (>500 mg/kg d.w. mussel) and significant B[a]P-induced changes were recorded for each enzymatic biomarkers. BPH and CAT activities were significantly increased by B[a]P exposure, whereas GST in the gills, DTD and AChE were significantly depressed. On the other hand, no change in CYP1A-like immunopositive protein content was observed. Induction and increase with time of bulky B[a]P-related DNA adducts were demonstrated in the digestive gland, although at low levels (0.269±0.082 adduct/10e8 dNps at maximum) by the 32P-postlabeling assay. DNA adduct level was significantly correlated with whole mussel tissue B[a]P concentration, so were all the enzymatic biomarkers measured except to GST activity in both gill and digestive gland tissues. BPH, DTD, CAT and AChE displayed a strong correlation with adduct levels. These results demonstrate the neurotoxicity and the genotoxicity of B[a]P exposure in the mussel. The induction of bulky DNA adducts in mussels demonstrates the existence of activation pathways already identified in vertebrates. It validates also the suitability of this model for further studies on B[a]P metabolism in mussels. Our results support the proposal of BPH, AChE, DTD and CAT activities as suitable biomarkers of PAH exposure for these sentinel species.

Biochemical indicators of pollution exposure in shorthorn sculpin ( Myoxocephalus scorpius), caught in four harbours on the southwest coast of Iceland

Shorthorn sculpins ( Myoxocephalus scorpius) were caught in four Icelandic harbours, differing in size, use and traffic. Biochemical responses in liver were measured and chemicals analysed in bile. Eyrarbakki harbour, which has not been in use for many years was chosen as a control site. Njar∂vı́k harbour is a small fishing harbour and a marina, Sandger∂i harbour is a large fishing harbour, and Reykjavı́k harbour is a large fishing harbour and an international transport harbour. Higher levels of DNA-adducts and cytochrome P4501A (CYP1A) in the fish from the harbours in Sandger∂i, Njar∂vı́k and Reykjavı́k, compared to Eyrarbakki harbour, indicate PAH exposure. This was confirmed by PAH analysis in bile. The higher activities of the antioxidant enzymes catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR) in fish caught in Sandger∂i, than in fish caught in the other harbours, indicate exposure of sculpin to prooxidative compounds in Sandger∂i harbour. Shorthorn sculpin seems to be a convenient species for monitoring pollution in northern coastal areas.

尿中PHA代謝物を用いたコークス炉作業者におけるPHA暴露への介入の評価

尿中PHA代謝物を用いたコークス炉作業者におけるPHA暴露への介入の評価

Polycyclic Aromatic Hydrocarbon Biomarkers of Internal Exposure in U.S. Army Soldiers Serving in Kuwait in 1991

Biomarkers of exposure were applied to a cohort of U.S. Army soldiers who were deployed to Kuwait and Saudi Arabia in 1991 in the aftermath of the Persian Gulf War. The U.S. Army Environmental Hygiene Agency (currently the U.S Army Center for Health Promotion and Preventive Medicine) monitored air and soil for ambient PAHs. In addition, a group of 61 soldiers kept diaries of daily activities. These soldiers provided blood and urine samples in June in Germany before deployment to Kuwait, in August after 8 weeks in Kuwait, and in October, one month after the return to Germany. DNA, prepared from white blood cells, was assayed for PAH-DNA adducts by immunoassay and bulky aromatic adducts by 32P-postlabeling. Urinary 1-hydroxypyrene-glucuronide (1-OH-PG) was determined by synchronous fluorescence spectrometry. Contrary to expectations, environmental monitoring showed low ambient PAH levels in the areas where these soldiers were working in Kuwait. In addition, literature values for ambient PAH monitoring in Germany in 1990 suggest that the soldiers may have been exposed to higher levels of ambient PAHs in Germany than in Kuwait. Blood cell DNA adduct levels were lowest in Kuwait and increased significantly after the return to Germany. Also, urinary 1-OH-PG levels were lowest in Kuwait and highest in Germany. This study demonstrates modulations in PAH exposure biomarker levels that appear to correlate with ambient PAH exposure.

Influence of GSTM1, GSTT1, GSTP1, and EPHX gene polymorphisms on DNA adduct level and HPRT mutant frequency in coke-oven workers

To evaluate the influence of individual susceptibility factors on the level of polyaromatic (PAH) hydrocarbon DNA adducts and hypoxanthine guanine phosphoribosyl transferase ( HPRT) mutants in peripheral lymphocytes, 70 coke-oven workers exposed to PAH were genotyped for four metabolic enzyme polymorphisms of potential importance in PAH metabolism. The examined genetic polymorphisms concerned glutathione S-transferases M1 ( GSTM1; gene deletion; 96 workers), T1 ( GSTT1; gene deletion), P1 ( GSTP1; Ile→Val substitution at codon 104 or Ile→Val at codon 104 and Val→Ala at codon 113), and microsomal epoxide hydrolase ( EPHX; Tyr→His substitution at codon 113 and His→Arg at codon 139). The workers were classified in a high- and low-exposure group on the basis of urinary concentration of 1-pyrenol. The GSTM1 null genotype increased the number of DNA adducts in smoking coke-oven workers with high PAH exposure. DNA adducts were affected by PAH-exposure in non-smokers and in GSTM1 null smokers and by smoking in GSTM1 null individuals. In a multiple linear regression analysis, the interaction of the GSTM1 genotype was statistically significant ( p=0.04) with smoking (yes/no) and of borderline significance ( p=0.06) with PAH-exposure (high/low). As smoking also increased urinary 1-pyrenol, the genotype modification seemed to concern DNA adducts due to smoking rather than occupational exposure. GSTT1 positive individuals showed an elevated level of DNA adducts in comparison with GSTT1 null subjects ( p=0.04), and EPHX genotypes associated with slow hydroxylation reaction yielded a higher ( p=0.05) HPRT mutant frequency than fast EPHX genotypes; these findings were, however, based on small numbers of subjects and need to be clarified in further studies. In conclusion, our findings indicate that homozygous deletion of GSTM1 results in an increased sensitivity to genotoxic PAHs in tobacco smoke, which is seen as an increase in aromatic DNA adducts in blood mononuclear cells.

Monohydroxylated Pah Metabolites as Biomarkers for Occupational Pah Exposure

The exposure to PAH of 21 workers occupied in a fire-proof stones producing plant has been investigated. Beside conventional ambient monitoring biological monitoring was performed using a coupled-column HPLC method for the determination of 10 mono-hydroxylated PAH metabolites including 3-hydroxybenzo[a]pyrene and 3-hydroxybenz[a]anthracene.

A new Indirect method for Detection of Metabolites of Polycyclic Aromatic Compounds in Urine

A frequently used means of assessing exposure to PAHs is measurement of urinary 1-hydroxypyrene and its metabolites following enzymatic deconjugation. Singh et al. [1] have reported an HPLC method which eliminates the deconjugation step, and directly measures levels of metabolites as well as free 1-hydroxypyrene. Using the samples from their study, including urine from mice fed coal tar, and urine from workers occupationally exposed to coal tar, we have developed a rapid, sensitive method for determining exposures to PAHs. The method takes advantage of the extreme mutagenicity of nitro-PAH in the Ames Assay. Urine samples were chemically nitrated and tested in Ames strain TA98. For the mouse urines, correlation of mutagenic potency with various measures of coal tar intake and excretion gave R2 values greater than 0.97. There was more scatter in the worker study, possibly reflecting differential responses of the two methods to non-coal tar PAH exposures.

Influence of parasitism in controlling the health, reproduction and PAH body burden of petroleum seep mussels

Petroleum seep mussels are often exposed to high hydrocarbon concentrations in their natural habitat and, thus, offer the opportunity to examine the relationship between parasitism, disease and contaminant exposure under natural conditions. This is the first report on the histopathology of cold-seep mussels. Seep mussels were collected by submersible from four primary sites in the Gulf of Mexico, lease blocks Green Canyon (GC) 184, GC-234, GC-233, and Garden Banks 425 in 550–650 m water depth. Five types of parasites were identified in section: (1) gill “rosettes” of unknown affinity associated with the gill bacteriocytes, (2) gill “inclusions” similar to chlamydia/rickettsia inclusions, (3) extracellular gill ciliates, (4) body “inclusions” that also resemble chlamydial/rickettsial inclusions, and (5) Bucephalus-like trematodes. Comparison to shallow-water mytilids demonstrates that: (1) both have similar parasite faunas; (2) seep mytilids are relatively heavily parasitized; and (3) infection intensities are extremely high in comparison to shallow-water mytilids for Bucephalus and chlamydia/rickettsia. In this study, the lowest prevalence for chlamydia/rickettsia was 67%. Prevalences of 100% were recorded from three populations. Bucephalus prevalence was ⩾70% in three of 10 populations. The parasite fauna was highly variable between populations. Some important parasites were not observed in some primary sites. Even within primary sites, some important parasites were not observed in some populations. Bucephalus may exert a significant influence on seep mussel population dynamics. Forty percent of the populations in this study are severely reproductively compromised by Bucephalus infection. Only a fraction of petroleum seep mussel populations are maintaining the entire beta-level population structure of this species. Variation in two parasites, gill ciliates and Bucephalus, explained most of the variation in PAH body burden between mussel populations. PAHs are known to be sequestered preferentially in gametic tissue. Bucephalus would be expected to reduce overall body burden, at high infection intensities, by replacing gametic tissue. PAH concentrations exceeded 1 ppm in 4 of 9 populations, a ratio significantly higher than the 8 of 30 mussel locales in the NOAA Mussel Watch Program. Only five Mussel Watch locales exceeded the highest value for a petroleum seep population. Digestive gland and gill tissue atrophy were not significantly correlated with PAH body burden, even though some populations were characterized by body burdens exceeding 1 ppm, suggesting that seep mussels may not be as sensitive to PAH exposure as are some shallow-water mytilid populations.

Elimination of 1-Hydroxypyrene in the Urine of Workers from Different Workplaces as an Indicator of Occupational PAH Exposure

The exposure to PAH of 327 workers occupied at 12 different workplaces has been investigated. Beside conventional ambient monitoring 1-hydroxypyrene was determined in the urine of the employees. Our investigations show that at certain workplaces the internal exposure to PAH of the employees is inadmissibly high although the PAH air concentrations determined at most of the workplaces were below the current threshold limits. Workers from different work places have different 1-hydroxypyrene concentrations in urine. Even employees working at work places with comperable PAH concentrations in air eliminate different 1-hydroxypyrene concentrations in urine. Our results indicate that the determination of PAH in air is not sufficient for health surveillance. To ensure health surveillance a biological monitoring is necessary.

Comparison of three analytical methods for 1-hydroxypyrene glucuronide in urine after non-occupational exposure to polycyclic aromatic hydrocarbons

Urinary pyrene metabolites, 1-OHP and 1-OHPG, have been used as biomarkers for the assessment of occupational and environmental exposure to PAHs. This study compares the sensitivity and applicability of the different analytical methods of 1-OHPG for human biomonitoring of low level exposure to PAHs. Three analytical methods were compared: (1) HPLC method from that reported by Singh et al. (Singh, R., Tucek, M., Maxa, K., Tenglerova, J., Weyand, E.H., 1995. A rapid and simple method for the analysis of 1-hydroxypyrene glucuronide: a potential biomarker for polycyclic aromatic hydrocarbon exposure. Carcinogenesis 16, 2909–2915); (2) IAC-SFS method: the rapid and simple assay using IAC purification using monoclonal antibody specific for PAH-DNA adduct and PAH metabolites and SFS quantitation; and (3) IAC-HPLC method: IAC and HPLC separation and quantitation. The correlation between the IAC-SFS method, HPLC method, and the IAC-SFS method was determined in 20 first year-grade junior high school students (age 12–13) from Yochon, Korea who participated in a nationwide survey for the environmental disease surveillance projects in Korea. Chromatograms obtained by the IAC purification and HPLC quantitation method were clear with no interfering peaks adjacent to 1-OHPG, thus 1-OHPG could be easily quantitated. However, the HPLC method produced chromatogram profiles with many interfering peaks adjacent to 1-OHPG peak. The concentrations of 1-OHPG in 20 urine samples were similar when analyzed by all three analytical methods. The correlation coefficient between the IAC-HPLC and IAC-SFS methods was 0.915, and between the IAC-HPLC and HPLC methods was 0.844, and between the IAC-SFS and HPLC methods was 0.805. The analytical methods for 1-OHPG compared in this study showed a good correlation with one another. These results suggest that any of the methods can be applied to human biomonitoring of PAH exposure. However, SFS quantitation after IAC purification is rapid and simple because this method does not need HPLC separation of 1-OHPG.

Influence of GSTM1 genotypes on anti-BPDE-DNA adduct levels in mononuclear white blood cells of humans exposed to PAH.

Association between genetic deletion polymorphism of GSTM1 (*0/*0 or active) and levels of anti (+/-)-r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE)-DNA adducts in the peripheral blood lymphocyte plus monocyte fraction (LMF) of PAH-exposed subjects was investigated. A total of 94 Caucasian subjects comprised the sample population: 13 coke-oven workers, 19 chimney sweeps, 36 aluminum-anode plant workers, and 26 non-occupationally PAH-exposed subjects (controls). PAH exposure was assessed in each group by means of the urinary excretion of 1-pyrenol (mean group levels 1.2, 0.7, 0.3, and 0.1 mumol/mol creatinine in coke-oven workers, chimney sweeps, aluminum-anode plant workers, and control subjects, respectively). Anti-BPDE-DNA adducts were detected by HPLC/fluorescence analysis of anti-BPDE tetrols (tetrol I-1) released after acid hydrolysis of DNA samples. In coke-oven workers the percentage of cases with adduct levels exceeding the 95th percentile control value (4.4 adducts/10(8) nucleotides) was significantly higher in the subgroup with the null GSTM1 genotype (*0/*0) (100%) than in that with active GSTM1 (43%; chi 2 test, P < 0.05). In the other groups with different and lower levels of PAH exposure the percentages of positive samples were always higher in the subgroup with GSTM1 *0/*0 than in the active one, although the differences were not statistically significant. Univariate (odds ratio) and multivariate (relative risk) analyses showed that the risk of having high anti-BPDE-DNA levels increased with occupational exposure to PAH. Such risks, moreover, were further significantly increased by the lack of GSTM1 activity (RR = 5.94; CI = 1.15-30.7; P < 0.05). In coke-oven workers, chimney sweeps, and aluminum workers, respectively, the multiplicative effect of the null genotype with occupational PAH exposure gives risks of 162 (= 27.2 x 5.94), 10 (= 1.70 x 5.94), and 3 (= 0.50 x 5.94) times higher probability (risk) of high BPDE-DNA adduct formation than that of non-exposed subjects with the active GSTM1 genotype. Our results indicate a greater risk of anti-BPDE-DNA adduct formation resulting from occupational high-level PAH-exposure in GSTM1 null (GSTM1 *0/*0) workers.

Benzo(a)pyrene diol-epoxide-DNA adduct levels and aryl hydrocarbon hydroxylase (AHH) in human white blood cells from smokers and non-smokers

Benzo(a)pyrene diol-epoxide (BPDE)-DNA adducts and aryl hydrocarbon hydroxylase (AHH) activity were determined in white blood cells (WBCs) obtained from non-smokers, ex-smokers and smokers. DNA adduct levels ranged from non-detectable (ND) to 8.8 per 10 8 nucleosides in all samples analysed. The mean level in WBC from smokers (1.93±0.55 mean±SE) was similar to that from non-smokers (1.18±0.40) and ex-smokers (1.83±0.50). The distribution frequency of AHH activity in non-smokers was different from that in smokers, while 50% of non-smokers had AHH activity ranging from ND to 20 fmol mg -1 min -1 , This low AHH activity was not found in smokers; no correlation was found between AHH activity and tobacco consumption (r=0.097; P = 0.76; n=12). On the contrary, AHH activity was correlated with BPDE-DNA adduct levels in WBCs from the same subjects (r=0.457; P=0.019; n=26). Our findings indicate that there are limitations to the use of WBCs as surrogate cells for determining internal PAH exposure in target organs.

Influence of metabolic genotype GSTM1 on levels of urinary mutagens in patients treated topically with coal tar

Fifteen hospitalized, non-smoking, dermatological patients were treated with ointment containing 2% coal tar (CT) in order to assess the influence of metabolic genotype GSTM1 on urinary mutagen levels. Urinary 1-pyrenol, the main metabolite of pyrene, was used to check the high exposure to PAH of this population. The mean levels of urinary 1-pyrenol found in the 24-h urine of our patients were 467.8±211.0 nmoles-24 h (range 94.6–890.1 nmoles-24 h). Mutagenicity was assessed on urine samples collected over a period of 24 h, after three consecutive days of topical application, using the bacterial mutagenesis test on Salmonella typhimurium strains TA98 and YG1024 in the presence of microsomal enzymes. The latter strain turned out to be more sensitive than the former in revealing urinary mutagens in these patients (42 693±30 867 vs. 6877±6040 net revertants-24 h). The mutagenicity on YG1024 strain and 1-pyrenol levels of urine samples were correlated (Spearman's rank correlation coefficient=0.6678, P<0.01, z=2.795). The influence of genotype GSTM1 on urinary mutagen levels was assessed on strain YG1024. The values of urinary mutagenicity of subjects with genotype GSTM1-null ( n=6) were on average higher than those of GSTM1-positive subjects ( n=9) (55 498±45 957 vs. 34 156±11 933 net rev.-24 h), a non-significant statistical difference. The mean total excretion of mutagens corrected for PAH exposure (net rev./nmoles of urinary 1-pyrenol) in GSTM1-null patients was double that of GSTM1-positive ones (136.8±34.7 vs. 70.8±23.3 net rev./nmoles of urinary 1-pyrenol; one-tailed Mann–Whitney U-test, U=11.5, P<0.05). These results indicate a greater body burden of promutagens, resulting from skin application of CT, in GSTM1-null subjects.

Profile of urinary phenanthrene metabolites in smokers and non-smokers

Phenanthrene metabolites (phenols and dihydrodiols) and 1-hydroxypyrene excreted in the 24-h urine of smokers, non-smokers and lung cancer patients, who after heavy smoking became light smokers, were determined and compared. In contrast to 1- hydroxypyrene, no significant differences of the absolute amounts of phenanthrene metabolites were found between smokers and non-smokers. A ratio phenanthrene metabolites/l-hydroxypyrene of 10.4 was observed for non-smokers and 9.9 for lung cancer patients, but 4.2 for smokers. Significantly different ratios for the regiospecific oxidation of phenanthrene were found for smokers when compared with non-smokers (1,2-oxidation vs 3,4-oxidation was 1.45 in the case of smokers, but 2.34 in the case of non-smokers) indicating a cigarette smoke - but not PAH - caused induction of CYP 1A2 in smokers. As a consequence of the degree of PAH exposure the ratio dihydrodiols/phenols depends on the total amount of metabolites excreted. Phenols predominate, equally in smokers and non-smokers after low exposure, while dihydrodiols become more prominent in highly exposed persons (coke plant workers). Both (i) the regiospecific oxidation of PAH and (ii) the ratio of dihydrodiol vs phenol formation may be recognized from the urinary phenanthrene metabolite profile. This pattern mirrors the enzymatic status (balance of the CYP isoforms and epoxide hydrolase) in individuals. Accordingly, more detailed information may be obtained from the urinary metabolite pattern than from 1- hydroxypyrene, commonly used in PAH biomonitoring.

Epizootics of cancer in fish associated with genotoxins in sediment and water.

Neoplasm epizootics in fish from a wide variety of freshwater, marine, and estuarine locations have been associated with genotoxins in sediment or water. The majority of cases have involved benthic or bottom feeding fish living in habitats with sediment contaminated by PAHs. The most common lesions involved in such epizootics include liver neoplasms, both biliary and hepatic, and skin neoplasms. Laboratory research has demonstrated the ability of fish to metabolize carcinogenic PAHs such as B(a)P into the ultimate carcinogen with the resulting formation of DNA adducts. Fish dosed with B(a)P or sediment extracts containing carcinogenic PAHs have developed skin and liver neoplasms. In the Black River, OH, neoplasm prevalence in wild brown bullhead has reflected PAH exposure as the latter has changed due to coke plant closures and remedial dredging activity. The weight of evidence supports a cause-and-effect relationship between exposure to genotoxins in sediment and water and neoplasm epizootics in wild fish populations.

Modulation of DNA adduct levels in human mononuclear white blood cells and granulocytes by CYP1A1, CYP2D6 and GSTM1 genetic polymorphisms

The CYP1A1, CYP2D6 and GSTM1 genes encode biotransforming enzymes involved in activation and detoxification of xenobiotics. Metabolically activated chemical compounds may interact with DNA and form adducts. In this study, the effect of the GSTM1, CYP1A1 exon 7 and CYP2D6 polymorphisms on DNA adduct levels was studied in 170 healthy volunteers. DNA adducts levels were measured by 32P-postlabelling in mononuclear white blood cells (WBC, lymphocytes and monocytes) and granulocytes collected in summer and winter. The influence of the genotype on the level of DNA adducts in both types of WBCs was observed only in summer samples. Individuals with GSTM1 deficient (null) genotype had significantly elevated level of adducts in mononuclear WBCs (p=0.045) and granulocytes (p=0.031) compared to GSTM1 positives. Higher adduct levels in carriers of combined GSTM1(null)/CYP1A1-Ile/Val genotype were found in both types of WBCs when compared to GSTM1(+)/CYP1A1-Ile/Ile genotype carriers (p=0.046 in granulocytes, p=0.092 in mononuclear WBCs). CYP2D6 wild-type homozygotes (EMs) and heterozygotes (HEMs) were shown to have significantly higher mononuclear WBC DNA adduct levels than mutant homozygotes (PMs) (p=0.037 and p=0.014). When confounding factors associated with PAH exposure were taken into account a statistically significant effect of CYP1A1 exon 7 polymorphism on DNA adduct levels was found (p=0.012 in mononuclear WBCs, p=0.043 in granulocytes). In a subgroup of current smokers (n=95) high DNA adduct levels in granulocytes were associated with GSTM1-(null) genotype, and increased adduct levels in mononuclear WBCs correlated with CYP2D6 EM and HEM genotypes. In winter samples the association between the genotype and DNA adduct levels was not observed.