Biomarker For PAH Exposure Research Articles

Facile fabrication of composited solid phase microextraction thin membranes for sensitive detections of trace hydroxylated polycyclic aromatic hydrocarbons in human urine

Solid phase microextraction (SPME) has potential to be used for the high-performance enrichments of hydroxylated polycyclic aromatic hydrocarbons (OH-PAHs), which are important biomarkers of PAH exposure. By choosing suitable adsorbent, it is conducive to fabricate new-type of SPME device for improved extraction efficiencies towards OH-PAHs. In this study, a novel method of surface solvent evaporation has been proposed to fabricate SPME thin membrane, integrating the advantages of polydimethylsiloxane (PDMS) and different porous adsorbents. The powdery metal organic framework (Uio66-NH2), porous polymer (powdery polymer aerogel, PPA) and ordered mesoporous carbon (OMC) have been chosen as typical adsorbents and fabricated as thin membranes successfully, indicating the universality of the proposed method for membrane fabrication. Comparing the extraction efficiencies of three prepared membranes towards OH-PAHs, the OMC-PDMS membrane has demonstrated best enrichment efficiencies. The OMC-PDMS membrane was used for the enrichments of trace OH-PAHs in human urine of both smokers and nonsmokers, combining with liquid chromatographic tandem mass spectrometry (LC-MS/MS). The detection limits were in the range of 0.15–0.39 ng L−1, and satisfactory recoveries were found to be 82.1%–115%. It can be seen that the universal strategy to fabricate SPME membrane is helpful to achieve broad-spectrum or selective enrichments of target analytes from complex matrix by simple modulation of membrane components.

Analysis of urinary metabolites of polycyclic aromatic hydrocarbons and cotinine in pooled urine samples to determine the exposure to PAHs in an Australian population.

Our previous biomonitoring study of hydroxylated polycyclic aromatic hydrocarbons (OH-PAHs) in a population in Australia found high levels of 1-naphthol, a metabolite of both naphthalene and carbaryl, in some adult samples. Here, we conducted a follow-up study to collect and analyse pooled urine samples, stratified by age and sex, from 2014 to 2017 using a GC-MS method. Geometric mean concentrations of 1-hydroxypyrene, the most common biomarker of PAH exposure, were 100 and 120 ng/L urine in 2014–2015 and 2016–2017, respectively. The concentrations of most OH-PAHs in this study except 1-naphthol are in line with those reported by biomonitoring programs in the US and Canada. In general, concentrations of OH-PAHs are lower in samples from small children (0–4 years) and school-aged children (5–14 years) compared with samples from the older age groups, except for some cases in the recent monitoring period. The concentrations of 1-naphthol in some adult samples of both sexes are very high, which is consistent with our previous findings. Such high concentrations of 1-naphthol together with the high 1-naphthol/2-naphthol ratio suggest potential exposure to the insecticide carbaryl in this population but other exposure sources and different rates of naphthalene metabolism should also be investigated.

Identification of new tetrahydroxylated metabolites of Polycyclic Aromatic Hydrocarbons in hair as biomarkers of exposure and signature of DNA adduct levels

The present study addresses the hypothesis that the concentration of tetrahydroxylated Polycylic Aromatic Hydrocarbons (tetra-OH-PAHs) in hair might be a useful biomarker of human exposure to PAHs, providing quantitative assessment of the internal dose, as well as information on the associated toxicity in relation to individual's specific metabolism. By means of animal models, this work aimed at identifying new tetra-OH-PAHs which can be released from the hydrolysis of DNA-adducts and can also be directly detected in biological matrices usually used in the field of biomonitoring such as hair and urine. Results obtained from a targeted gas chromatography coupled with tandem mass spectrometry (GC-MS/MS) approach, demonstrated the presence of 8 tetrahydroxylated metabolites in DNA and 23 in hairs of rats exposed to mixtures of PAHs, which had never been analyzed before. Ten tetra-OH-PAHs were clearly characterized by using their analytical standards, corresponding to 4 parent PAHs (phenanthrene, chrysene, benz[a]anthracene and benzo[a]pyrene) whereas 13 tetra-OH-PAHs from 3 other parents (anthracene, fluoranthene and benz[k]fluoranthene) were detected but not yet characterized. No tetrahydroxylated metabolite has been clearly identified for naphthalene, fluorene, benzo[b]fluoranthene, benzo[g,h,i]perylene, or dibenzo[a,h]anthracene, which can all potentially form adducts.The relevance of tetra-OH-PAH analysis in hair as biomarkers of PAH exposure was evaluated in a dose-response study conducted on 64 rats (Long Evans females/n = 8 per groups) under repeated exposure (3 times per week) to a mixture of 16 PAHs at low doses (0.01–0.8 mg/kg) for 90 days. Most of the tetra-OH-PAHs targeted in the method were detected in the hairs of the rats, regardless of the dose of exposure. Significant linear relationships (R2 ranging from 0.558 to 0.964, p < 0.001) were observed between the administered dose and the tetra-OH-PAH concentrations in the hairs for 20 out of the 23 metabolites. By widening the range of PAH metabolites used as biomarkers of exposure so as to include the analysis of PAH tetrahydroxylated forms (especially those exhibiting more than 5 aromatic rings), the present methodology will enable multi-exposure assessments which are more accurately representative of actual situations of exposure to PAHs.

Tracing biomarker of PAH-exposure and susceptibility factor (GSTM-polymorphism) among cancer patients in Pakistan

We studied cancer patients for possible PAH exposure, using urinary concentration of 1-hydroxypyrene (1-OHP) as a biomarker of internal dose of polycyclic aromatic hydrocarbons (PAHs). The subjects included in this study belonged to various socio-demographic backgrounds, and were diagnosed with cancer (i.e. lung, head and neck or digestive tract cancer). In general, we observed high concentration of urinary 1-OHP among digestive tract cancer patients, compared with the controls (CN) (mean 1.06, median 1.03 and mean 0.62, median 0.63 μmol/mol-Cr in digestive tract cancer patients and controls respectively). The concentrations of urinary 1-OHP were higher than the background level of PAHs; therefore, these groups could have been exposed to PAHs. Highest urinary 1-OHP concentration was observed in digestive tract cancer patients (median 1.25 μmol/mol-Cr) with GSTM-1 genotype. The results of PCA were consistent with qualitative and quantitative data analysis. The contribution of urinary 1-OHP eigenvector revealed a relatively high PAH-exposure among cancer patients compared with CN, while diet and age were influential parameters among cancer patients, which could have a strong link in cancer etiology in the selected exposure groups.

Prenatal airborne polycyclic aromatic hydrocarbon exposure, LINE1 methylation and child development in a Chinese cohort

BackgroundPolycyclic aromatic hydrocarbons (PAH) are carcinogenic, neurotoxic environmental pollutants generated during incomplete combustion of fossil fuel and other organic material. PAH exposure has been associated with adverse fetal development and epigenetic alterations in cord blood. Several molecular epidemiology studies have established PAH-DNA adducts as biomarkers of PAH exposure. ObjectivesWe investigated the relationship between LINE1 DNA methylation and PAH-DNA adduct levels in cord blood, and with neurodevelopmental outcomes. MethodsIn Tongliang County, China, the current study enrolled two population-based cohorts of nonsmoking pregnant women before (2002) and after (2005) the closure of a local coal-fired power plant in May 2004. We analyzed cord blood samples collected from mothers in the two cohorts (n=110 from 2002 cohort and n=107 from 2005 cohort) for PAH-DNA adducts and genomic LINE1 DNA methylation. Neurodevelopmental data on children were collected using the Gesell Developmental Scales (GDS) at age 2 and using the Wechsler Intelligence Scale for Children (WISC) at age 5. ResultsA significant inverse relationship was observed between PAH-DNA adducts and LINE1 DNA methylation (β=−0.010, p<0.038). A significant, positive association between LINE1 methylation and scores on WISC full scale and verbal (β=85.31, p<0.005; β=94.36, p<0.003) but not on the GDS. Mediation analysis did not find LINE1 to be a direct mediator between PAH-DNA adducts and IQ score. ConclusionLINE1 methylation in cord blood DNA was a positive predictor of IQ at age 5 and was decreased at higher levels of prenatal PAH exposure measured by PAH-DNA adducts in cord blood. However, the adverse effects of prenatal exposure to PAH on IQ scores did not appear to be directly mediated by altered LINE1 methylation.

First evidence of high urinary 1-hydroxypyrene level among rural school children during smoke haze episode in Chiang Mai Province, Thailand

Smoke haze from biomass burning has been a crisis in northern Thailand. There have been plenty of reports on ambient PM10 and PAHs but not on individual exposure during smoke haze episodes. The present study aimed to quantify urinary 1-OHP, a predominant biomarker of PAH exposure, among 200 rural school children in Om Koi and Mae Chaem districts, Chiang Mai Province, northern Thailand. Questionnaires for information regarding environmental exposure and health diaries were given out. Urine samples were collected to measure 1-OHP using high-performance liquid chromatography-fluorescence detection, HPLC-FLD (HP1100 Series/HP1046A). Proficiency testing materials were obtained from the participation of German External Quality Assessment Scheme (G-EQUAS, Erlangen, Germany) and analyzed as part of the method validation of our urinary 1-OHP method. The median urinary 1-OHP level in the school children from Om Koi District was significantly higher than that in Mae Chaem District (0.70 versus 0.24 μmol/mol cr). The present study showed the evidence that urinary 1-OHP level among school children from the selected study sites during haze episode in northern Thailand is higher than those reported in other region of Thailand and other countries.

Quantitative determination of hydroxy polycylic aromatic hydrocarbons as a biomarker of exposure to carcinogenic polycyclic aromatic hydrocarbons

A high-resolution gas chromatography/high-resolution mass spectrometry (HRGC/HRMS) method was developed for quantitative analysis of hydroxy polycyclic aromatic hydrocarbons (OH-PAHs). Four hydroxy metabolites of known and suspected carcinogenic PAHs (benzo[a]pyrene (B[a]P), benz[a]anthracene (B[a]A), and chrysene (CRY)) were selected as suitable biomarkers of PAH exposure and associated risks to human health. The analytical method included enzymatic deconjugation, liquid − liquid extraction, followed by derivatization with methyl-N-(trimethylsilyl) trifluoroacetamide and instrumental analysis. Photo-induced oxidation of target analytes − which has plagued previously published methods − was controlled by a combination of minimizing exposure to light, employing an antioxidant (2-mercaptoethanol) and utilizing a nitrogen atmosphere. Stability investigations also indicated that conjugated forms of the analytes are more stable than the non-conjugated forms. Accuracy and precision of the method were 77.4–101% (<4.9% RSD) in synthetic urine and 92.3–117% (<15% RSD) in human urine, respectively. Method detection limits, determined using eight replicates of low-level spiked human urine, ranged from 13 to 24pg/mL. The method was successfully applied for analysis of a pooled human urine sample and 78 mouse urine samples collected from mice fed with PAH-contaminated diets. In mouse urine, greater than 94% of each analyte was present in its conjugated form.

Gender differences in detoxification metabolism of polycyclic aromatic hydrocarbon (chrysene) in scallop Chlamys farreri during the reproduction period

This study aims to investigate the effects of chrysene (CHR) on biotransformation and detoxification responses of mature scallop Chlamys farreri during the reproduction period. Scallops were exposed to 0.2, 0.8 and 3.2μg/L CHR for 21days; at day 10 scallops were induced to spawn. At days 1, 3, 6, 10, 11, 15 and 21, enzymatic activities of 7-ethoxyresorufin O-deethylase (EROD) and glutathione-s-transferase (GST), related mRNA expression levels of CYP1A1, GST-pi and P-glycoprotein (P-gp) in digestive glands and CHR bioaccumulation in tissues were examined by separately analyzing male and female scallops. During the pre-spawn period, CHR concentrations of the treated groups in tissues except the hemolymph increased rapidly. Levels of enzymatic activities and related gene expressions were all induced by the exposure to CHR for females and males. GST activity and GST-pi mRNA expression showed a good time- and dose-dependent relationship only in males, and P-gp mRNA expression exhibited a dose-dependent manner in both sexes. During the post-spawn period, spawning caused significant reductions of bioaccumulation in tissues but the gill and hemolymph. Enzymatic activities and related gene expressions were for females significantly depressed at day 21 at 0.8 or 3.2μg/L CHR. Overall, females accumulated more CHR than males, while males were more sensitive than females to CHR exposure in gene expressions and enzyme activities. P-gp mRNA expression seemed to be a potential biomarker for PAH exposure. These results will offer the information on CHR biotransformation in this species, and ensure the influence of gender and reproductive status on PAH detoxification metabolism.

Molecular and Neurodevelopmental Benefits to Children of Closure of a Coal Burning Power Plant in China

Polycyclic aromatic hydrocarbons (PAH) are major toxic air pollutants released during incomplete combustion of coal. PAH emissions are especially problematic in China because of their reliance on coal-powered energy. The prenatal period is a window of susceptibility to neurotoxicants. To determine the health benefits of reducing air pollution related to coal-burning, we compared molecular biomarkers of exposure and preclinical effects in umbilical cord blood to neurodevelopmental outcomes from two successive birth cohorts enrolled before and after a highly polluting, coal-fired power plant in Tongliang County, China had ceased operation. Women and their newborns in the two successive cohorts were enrolled at the time of delivery. We measured PAH-DNA adducts, a biomarker of PAH-exposure and DNA damage, and brain-derived neurotrophic factor (BDNF), a protein involved in neuronal growth, in umbilical cord blood. At age two, children were tested using the Gesell Developmental Schedules (GDS). The two cohorts were compared with respect to levels of both biomarkers in cord blood as well as developmental quotient (DQ) scores across 5 domains. Lower levels of PAH-DNA adducts, higher concentrations of the mature BDNF protein (mBDNF) and higher DQ scores were seen in the 2005 cohort enrolled after closure of the power plant. In the two cohorts combined, PAH-DNA adducts were inversely associated with mBDNF as well as scores for motor (p = 0.05), adaptive (p = 0.022), and average (p = 0.014) DQ. BDNF levels were positively associated with motor (p = 0.018), social (p = 0.001), and average (p = 0.017) DQ scores. The findings indicate that the closure of a coal-burning plant resulted in the reduction of PAH-DNA adducts in newborns and increased mBDNF levels that in turn, were positively associated with neurocognitive development. They provide further evidence of the direct benefits to children's health as a result of the coal plant shut down, supporting clean energy and environmental policies in China and elsewhere.

CDNA fingerprint from the hepatopancreatic glands of pond snails (Lymnaea stagnalis) exposed to benzo[a]pyrene

Identification of differentially expressed genes that could be potentially used as biomarkers of PAH exposure of common invertebrate animal (like freshwater snail) would be a valuable resource for investigators interested in toxicology and biomonitoring of aquatic environments. Therefore, the aim of this research was to investigate effects of waterborne benzo[ a]pyrene (B[ a]P) exposure on mRNA expression in the pond snail’s (Lymnaea stagnalis ) hepatopancreatic gland. Toward this end, mature individuals of pond snail ( L. stagnalis ) were treated with 50µM B[ a]P solution in a short 36h static exposure test. Differential Display PCR (DD-PCR) was used to generate a unique cDNA fingerprint of genes that were differentially expressed in the tissues of exposed and unexposed snails. To assess the putative identity of the isolated cDNA amplicons (ESTs), BLAST queries were performed to find similarities in their nucleotide sequence. Real-Time qPCR analysis was used to verify the DDPCR expression profile. Finally, an additional independent exposure study, including higher dose of B[ a]P (100µM), was conducted to validate the expression of selected ESTs. BLAST

Repeated measurements for assessment of urinary 2-naphthol levels in individuals exposed to polycyclic aromatic hydrocarbons

A repeated measurement cohort study was conducted to determine whether 2-napthol can be a suitable biomarker for evaluating the magnitude of exposure to PAHs from coke oven emissions. Time-course patterns of urinary 2-naphthol levels in coke-oven workers were examined. Also, the correlation between urinary 2-naphthol levels and PAHs from personal breathing zone samples was analyzed while examining and adjusting possible confounding factors, such as smoking alcohol consumption, and age of human subjects. A total of 8 spot urine samples were collected from each high-exposure group (topside-oven workers, n = 17) and low-exposure group (side-oven workers, n = 25) during the whole working cycle, which consists of six consecutive working days followed by 2 days off. Personal breathing zone samples were collected to quantify PAH intake. A questionnaire was distributed and collected from each worker for assessment of demographic parameters. Our results confirmed that the topside-oven area contained significantly higher PAH levels than the side-oven area. Urinary 2-naphthol levels correlated with the levels of PAH species, including pyrene, benzo[k]fluoranthene, benezo[g,h,i]pyrene, naphthalene, phenanthrene, anthracene, indeno[1,2,3-cd]pyrene, benzo[k]fluoranthene, and total PAHs. During the working cycle, urinary 2-naphthol levels remained stable at around 46–97 ng/mg creatinine during the working days and dramatically increased during the off days. After stratification of data based on smoking status, smokers had significantly higher urinary 2-naphthol levels than non-smokers, and 2-naphthol levels positively correlated with smoking status. Coke-oven emissions are a source of exposure to naphthalene. Also, smoking is a significant source of exposure to naphthalene and served as a confounder factor. Due to its abundance, positive association with total PAHs and certain carcinogenic PAH compounds, and slower elimination kinetics, urinary 2-naphthol may have the potential to serve as a biomarker for PAH exposure, when smoking status is carefully adjusted.

Biomarkers of oxidative stress and its association with the urinary reducing capacity in bus maintenance workers

BackgroundExposure to particles (PM) induces adverse health effects (cancer, cardiovascular and pulmonary diseases). A key-role in these adverse effects seems to be played by oxidative stress, which is an excess of reactive oxygen species relative to the amount of reducing species (including antioxidants), the first line of defense against reactive oxygen species. The aim of this study was to document the oxidative stress caused by exposure to respirable particles in vivo, and to test whether exposed workers presented changes in their urinary levels for reducing species.MethodsBus depot workers (n = 32) exposed to particles and pollutants (respirable PM4, organic and elemental carbon, particulate metal content, polycyclic aromatic hydrocarbons, NOx, O3) were surveyed over two consecutive days. We collected urine samples before and after each shift, and quantified an oxidative stress biomarker (8-hydroxy-2'-deoxyguanosine), the reducing capacity and a biomarker of PAH exposure (1-hydroxypyrene). We used a linear mixed model to test for associations between the oxidative stress status of the workers and their particle exposure as well as with their urinary level of reducing species.ResultsWorkers were exposed to low levels of respirable PM4 (range 25-71 μg/m3). However, urinary levels of 8-hydroxy-2'-deoxyguanosine increased significantly within each shift and between both days for non-smokers. The between-day increase was significantly correlated (p < 0.001) with the concentrations of organic carbon, NOx, and the particulate copper content. The within-shift increase in 8OHdG was highly correlated to an increase of the urinary reducing capacity (Spearman ρ = 0.59, p < 0.0001).ConclusionsThese findings confirm that exposure to components associated to respirable particulate matter causes a systemic oxidative stress, as measured with the urinary 8OHdG. The strong association observed between urinary 8OHdG with the reducing capacity is suggestive of protective or other mechanisms, including circadian effects. Additional investigations should be performed to understand these observations.

Fluorescence Spectroelectrochemical Sensor for 1-Hydroxypyrene, a Biomarker for PAH Exposure

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Concentration and profile of 22 urinary polycyclic aromatic hydrocarbon metabolites in the US population

Urinary monohydroxy polycyclic aromatic hydrocarbons (OH-PAHs) are a class of PAH metabolites used as biomarkers for assessing human exposure to PAHs. The Centers for Disease Control and Prevention's National Health and Nutrition Examination Survey (NHANES) uses OH-PAHs to establish reference range concentrations for the US population, and to set benchmarks for future epidemiologic and biomonitoring studies. For the years 2001 and 2002, 22 OH-PAH metabolites were measured in urine specimens from 2748 NHANES participants. Percentages of samples with detectable levels ranged from nearly 100% for metabolites of naphthalene, fluorene, phenanthrene, and pyrene, to less than 5% for metabolites from parent compounds with higher molecular weight such as chrysene, benzo[ c]phenanthrene, and benz[ a]anthracene. The geometric mean for 1-hydroxypyrene (1-PYR)—the most commonly used biomarker for PAH exposure—was 49.6 ng/L urine, or 46.4 ng/g creatinine. Children (ages 6–11) generally had higher levels than did adolescents (ages 12–19) or adults (ages 20 and older). Model-adjusted, least-square geometric means for 1-PYR were 87, 53 and 43 ng/L for children, adolescents (ages 12–19) and adults (ages 20 years and older), respectively. Log-transformed concentrations for major detectable OH-PAHs were significantly correlated with each other. The correlation coefficients between 1-PYR and other metabolites ranging from 0.17 to 0.63 support the use of 1-PYR as a useful surrogate representing PAH exposure.

PAH-DNA adducts in a Chinese population: relationship to PAH exposure, smoking and polymorphisms of metabolic and DNA repair genes

The present study was conducted in a Chinese population to evaluate the usefulness and sensitivity of PAH-DNA adduct as a biomarker of PAH exposure, and to examine the potential effects of smoking and polymorphisms of responsive genes on DNA adduct formation induced by PAH exposure. The polymorphisms of genes examined include GSTM1, GSTT1, CYP1A1, microsomal epoxide hydrolase (mEH) and excision repair cross-complementary group 2 (ERCC2). A total of 194 subjects with a broad range of PAH exposures were recruited, including 116 occupationally exposed workers, 49 metropolitan residents and 29 suburban gardeners. A significant exposure–response relationship was observed between PAH exposure and DNA adducts in leukocytes across the entire group of subjects (p<0.0001). The levels of PAH-DNA adducts in the subgroup with lowest occupational exposure to PAHs (<0.1µg BaP m−3) was significantly higher than that in metropolitan residents and suburban gardeners. However, no significant difference was detected between residents and gardeners, with mean BaP concentrations of 0.028 and 0.011µg m−3, respectively. The polymorphisms of genes examined failed to show significant effects on PAH-induced adduct formation except ERCC2 Lys751Gln genotypes. A significantly higher level of PAH-DNA adduct was found in subjects with wild-type ERCC2 than those who have either heterozygous or homozygous variant alleles (p<0.01). Smoking, age and gender did not substantially contribute to PAH-induced DNA adduct formation in this study. The study suggests that PAH-DNA adducts may serve as a reliable biomarker of PAH exposure in occupational settings but may not be sensitive enough to be used in populations with environmental exposures to PAHs.

Design issues in cross-sectional biomarkers studies: Urinary biomarkers of PAH exposure and oxidative stress

Cross-sectional biomarker studies can provide a snapshot of the frequency and characteristics of exposure/disease in a population at a particular point in time and, as a result, valuable insights for delineating the multi-step association between exposure and disease occurrence. Three major issues should be considered when designing biomarker studies: selection of appropriate biomarkers, the assay (laboratory validity), and the population validity of the selected biomarkers. Factors related to biomarker selection include biological relevance, specificity, sensitivity, biological half-life, stability, and so on. The assay attributes include limit of detection, reproducibility/reliability, inter-laboratory variation, specificity, time, and cost. Factors related to the population validity include the frequency or prevalence of markers, greater inter-individual variation than intra-individual variation, intra-class correlation coefficients (ICC), association with potential confounders, invasiveness of specimen collection, and subject selection. Three studies are selected to demonstrate different features of cross-sectional biomarker studies: (1) characterizing the determinants of the biomarkers (study I: urinary PAH metabolites and environmental particulate exposure), (2) relationship of multiple biomarkers of exposure and effect (study II: relationship between urinary PAH metabolites and oxidative stress), and (3) evaluating gene–environmental interaction (study III: effect of genetic polymorphisms of GSTM1 on the association of green tea consumption and urinary 1-OHPG levels in shipbuilding workers).

EROD activity and biliary fluorescence in Schroederichthys chilensis (Guichenot 1848): Biomarkers of PAH exposure in coastal environments of the South Pacific Ocean

Schroederichthys chilensis is a common shark that lives in Chilean coastal environments. In this work, the relationship between liver 7-ethoxyresorufin- O-deethylase dealkylation (EROD) activity and Fluorescent Aromatic Compounds (FAC) in bile of S. chilensis sampled in three bays with different degrees of pollution were performed including a reference area. Sixty individuals were collected, 20 for each site; (10 males and 10 females per site) livers and bile samples were obtained and immediately frozen. EROD activity and FAC were measured according to three standard methods. EROD activity and FAC were higher in polluted areas than in the reference area. Synchronous Fluorescence Spectra of the bile from the fish collected at the most polluted area showed a peak at 347 nm representing a metabolite corresponding to 1-hydroxypyrene. The low EROD activity in the reference area is likely related to the low level of PAH in sediments. We propose that this species is a good indicator of exposure to FACs, since it presents a series of characteristics that make it suitable for monitoring PAH exposure in coastal zones.

Metabolism of 1-fluoropyrene and pyrene in marine flatfish and terrestrial isopods

Monofluorinated polycyclic aromatic hydrocarbons (F-PAHs) are useful reference compounds for a broad spectrum of PAH studies. The pyrene metabolite 1-hydroxypyrene is often used as a biomarker of PAH exposure. Two species, isopod ( Porcellio scaber) and flatfish ( Platichthys flesus), that produce 1-hydroxypyrene as the major intermediary metabolite and have distinct phase-II conjugates, were selected to investigate the cytochrome P450 catalyzed metabolism of 1-fluoropyrene. The fluorine atom blocks one of the four most favored carbon atoms, yielding different metabolite patterns and the results obtained in the selected species were compared with metabolite profiles obtained for unsubstituted pyrene. Charge densities in 1-fluoropyrene measured with 13C-NMR were used to predict possible positions of hydroxylation, which were confirmed by 19F-NMR. Additionally, the retention behaviour of conjugated 1-fluoropyrene metabolite isomers in reversed-phase liquid chromatography on a polymeric alkyl-bonded phase was interpreted based on the slot model. Whereas three phase-I metabolites were found in isopod hepatopancreas, only two were observed in flatfish bile. Phase-II metabolism appeared unaffected by the fluorine substituent. It was concluded that the phase-I enzyme cytochrome P450 is non-regioselective in the isopod: the activation is mostly influenced by the electron density distribution. In contrast, the enzymatic oxidation in the flounder is more selective. These differences will affect to what extent pyrene metabolite measurements can be used to assess the impact of PAHs to different species.

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.

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.