Bioavailability of PCBs to Male Laboratory Rats Maintained on Litters of Contaminated Soils: PCB Burden and Induction of Alkoxyresorufin O-Dealkylase Activities in Liver and Lung

Male rats from the Sprague-Dawley laboratory strain were maintained in the laboratory during 3 days and 1 night on litters containing a reference soil and different amounts of a soil, mainly polluted by PCBs (207 ppm expressed in Aroclor? 1254; SIII soil). Two categories of biomarkers of exposure were measured in both liver and lung of these rats: PCB burdens and activities of microsomal liver and lung cytochrome P450–dependent mono-oxygenases, namely ethoxy-, pentoxy-, and benzoxy-resorufin O-dealkylase activities (EROD, PROD, and BROD, respectively). PCB burdens in liver and lung of rats exposed to SIII soil were 1,845 and 241 ppb, respectively (expressed in Aroclor? 1254 equivalents). EROD, PROD, and BROD were significantly induced in the liver of rats exposed to SIII soil, while only EROD activity was induced in the lung. Induction of hepatic EROD activity was ∼3- to 5.4-fold; pulmonary EROD activity was induced by 9- to 12-fold. In the lung, PROD and BROD activities were inhibited. When rats were exposed to SIII soil diluted with various amounts of standard ISO soil, a nearly linear dose-response relationship was found between the level of PCBs in the litter and EROD activity in both liver and lung. A nonlinear dose-response relationship exists with hepatic BROD activity; no dose-response relationship was observed with hepatic PROD and pulmonary PROD and BROD activities. EROD activity measurement in both liver and lung of rats maintained on a litter of PCB polluted soil was used to assess the bioavailability to mammals of PCBs. Received: 16 December 1996/Accepted: 18 November 1997     

Liver microsomal levels of cytochrome P450IA1 as biomarker for exposure and bioavailability of soil-bound polycyclic aromatic hydrocarbons

The bioavailability of soil-bound polycyclic aromatic hydrocarbons (PAHs) for mammalian species was studied with rats fed with a diet containing contaminated soil preparations. The extent of cytochrome P450IA1 (CYP1A1) induction in the liver correlated with the amount of 5- and 6-ring PAHs in the soil samples but not with the total PAH content. Other cytochromes P450 were much less affected by the soil-contaminants. The highest induction of CYP1A1 was obtained with a sample containing 274 mg 5- and 6-ring PAH/kg soil, resulting in a nearly 360-fold increase in the ethoxyresorufin deethylase (EROD) activity. In a semilogarithmic plot, a linear correlation was found between the 5- and 6-ring PAH concentration in the soil and the microsomal CYP1A1 content. As a model for the action of intestinal fluids, soil samples were extracted by bile acid solution. In these experiments, the selectivity in the solubilization of individual PAHs parallels that of toluene extraction, although the yield is lower than the latter and varies with the soil sample. The bioavailability of PAHs for microorganisms, but not for mammals, was shown to be considerably reduced in the presence of high total organic carbon (TOC) values of the soil samples. This may have implications for decontamination strategies, diminishing the effectiveness of biological decontamination in cases with high TOC values. The data suggest that CYP1A1 induction in rats is a parameter that may be useful in risk assessments of contaminated soils for mammalian species.Part of this work was presented at the International Congress on Cytochrome P450: Biochemistry, Biophysics and Molecular Biology, Lisbon, 1993 (Roos et al. 1994a) and as a lecture on ECOINFORMA, Wien, 1994 (Roos et al. 1994b, 1994c)     

Lack of a distinct gradient in biomarker responses in small mammals collected at different distances from a highway

This study describes biomarker effects in small mammals exposed to traffic emissions. Animals were collected at 10–50 m (site 1), 150–200 m (site 2), and 5 km (site 3) from a very busy highway (A2). To distinguish between routes of exposure, strictly carnivorous common shrews (Sorex araneus) and predominantly herbivorous bank voles (Clethrionomys glareolus) were collected. As a measure of exposure to polycyclic aromatic hydrocarbons (PAHs), aromatic DNA adduct levels were determined by ³²P-postlabeling techniques in tissue from heart, lung, and liver. Lead (Pb), cadmium (Cd), and copper (Cu) levels were analyzed in kidney as a measure of exposure to heavy metals. EROD and PROD activity and retinoid levels were determined in liver as effect biomarkers for exposure to PAHs and polyhalogenated aromatic hydrocarbons (PHAHs). Relatively high Cd levels in S. araneus and in particular elevated DNA adduct levels in C. glareolus indicated that small mammals at site 3 were exposed to more compounds than at sites 1 and 2 (3 ≥ 1 > 2). The latter effect is probably due to an incidental and actual input of airborne pollutants that is deposited on plant surfaces. By consumption of above-ground vegetation, voles are chronically exposed to this pollution. Relatively high background input of PAHs probably hinders that the traffic-related gradient of airborne PAH concentrations found in an earlier study is reflected in DNA adduct levels in small mammals in the present study. Moreover, historical biomarkers for exposure to traffic emissions, such as increased kidney Pb levels, increased hepatic EROD activity, and disturbed hepatic vitamin A homeostasis are no longer applicable to indicate differences in exposure. This is a result of the ban on addition of Pb and chlorinated scavengers to gasoline and of cleaner combustion techniques, which were enforced by law over the past decade. Finally, it is advisable to use only juvenile small mammals for in situ monitoring of diffuse pollution because DNA adduct levels increased with age. Received: 5 November 2001/Accepted: 11 March 2002     

Activities of liver and lung cytochrome P450-dependent monooxygenases and antioxidant enzymes in laboratory and wild Norway rats exposed to reference and contaminated soils

Laboratory and wild Norway rats were exposed in the laboratory to an uncontaminated soil and to a soil from a site contaminated with petrochemical waste. Activities of microsomal lung and liver cytochrome P450-dependent monooxygenases, including 7-ethoxyresorufin O-deethylase (EROD), 7-pentoxyresorufin O-depentylase (PROD) and 7-benzoxyresorufin O-debenzylase (BROD) were measured at selected times during the course of the study. The highest degree of induction of hepatic EROD (7-fold) was shown after 3 days of exposure to the contaminated soil. However, two months later, the EROD activity declined to fourfold increase over the control. The PROD and BROD activities displayed a similar time course of induction, but the degree of induction was lower. The induction of hepatic monooxygenase activities was observed in both laboratory and wild rats. Lung monooxygenase EROD was highly induced (up to 28-fold) after 3 days of exposure, and the activity remained elevated throughout the two-month experiment. BROD and PROD activities were not induced. The activities of three antioxidant enzymes, namely superoxide dismutase, glutathione peroxidase (Se- and non-Se-dependent) and catalase also were measured in lung and liver cytosol, but no significant changes were observed after two months of exposure to contaminated soil.     

Cell-specific CYP1A expression and benzo[a]pyrene adduct formation in gills of rainbow trout (Oncorhynchus mykiss) following CYP1A induction in the laboratory and in the field

The effect of cytochrome P4501A (CYP1A) induction on cell-specific benzo[a]pyrene (BaP) adduct formation was studied in rainbow trout (Oncorhynchus mykiss) gills. Fish preexposed to beta-naphthoflavone (betaNF) or caged in a polluted river were exposed to waterborne 3H-benzo[a]pyrene (3H-BaP). The 3H-benzo[a]pyrene adducts in the gill filaments were localized by autoradiography and CYP1A protein by immunohistochemistry. Ethoxyresorufin O-deethylase (EROD) activity was measured using a gill filament-based ex vivo assay. Branchial 3H-BaP binding and EROD activity were enhanced by exposure to betaNF or to the river water, and completely blocked by the CYP1A inhibitor ellipticine. The predominant sites of adduct formation were in epithelium of the secondary lamellae and in epithelium of the efferent edge of the gill filament. In betaNF-exposed fish, the strongest CYP1A immunoreactivity was observed in differentiating cells and in pillar cells. In fish caged in the polluted river, strong CYP1A immunoreactivity was found in most cells in the secondary lamellae, whereas the primary lamellae were almost devoid of immunoreactivity. Our results reveal a discrepancy between the localization of CYP1A protein and BaP adducts in the gill. Consequently, other factors, such as bioavailability of waterborne polycyclic aromatic hydrocarbons (PAHs) to the target cells, are important for the localization of PAH adducts in the gill.     

Polycyclic aromatic hydrocarbon exposure in Steller's eiders (Polysticta stelleri) and Harlequin ducks (Histronicus histronicus) in the eastern Aleutian Islands, Alaska, USA

Seaducks may be affected by harmful levels of polycyclic aromatic hydrocarbons (PAHs) at seaports near the Arctic. As an indicator of exposure to PAHs, we measured hepatic enzyme 7-ethoxyresorufin-O-deethylase activity (EROD) to determine cytochrome P4501A induction in Steller's eiders (Polysticta stelleri) and Harlequin ducks (Histronicus histronicus) from Unalaska, Popof, and Unga Islands (AK, USA) in 2002 and 2003. We measured PAHs and organic contaminants in seaduck prey samples and polychlorinated biphenyl congeners in seaduck blood plasma to determine any relationship to EROD. Using Akaike's information criterion, species and site differences best explained EROD patterns: Activity was higher in Harlequin ducks than in Steller's eiders and higher at industrial than at nonindustrial sites. Site-specific concentrations of PAHs in blue mussels ([Mytilus trossilus] seaduck prey; PAH concentrations higher at Dutch Harbor, Unalaska, than at other sites) also was important in defining EROD patterns. Organochlorine compounds rarely were detected in prey samples. No relationship was found between polychlorinated biphenyl congeners in avian blood and EROD, which further supported inferences derived from Akaike's information criterion. Congeners were highest in seaducks from a nonindustrial or reference site, contrary to PAH patterns. To assist in interpreting the field study, 15 captive Steller's eiders were dosed with a PAH known to induce cytochrome P4501A. Dosed, captive Steller's eiders had definitive induction, but results indicated that wild Steller's eiders were exposed to PAHs or other inducing compounds at levels greater than those used in laboratory studies. Concentrations of PAHs in blue mussels at or near Dutch Harbor (approximately 1,180-5,980 ng/g) approached those found at highly contaminated sites (approximately 4,100-7,500 ng/g).     

Effect of simulated rhizodeposition on the relative abundance of polynuclear aromatic hydrocarbon catabolic genes in a contaminated soil

Microcosms were used to investigate whether soil exposure to mulberry root extracts (rich in phenolic compounds) select for bacteria that degrade polynuclear aromatic hydrocarbons (PAHs). Unlike previous studies with freshly spiked soil, the present experiments were conducted with soils aged for 518 d with [14C]phenanthrene to decrease bioavailability and avoid exaggerating the selective pressure exerted by PAHs relative to the rhizosphere effect. Microcosms simulating contaminated planted soil were exposed to carbon at 20 mg/L/week of mulberry root extract for 211 d to simulate rhizodeposition. Contaminated bulk soils microcosms were amended with a C-free mineral medium to discern the effect of rhizodeposition. Uncontaminated soil controls also were exposed to similar dose regimes. Real-time quantitative polymerase chain reaction was used to enumerate total bacteria and PAH degraders harboring the genes nahAc (coding for naphthalene dioxygenase), todC1 (coding for toluene/benzene/chlorobenzene dioxygenase), bmoA (coding for hydroxylating monooxygenases), and dmpN (coding for phenol hydroxylase). Exposure to root extracts enhanced the growth of total bacteria and PAH degraders in both contaminated and uncontaminated rhizosphere microcosms. The relative abundance of PAH-degrader gene copies (as a fraction of the total bacteria) was similar for different treatments, suggesting that the root extracts did not select for PAH degraders. Overall, these results suggest that rhizodeposition from phenolic releasers contributes to the fortuitous (but not selective) proliferation of PAH degraders, which may enhance phytoremediation.     

Effects of the polycyclic aromatic hydrocarbon heterocycles, carbazole and dibenzothiophene, on in vivo and in vitro CYP1A activity and polycyclic aromatic hydrocarbon-derived embryonic deformities

Heterocyclic derivatives of polycyclic aromatic hydrocarbons (PAHs) are often significant components of environmental contaminant mixtures; however, their contribution to the toxicity of these mixtures is not well characterized. These heterocycles commonly co-occur in PAH mixtures, which contain agonists for the aryl hydrocarbon receptor (AHR). Our goal for these studies was to explore the effects of two PAH heterocycles, carbazole (CB) and dibenzothiophene (DBT), alone and in combination with a PAH-type agonist for the AHR (beta-naphthoflavone [BNF]) on AHR-mediated cytochrome P4501A (CYP1A) activity and on fish embryotoxicity. Embryos of Fundulus heteroclitus were exposed to CB or DBT, with and without coexposure to BNE Carbazole alone slightly induced, whereas DBT alone slightly reduced, in ovo CYP1A-mediated ethoxyresorufin-O-deethylase (EROD) activity compared to control values. However, exposure to CB or DBT reduced in ovo EROD activity in embryos coexposed to BNE Carbazole and DBT were characterized in vitro as noncompetitive CYP1A inhibitors. Carbazole and DBT enhanced the embryotoxicity of BNF, although neither compound was embryotoxic by itself. The co-occurrence of CB and DBT with PAH-type AHR inducers in contaminated ecosystems may increase the toxicity of PAH-type AHR agonists in these settings and may need to be considered when estimating the embryotoxicity of PAH mixtures.     

Assessment of the bioactivity of creosote-contaminated sediment by liver biotransformation system of rainbow trout

A sediment site in the Lake J?ms?nvesi (municipality of Pet?j?vesi, Finland) contaminated by creosote was investigated to assess the possible ecotoxicological risks it may cause to benthic animals, including ones which may arise due to physical measures in remediating the site. It is suggested that polycyclic aromatic hydrocarbons (PAHs) are bioavailable to fish and other aquatic animals during exposure to contaminated water, sediment, and food. In order to assess toxicological risks of sediment contents to fish, juvenile rainbow trout (Onchorhynchus mykiss) were intraperitoneally dosed with extracts of the creosote-contaminated sediments and their elutriates. This was compared to pristine lake sediment spiked with creosote. Activity of CYP1A1 in trout liver was measured as ethoxyresorufin O-deethylase (EROD). When compared to vehicle controls and the pristine reference sediment (0.9-1.3 pmol/min/mg PMS protein), the extract of creosote-contaminated sediment of Lake J?ms?nvesi induced EROD activity up 20-30 times with a dose of 100 mg/kg [total PAHs (mg)/(kg) in fish]. The rise of EROD was associated with increasing levels of PAH metabolites in bile, analyzed as 1-OH pyrene equivalents.     

Apoptosis and Bax expression are increased by coal dust in the polycyclic aromatic hydrocarbon-exposed lung

BACKGROUND: Miners inhaling respirable coal dust (CD) frequently develop coal workers' pneumoconiosis, a dust-associated pneumoconiosis characterized by lung inflammation and variable fibrosis. Many coal miners are also exposed to polycyclic aromatic hydrocarbon (PAH) components of diesel engine exhaust and cigarette smoke, which may contribute to lung disease in these workers. Recently, apoptosis was reported to play a critical role in the development of another pneumoconiosis of miners, silicosis. In addition, CD was reported to suppress cytochrome P450 1A1 (CYP1A1) induction by PAHs. METHODS: We investigated the hypothesis that apoptosis plays a critical role in lung injury and down-regulation of CYP1A1 induction in mixed exposures to CD and PAHs. We exposed rats intratracheally to 0.0, 2.5, 10.0, 20.0, or 40.0 mg/rat CD and, 11 days later, to intraperitoneal beta-naphthoflavone (BNF) , a PAH. In another group of rats exposed to CD and BNF, caspase activity was inhibited by injection of the pan-caspase inhibitor Q-VD-OPH [quinoline-Val-Asp (OMe) -CH2-OPH]. RESULTS: In rats exposed to BNF, CD exposure increased alveolar expression of the proapoptotic mediator Bax but decreased CYP1A1 induction relative to BNF exposure alone. Pan-caspase inhibition decreased CD-associated Bax expression and apoptosis but did not restore CYP1A1 activity. Further, CD-induced lung inflammation and alveolar epithelial cell hypertrophy and hyperplasia were not suppressed by caspase inhibition. CONCLUSIONS: Combined BNF and CD exposure increased Bax expression and apoptosis in the lung, but Bax and apoptosis were not the major determinants of early lung injury in this model.     

A biomarker approach to measure biological effects of contaminant exposure in largemouth bass from Lake Conestee, South Carolina, U.S.A

Sediments from Lake Conestee, a former reservoir now filled with pollutant-enriched sediments, located south of Greenville, South Carolina, USA, and other nearby reservoirs were collected and analyzed for lead and polycyclic aromatic hydrocarbons (PAHs). Hepatic ethoxyresorufin-O-deethylase (EROD), glutathione S-transferase (GST), UDP-glucuronosyltransferase (UGT), sulfotransferase (SULT), and erythrocyte delta-aminolevulinic acid dehydratase (ALAD) were measured in largemouth bass (Micropterus salmoides) to characterize biological effects of these contaminants over three seasons. Results showed that total PAH concentrations in Lake Conestee sediments were significantly greater than the control during each season. An average 10-fold induction in EROD activity was observed at Lake Conestee compared with the control over all three seasons, indicating that PAHs present in sediment were bioavailable to fish. Significant gender effects were observed in EROD activity during the spring, in which activity in reproductively active female fish was significantly suppressed compared with the male fish. Sediments from Lake Conestee had elevated lead concentrations, but the lack of ALAD inhibition in bass indicated that lead was not biologically available. Total GST activity, UGT activity, and SULT activity were not significantly induced in fish from any of the affected sites compared with the reference site. Both EROD and UGT activities were highest during the winter, as were sediment PAH concentrations in Lake Conestee, possibly linked to seasonal resuspension events. The biomarkers measured in this study were useful as a first investigation into the biological effects of contaminant exposure, as well as in determining the bioavailability of contaminants in Lake Conestee.     

Polycyclic Aromatic Hydrocarbon Metabolites and 7-Ethoxyresorufin O-Deethylase Activity in Caged European Eels

This study investigated the contribution of two biomarkers, bile polycyclic aromatic hydrocarbon (PAH) metabolites and 7-ethoxyresorufin O-deethylase (EROD), activity in the assessment of PAH contaminated sites. European eels (Anguilla anguilla) were caged in a freshwater stream upstream and downstream from local industrial effluent outlets. Bile PAH metabolites were recorded as fluorescent aromatic compounds by synchronous fluorescence spectroscopy and as a marker for total PAH metabolism: 1-hydroxypyrene (1-OH Pyr) was isolated by high-pressure liquid chromatography and quantified. After 14 and 28 days of caging, EROD activity, bile fluorescence (synchronous fluorometric measurement), and 1-OH Pyr concentrations in bile were higher at the downstream site than at the upstream site. This increase was similar after 2 and 4 weeks of caging. During a reversibility study, EROD activity, bile fluorescence, and 1-OH Pyr concentrations decreased, and this trend was similar for the three markers. These results suggest that PAHs could be the main factor responsible for EROD induction in eels caged at the downstream site.     

The Dose–Response Relationships for EROD and GST Induced by Polyaromatic Hydrocarbons in <Emphasis Type="Italic">Carassius auratus</Emphasis>

Freshwater fish Carassius auratus were chosen as experimental animals, the hepatic biochemical responses to medium-term exposure of five PAHs were measured as ethoxyresorufin O-deethylase (EROD) activity (phase I) and glutathione S-transferase (GST) activity (phase II) to assess sub-lethal effects. The fold increases of EROD and GST activity were calculated and both increased in the order Fluoranthene < Fluorene < Benzo(b)fluoranthene < Benzo(g,h,i)perylene < Indeno(1,2,3-cd)-pyrene. The clear dose–response relationships were found for liver EROD and GST activity induced by PAHs. The enzyme EROD and GST in Carassius auratus were confirmed as useful biomarkers of exposure to both PAH and PAH-like compounds.     

Cytochrome P450 CYP1A1 enzyme activity and DNA adducts in placenta of women environmentally exposed to organochlorines.

Organochlorine compounds bioaccumulate in fishing and hunting products included in the daily diet of many coastal populations. Prenatal and perinatal exposure to large doses of PCBs and PCDFs was shown to be deleterious on fetal and neonatal development, but information is scarce regarding possible effects of chronic low-dose exposure. This study investigates biomarkers of early effects in newborns from women exposed to organochlorines through the consumption of species from marine food chains, in two remote coastal regions of the province of Quebec (Canada). A CYP1A1-dependent enzyme activity (EROD) and DNA adducts were measured in placenta samples obtained from 30 women living on the Lower-North-Shore of the St. Lawrence River and 22 Inuit women from Nunavik (Arctic Quebec). These biomarkers were also assessed in 30 women from a Quebec urban center (Sept-Iles) as a reference group. Prenatal organochlorine exposure was determined by measuring these compounds in umbilical cord plasma. The amount of bulky polycyclic aromatic hydrocarbon (PAH)-related DNA adducts was significantly greater in the Lower-North-Shore group than in the reference group. Placental EROD activity and the amount of less bulky (OC-related) DNA adducts were significantly higher in the Nunavik group than in the reference group. For both biomarkers, smoking was found to be an important confounding factor. Organochlorine exposure was significantly associated with EROD activity and DNA adduct levels when stratifying for smoking. This study confirms that CYP1A1 enzyme induction and DNA adducts in placental tissue constitute useful biomarkers of early effects induced by environmental exposure to organochlorines.     

Using Semipermeable Membrane Devices, Bioassays, and Chemical Analysis for Evaluation of Bioavailable Polycyclic Aromatic Hydrocarbons in Water

Meiliang Bay is a sublake of Taihu Lake and has been polluted by domestic and industrial effluents. As part of a comprehensive risk assessment project in this region, semipermeable membrane devices (SPMDs) were applied to evaluate the levels and potential toxic potency of polycyclic aromatic hydrocarbons (PAHs) in lakewater, in combination with chemical analysis and in vitro bioassay using H4IIE rat hepatoma cells. In addition, induction of hepatic ethoxyresorufin-O-deethylase (EROD) activity, in crucian carp (Carassius auratus), caged in the vicinity of SPMD sampling sites was also used as a biomarker of exposure to PAHs and related chemicals. The caged crucian carp accumulated similar PAH profile patterns (p < 0.001) but at lower levels compared with SPMDs on a wet-weight basis. Total concentrations of PAHs in crucian carp muscle tissues ranged from 35.6 to 69.1 ng/g after the 32-day exposure, whereas total PAHs in SPMDs ranged from 716.9 to 1007.8 ng/g. Dialysates from SPMDs exposed to Meiliang Bay water caused marked EROD activity in H4IIE cells. Toxic potency of dialysates expressed as bioassay-derived 2,3,7,8-tetrachlorodibenzo-p-dioxin equivalents (TCDD-EQ) ranged from 3.8 to 6.2 pg TCDD-EQ/g SPMD for 32-day exposure samples. A linear correlation of total amount of PAHs and in vitro TCDD-EQ of SPMD dialysates yielded an R ² of 0.82. Empirical evidence suggests that aryl hydrocarbon receptor-active PAHs can account for about 42–56% of the potency observed. Significant induction of liver EROD activity was also observed in crucian carp caged alongside SPMDs and there was a good correlation between the results of EROD assays in vivo and in vitro (R ² = 0.62, p = 0.02). It is, therefore, suggested that the SPMD technique in connection with chemical analysis and specific in vitro bioassays might be a valuable tool to assess the levels and effects of bioavailable hydrophobic pollutants in water.     

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, ben-zo(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 μg/m³ and 2.7 μg/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. © 1995 Wiley-Liss, Inc.     

Cytochrome P450-Dependent Enzymes and Oxidant-Mediated Responses in Rainbow Trout Exposed to Contaminated Sediments

Hatchery-reared immature rainbow trout (Oncorhynchus mykiss) were exposed to different concentrations (2 and 4 liters) of contaminated sediment taken from a site receiving unbleached pulp mill effluents. The fish were held in aquaria and sampled three times during an experimental period of 21 days. The monooxygenase activity, measured as the deethylation of 7-ethoxyresorufin (EROD activity), increased three- to fourfold in the exposed fish relative to controls. The increase was not dependent on exposure concentration. Cytochrome P450IA1, the EROD catalyst, demonstrated proportional induction in the 2-liter exposed fish. However, exposure to 4 liters sediment strongly induced P450IA1 and did not reflect EROD activity. This may suggest inhibition of P450IA1 activity by the amount of chemicals discharged from pulp mills. UDPglucuronosyl-transferase increased at one stage of the experimental period, while glutathione S-transferase remained unchanged. Amounts of total glutathione in blood, liver, and muscle were slightly increased by exposure to contaminated sediments, but hepatic enzyme activities of superoxide dismutase and catalase were not affected. In conclusion, monooxygenase activities appear to be a sensitive tool in the monitoring of sediment toxicity.     

Cytochrome P4501A Induction and Porphyrin Accumulation in PLHC-1 Fish Cells Exposed to Sediment and Oil Shale Extracts

The present study describes the use of a fish hepatoma cell line (PLHC-1) in monitoring the biological effects of sediments collected from recipient waters of the oil shale industry. Sampling sites were located in River Purtse and River Kohtla in northeast Estonia. The effects of pure oil shale on the PLHC-1 cells were also studied. The cells were exposed to n-hexane–extracted samples in 48-well plates for 24 h, and 7-ethoxyresorufin O-deethylase (EROD) activity, total protein, and porphyrin content were measured in the exposed cells. Polycyclic aromatic hydrocarbon (PAH) contents in the samples were measured by high-performance liquid chromatography (HPLC). All the sediment and oil shale samples induced CYP1A activity and led to porphyrin accumulation in the cells. The most potent inducers were the sediments collected near the oil shale processing plants (site Lüganuse in River Purtse and Kohtla in River Kohtla), as well as those at the most downstream site in River Purtse (Purtse). These samples possessed high total PAH contents, ranging from 4,270 to nearly 150,000 μg/kg dry sediment. The presence of other lipophilic organic contaminants in the samples was not determined in this study. Both EROD activity and porphyrin content exhibited biphasic induction curves, and the ED₅₀ ¹ values for EROD activity were lower than the ED₅₀s for porphyrin content. 2,3,7,8-Tetrachlorodibenzo-p-dioxin induction equivalents (TCDD-EQs) calculated from EROD induction potencies correlated well with total PAHs (r ² = 0.827 and p = 0.003 for log-transformed data) and also with individual PAHs. TCDD-EQs for porphyrin content did not correlate significantly with total PAHs (log-log r² = 0.785, p = 0.116). The biological potency and PAH contamination of the samples showed the same rank order, except at Lüganuse, where sediment extracts induced CYP1A and porphyrins more than could have been expected based on PAH contents. Bioassay-derived induction EQs (normalized to dibenz(a,h)anthracene) were 20- to 3,200-fold greater than EQs calculated from the concentrations of five PAHs, suggesting important contributions from other compounds or nonadditive effects. The PLHC-1 cells proved to be a sensitive bioanalytical tool for sediments contaminated with PAH-type pollutants in the oil shale processing area. We suggest further use of this bioassay in screening and monitoring waters with similar background of pollution as in northeast Estonia. Received: 24 August 1998/Accepted: 22 July 1999     

Modulatory Effects of Curcumin in Conjunction with Piperine on Benzo(A)Pyrene-Mediated DNA Adducts and Biotransformation Enzymes

The antigenotoxic effects of curcumin alone and with piperine on benzo(a)pyrene-diol (BaP) epoxide DNA adducts (BaPDE-DNA adducts), and carcinogen biotransformation enzymes was investigated in liver and lung of mice. Male Swiss albino mice received curcumin (100 mgkg^?1 body weight) and piperine (20 mgkg^?1 body weight) separately as well as in combination orally in corn oil for 7 days as pretreatments and thereafter 2 h, BaP (125 mgkg^?1 body weight) was administered orally in corn oil. A single dose of BaP to normal mice increased the activities of ethoxyresorufin o-deethylase (EROD), pentoxyresorufin o-depentylase (PROD) and levels of BaPDE-DNA adducts in both the tissues. Quinone reductase (QR) activity was also elevated in the BaP-treated group in both liver and lung when compared with normal control group. Pretreatment of curcumin and curcumin plus piperine before administration of BaP significantly decreased the activities of EROD, PROD, and the level of BaPDE-DNA adducts with consequent increase in QR activities. The study clearly indicates that curcumin, when given in combination with piperine, is more effective in modulating BaPDE-DNA adducts (liver and lung), and activity of EROD (liver).     

Determination of Polycyclic Aromatic Hydrocarbons (PAH) and Their Metabolites in Blood, Feces, and Urine of Rats Orally Exposed to PAH Contaminated Soils

Polycyclic aromatic hydrocarbons (PAH) have become an ubiquitous upper soil component as a consequence of industrialization involving a multitude of combustion processes. Ingestion of PAH contaminated soil is considered to be a major exposure route, specifically for small children living on these soils. Health risk assessment is based on extrapolations from data obtained via studies performed with pure chemicals. Additionally it is assumed that after oral intake all PAH present in the soil will be absorbed by the human body. Interactions with the soil matrix, however, may modulate the bioavailability of PAH. In this study, we examined the absorption and excretion of PAH in rats orally exposed either to industrially contaminated soils or pure model compounds as anthracene, pyrene and benzo(a)pyrene (B[a]P). The model compounds and the metabolites, 1-hydroxypyrene (1-OH-pyrene) and 3-hydroxybenzo(a)pyrene (3-OH-B[a]P), were measured in blood, feces or urine by means of HPLC with fluorescence detection. Because of rapid biotransformation only minimal levels of unmetabolized anthracene, pyrene and B[a]P in blood could be detected. The pharmacokinetic parameters were nonlinear and suggestive of enterohepatic cycling. Only low levels of the compounds were excreted unchanged in feces whereas the levels of the metabolites were considerably higher in feces and urine. These results indicate that the dosed PAH are largely absorbed by the gastrointestinal tract, subsequently metabolized and excreted as metabolites via urine and feces. Significant differences between the soil-treated group and the pure mixture-treated group could be observed; the soil-treated group showed higher fecal excretion of unchanged pyrene (0.5 versus 0.2% of the original dose) and B[a]P (1 versus 0.3%), lower excretion of 1-OH-pyrene in feces (5.1 versus 17.0%), and lower excretion of 1-OH-pyrene in urine (0.2 versus 3.4%). The fecal excretion of 3-OH-B[a]P between the two groups was similar (8.8 versus 8.8%). These results suggest that the soil matrix is capable of reducing the absorption of at least pyrene. Therefore, exposure risk assessment models assuming complete bioavailability of soilmatrix-bound PAH probably overestimate the endogenous dose. Received: 18 April 1996/Accepted: 27 March 1997