Toxic responses in primary rat hepatocytes exposed with occupational dust collected from work environment of bone-based industrial unit

In this in vitro study we investigated the toxic responses in hepatocytes treated with occupational dust to which workers are exposed in bone-based industrial units. The present study investigated the toxicity mechanism of bone-based occupational dust, from a particular industrial unit, on isolated rat hepatocytes. The hepatocytes were isolated by collagenase perfusion method and cell viability was determined by trypan blue exclusion and MTT [3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay treated with occupational dust at 0.1-1.0 mgmL(-1), for 120 min. The cell viability decreased significantly in a concentration-dependent manner. Dust induced significant membrane damage measured by lactate dehydrogenase (LDH) and glutathione (GSH) release in culture media for 30-, 60- and 120 min treatment duration. The toxicity was found to be correlated with the induction of lipid peroxidation (LPO). In addition, nitric oxide (NO), and hydrogen peroxide (H(2)O(2)) generation by occupational dusts were also found to be time- and concentration-dependent. Over all the present study provides initial evidences for the toxic potential of occupational dust generated in bone-based industries and, therefore, the dust exposure to workers in unorganized industrial units should be controlled.     

Effects of benzo[a]pyrene as an environmental pollutant and two natural antioxidants on biomarkers of reproductive dysfunction in male rats

Benzo[a]pyrene (B[a]P) is an environmental toxicant and endocrine disruptor. Therefore, the aim of the present study was to investigate the toxicity of B[a]P in testis of rats and also to study the role of silymarin and thymoquinone (TQ) as natural antioxidants in the alleviation of such toxicity. Data of the present study showed that levels of testosterone, estrogen and progesterone were significantly decreased after treatment of rats with B[a]P. In addition, B[a]P caused downregulation of the expressions of steroidogenic enzymes including CYP17A1 and CP19A1, and decreased the activity of 17-β hydroxysteroid dehydrogenase (17β-HSD). Moreover, B[a]P decreased the activities of antioxidant enzymes including catalase (CAT), glutathione peroxidase (GP_X) and superoxide dismutase (SOD), and significantly increased free radicals levels in testis of male rats. However, pretreatment of rats with silymarin prior to administration of B[a]P was found to restore the level of free radicals, antioxidant status, and activities of steroidogenic enzymes to their normal levels in testicular tissues. Moreover, histopathological finding showed that silymarin recovered the abnormalities occurred in tubules caused by B[a] P in testis of rats. On the other hand, TQ showed pro-oxidant effects and did not ameliorate the toxic effects of B[a] P on the testicular tissue since it decreased antioxidant enzymes activities and inhibited the protein expression of CYP11A1 and CYP21A2 compared to control rats. Moreover, TQ decreased the levels of testosterone, estrogen, and progesterone either in the presence or absence of B[a]P. It is concluded that B[a]P decreased testosterone levels, inhibited antioxidant enzymes activities, caused downregulation of CYP isozymes involved in steroidogenesis, and increased free radical levels in testis. Moreover, silymarin was more effective than TQ in restoring organism health and alleviating the deleterious effects caused by B[a]P in the testis of rats. Due to its negative impact, it is highly recommended to limit the use of TQ as a dietary supplement since millions of people in the Middle East are using it to improve their health.

     

Antioxidant responses to benzo[a]pyrene and Aroclor 1254 exposure in the green-lipped mussel, Perna viridis

In this study, the green-lipped mussel, Perna viridis (L.), was exposed to two concentrations of benzo[a]pyrene (B[a]P) (0.3 microg l(-1); 3 microg l(-1)) and two concentrations of Aroclor 1254 (0.5 microg l(-1); 5 microg l(-1)). In addition, a mixture of the contaminants was used (0.3 microg l(-1) B[a]P+0.5 microg l(-1) Aroclor 1254; 3 microg l(-1) B[a]P+5 microg l(-1) Aroclor 1254). All concentrations were nominal. A suite of enzymes [glutathione S transferase (GST), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR)], glutathione (GSH) level and lipid peroxidation (LPO) in the mussel gill and hepatopancreas were monitored over 18 days. CAT and GSH in gill tissue were positively correlated with concentration of Aroclor 1254. Activity of hepatic GST and SOD was significantly related to body burden of Aroclor 1254. LPO, GR and GPx in gill and hepatopancreas and hepatic GST were positively correlated with B[a]P concentration. The results indicate the importance of using biomarkers specific to the type of contaminant(s) that are likely to be present. Controlled laboratory experiments, such as this study, are useful in ascertaining biomarkers suitable for use with complex contaminant mixtures in the marine environment.     

Expression alterations of cytochromes P4501A1, 2E1, and 3A,and their receptors AhR and PXR caused by 1-octyl-3-methylimidazolium chloride in mouse mammary carcinoma cells

Cytochrome P450s (CYPs) play a key role in the metabolism of a wide range of environmental xenobiotics and endogenous compounds. The expression and activity levels of CYPs can be elevated by a process of induction involving the activation of nuclear receptors. The effects of the ionic liquid 1-octyl-3-methylimidazolium chloride ([C₈mim][Cl]) on the expression of cytochrome P450 members, including CYP1A1, CYP2E1, and CYP3A, as well as on aryl hydrocarbon receptor (AhR) and pregnane X receptor (PXR) in mouse mammary carcinoma cells (EMT6) were investigated by using quantitative real-time PCR in the present study. The results reveal that [C₈mim][Cl]-exposure up-regulates the expressions of CYP1A1, CYP2E1, and CYP3A at mRNA level, suggesting that imidazolium-based ionic liquids can activate CYPs. Our results also suggest that [C₈mim][Cl]-mediated CYP3A induction be PXR-dependent. This result may be beneficial to evaluating the environmental toxicity of imidazolium-based ionic liquids and investigating the metabolism of imidazolium-derivative drugs.     

EROD and CYP1A protein in channel catfish (Ictalurus punctatus) from an urban estuary relative to that in benzo[a]pyrene-exposed hatchery specimens

Bottom-feeding fish such as flounder and killifish have been widely used in monitoring hepatic monooxygenase induction in polluted water bodies. While channel catfish are often utilized in tissue monitoring of fresh and estuarine water bodies, few data are available on their use in environmental monitoring of hepatic monooxygenase activity. In this project, the presence of CYP1A protein was verified in channel catfish through recognition by Mab 1-12-3, an antibody which recognizes the CYP1A homologue in a variety of teleost species. CYP1A protein levels and 7-ethoxyresorufin-o-deethylase (EROD) activity in laboratory control and benzo-a-pyrene (BaP)-challenged channel catfish were compared to those in feral channel catfish from Back River, an urban estuarine tributary to Chesapeake Bay. Though more variable, mean CYP1A protein levels in the field-collected fish were similar to those of the BaP-induced laboratory fish. However, EROD activity of the Back River fish was less than one half that observed in the BaP-induced laboratory fish. When normalized to CYP1A protein levels, EROD activity was slightly lower in the Back River fish than either the laboratory control or BaP-treated fish. This finding may indicate possible inhibition or inactivation of the CYP1A protein in the feral fish.     

New cytochrome P450 1B1, 1C1, 2Aa, 2Y3, and 2K genes from Chinese rare minnow (Gobiocypris rarus): Molecular characterization,basal expression and response of rare minnow CYP1s and CYP2s mRNA exposed to the AHR agonist benzo[a]pyrene

Cytochrome P450 (CYP450) genes play an important role in catalyzing oxidative metabolism of toxicants. Recently, CYP1 subfamily were discovered and reported in fish, however, little is known regarding the CYP2 isoforms in fish. In the present study, the cDNA fragments of CYP 1B1 and 1C1 and CYP2Aa, 2Y3, and 2K of rare minnow were cloned and exhibited a high amino acid sequence identity compared with their zebrafish orthologs. Basal expression showed CYP1C1 and CYP 2Aa expression were observed in all eight tissues analyzed (liver, gill, intestine, kidney, spleen, brain, skin, and muscle). CYP 1A, and 1B1 expression was found in all tissues except for muscle and skin. However, CYP 2Y3 was expressed in liver, spleen, intestine and muscle whereas CYP 2 K in liver, kidney and intestine. 4 and 100 μg L⁻¹ Benzo[a]pyrene (BaP) induced patterns showed that CYP 1A, 1B1 and 1C1 expression in liver, gill, and intestine was strongly up-regulated (p < 0.05). Furthermore, CYP 2Y3 was strongly induced in liver from BaP treatments (p < 0.05). The high induction on mRNA level of CYP1s and CYP 2Y3 by BaP could be associated with catalyzing detoxification and indicated that CYP2s may also be potential biomarker to screen AHR agonist. The high responsiveness of CYP1 and 2 genes suggested Chinese rare minnow is feasible to screen and assess pollution with AHR agonist.     

Biotransformation of metamitron by human p450 expressed in transgenic tobacco cell cultures

In the present investigation, the oxidative metabolism of 14C-labeled metamitron was examined in plant cell cultures of tobacco overexpressing human P450 enzymes CYP1A1 or CYP1A2; special interest was in the aromatic hydroxylation of the herbicide. The oxidative metabolites deaminometamitron (DAM) and 4-hydroxydeaminometamitron (4-HDAM) were found in the untransformed control culture as well as in the transgenic culture. The transgenic cultures, however, exhibited higher turnover rates after 48 h of incubation with 20 microg 14C-metamitron per assay (untransformed: 40%, CYP1A1: 80%, CYP1A2: 100%). Primary metabolite 4-HDAM was partially found in glucosylated form in the transgenic cultures. As minor oxidative metabolites, 6-hydroxyphenyl-3-methoxymethyl-1,2,4-triazine-5(4H)-one and 3-hydroxymethyl-6-phenyl-1,2,4-triazine-5(4H)-one were identified in the transgenic cultures by GC-MS, LC-MS. Additionally, it could be demonstrated that both foreign enzymes (CYP1A1, CYP1A2) also catalyzed the deamination of metamitron. In a large-scale study (up to 400 microg per assay) with the transgenic culture expressing CYP1A2, the high efficiency of this P450 system toward metamitron was demonstrated: turnover of the xenobiotic was almost complete with 400 microg. Since large portions of unglucosylated 4-H-DAM were found, the activity of foreign CYP1A2 apparently exceeded that of endogenous O-glucosyltransferases of the tobacco cell culture. We concluded that in comparison to the nontransformed cell culture, the extent of metabolism was considerably higher in the transgenic cultures. The transgenic cell cultures expressing human CYP1A1 or CYP1A2 are thus suitable tools for the production of large quantities of primary oxidized metabolites of metamitron.     

Cytochrome P4501A induction and testosterone hydroxylation in cultured hepatocytes of four fish species

Primary hepatocytes of the rainbow trout (Oncorhynchus mykiss), flounder (Platychthis flesus), dab (Limanda limanda) and lemon sole (Microstomus kitt) were exposed to 3,3'4,4'5 pentachlorobiphenyl (PCB 126) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) for two days. This resulted in a dose-dependent induction of cytochrome P4501A (CYP1A) activity, measured as ethoxyresorufin O-deethylase (EROD), or methoxyresorufin O-deethylase (MROD) activity. In all species, a linear relationship was observed between EROD and MROD activities, suggesting that the same CYP1A enzyme metabolizes the two alkoxy-resorufin substrates. Exposures of hepatocytes of flounder or dab to TCDD, resulted in a 59-fold and 8.2-fold induction of EROD activity, respectively. This did not concur with a change in the in vitro testosterone hydroxylation profiles of both species. These and other in vitro data indicate that TCDD exposure does not influence monooxygenase activities involved in testosterone hydroxylation. Furthermore, CYP1A is of minor importance for testosterone hydroxylation in these fish species.     

Indoor/Outdoor relationships, trends, and carbonaceous content of fine particulate matter in retirement homes of the Los Angeles Basin

Hourly indoor and outdoor fine particulate matter (PM2.5), organic and elemental carbon (OC and EC, respectively), particle number (PN), ozone (O3), carbon monoxide (CO), and nitrogen oxide (NOx) concentrations were measured at two different retirement communities in the Los Angeles, CA, area as part of the Cardiovascular Health and Air Pollution Study. Site A (group 1 [G1]) was operated from July 6 to August 20, 2005 (phase 1 [P1]) and from October 19 to December 10, 2005 (P2), whereas site B (group 2 [G2]) was operated from August 24 to October 15, 2005 (P1), and from January 4 to February 18, 2006 (P2). Overall, the magnitude of indoor and outdoor measurements was similar, probably because of the major influence of outdoor sources on indoor particle and gas levels. However, G2 showed a substantial increase in indoor OC, PN, and PM2.5 between 6:00 and 9:00 a.m., probably from cooking. The contributions of primary and secondary OC (SOA) to measured outdoor OC were estimated from collected OC and EC concentrations using EC as a tracer of primary combustion-generated OC (i.e., "EC tracer method"). The study average outdoor SOA accounted for 40% of outdoor particulate OC (40-45% in the summer and 32-40% in the winter). Air exchange rates (hr(-1)) and infiltration factors (Finf; dimensionless) at each site were also determined. Estimated Finf and measured particle concentrations were then used in a single compartment mass balance model to assess the contributions of indoor and/or outdoor sources to measured indoor OC, EC, PM2.5, and PN. The average percentage contributions of indoor SOA of outdoor origin to measured indoor OC were approximately 35% (during G1P1 and G1P2) and approximately 45% (for G2P1 and G2P2). On average, 36% (G2P1) to 44% (G1P1) of measured indoor OC was composed of outdoor-generated primary OC.     

Structure dependent induction of CYP1A by polychlorinated biphenyls in hepatocytes of male castrated pigs

Hepatocytes cultures prepared from castrated pig hepatocytes (Great Yorkshire x Dutch Landrace), as a model for human liver, were used to study the effect of twenty polychlorinated biphenyls (PCBs) on CYP1A activity, measured as the dealkylation of either ethoxyresorufin or methoxyresorufin. The selection of the PCBs was based on their differences in physico-chemical properties. The non-ortho and mono-ortho substituted PCBs were the most potent CYP1A inducers in pig hepatocytes. In addition, several multiple-ortho substituted congeners, with five or more chlorine atoms, were inducers of CYP1A activity as well. Their relative effect potencies (REP) were proximately 10,000 times lower than the most potent congener, 3,3',4,4',5 PeCB (PCB#126). Using partial least-squares (PLS) modeling, predictions of CYP1A activity could be made for all tetra to hepta substituted congeners. Several multiple-ortho substituted PCBs, which are highly abundant in the biotic and abiotic environment, have been found to induce CYP1A activity in pig hepatocytes. Because induction of CYP1A activity is used as biomarker for Ah-receptor mediated responses, it is suggested to include these congeners in future risk assessment.     

Biotransformation of Metamitron by Human P450 Expressed in Transgenic Tobacco Cell Cultures

In the present investigation, the oxidative metabolism of ¹⁴C-labeled metamitron was examined in plant cell cultures of tobacco overexpressing human P450 enzymes CYP1A1 or CYP1A2; special interest was in the aromatic hydroxylation of the herbicide. The oxidative metabolites deaminometamitron (DAM) and 4-hydroxydeaminometamitron (4-HDAM) were found in the untransformed control culture as well as in the transgenic culture. The transgenic cultures, however, exhibited higher turnover rates after 48 h of incubation with 20 μg ¹⁴C-metamitron per assay (untransformed: 40%, CYP1A1: 80%, CYP1A2: 100%). Primary metabolite 4-HDAM was partially found in glucosylated form in the transgenic cultures. As minor oxidative metabolites, 6-hydroxyphenyl-3-methoxymethyl-1,2,4-triazine-5(4H)-one and 3-hydroxymethyl-6-phenyl-1,2,4-triazine-5(4H)-one were identified in the transgenic cultures by GC-MS, LC-MS. Additionally, it could be demonstrated that both foreign enzymes (CYP1A1, CYP1A2) also catalyzed the deamination of metamitron. In a large-scale study (up to 400 μ g per assay) with the transgenic culture expressing CYP1A2, the high efficiency of this P450 system toward metamitron was demonstrated: turnover of the xenobiotic was almost complete with 400 μ g. Since large portions of unglucosylated 4-H-DAM were found, the activity of foreign CYP1A2 apparently exceeded that of endogenous O-glucosyltransferases of the tobacco cell culture. We concluded that in comparison to the nontransformed cell culture, the extent of metabolism was considerably higher in the transgenic cultures. The transgenic cell cultures expressing human CYP1A1 or CYP1A2 are thus suitable tools for the production of large quantities of primary oxidized metabolites of metamitron.     

Gene expression of heat shock protein 70, interleukin-1β and tumor necrosis factor α as tools to identify immunotoxic effects on Xenopus laevis: a dose-response study with benzo[a]pyrene and its degradation products

The exposure to benzo[a]pyrene (B[a]P) results in an alteration of immune function in mammals and fish, and the analysis of cytokine mRNA levels has been suggested for predicting the immunomodulatory potential of chemicals. To obtain evidence of the innate immune responses to B[a]P in Xenopus laevis, the present study monitored the mRNA expression of interleukin 1-β (IL-1β), tumor necrosis factor α (TNF-α) and heat shock protein 70 (HSP70) in a laboratorial exposure. Tadpoles exposed to 8.36, 14.64, 89.06 and 309.47 μg/L of B[a]P,were used for detecting hsp70, IL-1β and TNF-α mRNA induction. A dose-response increase in the expression of hsp70 and IL-1β mRNA was found. The results of this study confirmed the use of hsp70 and IL-1β, but not TNF-α, as sensitive indicators of immunotoxic effect of B[a]P in X. laevis. Further research would be required for the validation of these endpoints.     

Metabolism of the14C-labeled herbicide clodinafop-propargyl in plant cell cultures of wheat and tobacco

The metabolism of ¹⁴C-clodinafop-propargyl (CfP) was examined in cell cultures of wheat (Triticum aestivum L. cv. ‘Heines Koga II’) and tobacco (Nicotiana tabacum L.). Besides the non-transgenic tobacco culture, cultures transformed separately with cDNA of human cytochrome P450-monooxygenases (P450s) CYP1A1, CYP1A2, CYP3A4, CYP2B6 and CYP2C19 were examined. Experiments with wheat were executed in the presence and absence of safener cloquintocet-mexyl (CqM). After 48 h of incubation, only about 10% of applied ¹⁴C was found in media (both tobacco and wheat). Non-extractable residues of ¹⁴C-CfP in wheat cells were 16.54% (without CqM) and 30.87% (with CqM). In all tobacco cultures, 82.41–92.46% of applied radioactivity was recovered in cell extracts. In contrast to wheat, non-extractable residues amounted only to 1.50–2.82%. As determined by radio-thin layer chromatography (TLC) and -high-performance liquid chromatography (HPLC), the parent CfP was not found in the cell extracts of wheat; in tobacco cell extracts, only traces of CfP were detected. After a hydrolysis of assumed carbohydrate conjugates of CfP derived polar ¹⁴C-labeled compounds, TLC and HPLC analysis showed that in wheat, a more complex pattern of metabolites of CfP were observed as compared to all tobacco cultures. In hydrolysates resulting from wheat, the identity of three primary products was confirmed by means of GC-EI-MS: free acid clodinafop (Cf), hydroxy-Cf hydroxylated at the pyridinyl moiety, and 4-(5-chloro-3-fluoropyridin-2-yloxy)phenol. In hydrolysates derived from all tobacco cultures, main metabolite was Cf besides only traces of further unidentified products. Differences among the different tobacco cultures (non-transgenic, transgenic) did not emerge. According to kinetics of disappearance of primary metabolite Cf as well as formation of polar soluble products and non-extractable residues, metabolization of CfP proceeded at a noticeably higher rate in wheat cells treated with safener CqM than in cells without CqM treatment. Thus, these results indicated a stimulation of CfP's metabolism by CqM, although metabolic profiles observed in CqM treated and non-treated cells (after hydrolysis) were qualitatively similar. The findings obtained from all tobacco cultures suggested that with the exception of ester cleavage to Cf, CfP cannot be metabolized by tobacco itself or by the human P450s examined.     

Alterations to proteome and tissue recovery responses in fish liver caused by a short-term combination treatment with cadmium and benzo[a]pyrene

The livers of soles (Solea senegalensis) injected with subacute doses of cadmium (Cd), benzo[a]pyrene (B[a]P), or their combination, were screened for alterations to cytosolic protein expression patterns, complemented by cytological and histological analyses. Cadmium and B[a]P, but not combined, induced hepatocyte apoptosis and Kupfer cell hyperplasia. Proteomics, however, suggested that apoptosis was triggered through distinct pathways. Cadmium and B[a]P caused upregulation of different anti-oxidative enzymes (peroxiredoxin and glutathione peroxidase, respectively) although co-exposure impaired induction. Similarly, apoptosis was inhibited by co-exposure, to which may have contributed a synergistic upregulation of tissue metalloproteinase inhibitor, β-actin and a lipid transport protein. The regulation factors of nine out of eleven identified proteins of different types revealed antagonistic or synergistic effects between Cd and B[a]P at the prospected doses after 24 h of exposure. The results indicate that co-exposure to Cd and B[a]P may enhance toxicity by impairing specific responses and not through cumulative damage.     

Benzo[b]fluoranthene,a Potential Alternative to Benzo[a]pyrene as an Indicator of Exposure to Airborne PAHs in the Vicinity of Söderberg Aluminum Smelters

ABSTRACT

Benzo[b]fluoranthene (B[b]F) was used in relative abundance ratios (RARs), a parameter obtained by dividing the concentration of individual polycyclic aromatic hydrocarbons (PAHs) found in a given sample by the concentration of B[b]F in the same sample. The B[b]F RARs were derived for PAH concentrations measured at stacks and sampling stations in the vicinity of two Söderberg aluminum horizontal stud smelters (HSSs). The samples collected were analyzed by high-performance liquid chromatography using UV and fluorescence detection. A total of 15 PAHs were analyzed, but, due to the inefficiency of the sampling method used in collecting gaseous PAHs, only particulate PAHs were considered. Comparisons between the B[b]F RARs obtained simultaneously at the source (stack) and those obtained at sampling stations at the two smelters showed that B[b]F degrades more slowly than or at the same rate as most other particulate PAHs monitored. Twenty-three months of urban sampling in the vicinity of one of the aluminum HSSs are also presented, and the results indicate that B[b]F is more stable than all other particulate PAHs investigated. Sampling conducted during a smelter shutdown period confirmed that B[b]F was a much better marker of this source than was benzo[a]pyrene (B[a]P), the usual indicator. The remarkable stability of the benzo[k]fluoranthene (B[k]F)/B[b]F ratio is also discussed.     

Benzo[b]fluoranthene, a potential alternative to benzo[a]pyrene as an indicator of exposure to airborne PAHs in the vicinity of Söderberg aluminum smelters

Benzo[b]fluoranthene (B[b]F) was used in relative abundance ratios (RARs), a parameter obtained by dividing the concentration of individual polycyclic aromatic hydrocarbons (PAHs) found in a given sample by the concentration of B[b]F in the same sample. The B[b]F RARs were derived for PAH concentrations measured at stacks and sampling stations in the vicinity of two S?derberg aluminum horizontal stud smelters (HSSs). The samples collected were analyzed by high-performance liquid chromatography using UV and fluorescence detection. A total of 15 PAHs were analyzed, but, due to the inefficiency of the sampling method used in collecting gaseous PAHs, only particulate PAHs were considered. Comparisons between the B[b]F RARs obtained simultaneously at the source (stack) and those obtained at sampling stations at the two smelters showed that B[b]F degrades more slowly than or at the same rate as most other particulate PAHs monitored. Twenty-three months of urban sampling in the vicinity of one of the aluminum HSSs are also presented, and the results indicate that B[b]F is more stable than all other particulate PAHs investigated. Sampling conducted during a smelter shutdown period confirmed that B[b]F was a much better marker of this source than was benzo[a]pyrene (B[a]P), the usual indicator. The remarkable stability of the benzo[k]fluoranthene (B[k]F)/B[b]F ratio is also discussed.     

Effects of dietary habits and CYP1A1 polymorphisms on blood dioxin concentrations in Japanese men

The major source of dioxin impurities in Japan in the past was agrochemical formulations; more recently, it has been exhaust from waste incinerators. To examine the environmental and genetic factors that influence blood dioxin concentration, we investigated the relationship among dioxin concentrations, dietary habits and cytochorome P450 1A1 (CYP1A1) polymorphisms (MspI type and Ile-Val type) in Japanese fishermen and farmers, including also a group of office workers as controls. The mean dioxin concentrations in the fishermen, the farmers and the controls were 161369, 79079 and 100500 pg/g fat, respectively. The elevated dioxin concentration with polychlorinated dibenzo-p-dioxins, polychlorinated dibenzofurans and coplanar-PCBs found in the fishermen may be due to the frequent consumption of fish; no such relationship was found both in the farmers and the controls. We found that the concentrations of congeners found as impurities in certain chemicals such as those previously used in agriculture showed no significant differences among the three groups; we consider it unlikely that the farmers would be directly exposed to dioxins from such chemicals. Thus, it is probable that the primary route of dioxin exposure in the Japanese population is through the food chain via fish consumption, regardless of occupation. No meaningful relationship between blood dioxin concentration and CYP1A1 polymorphisms was found in this study, although there was a significant difference between the concentration of total non-ortho-PCBs in genotypes A and B. Further studies on more subjects, including those of genotype C, are needed to confirm the relationship between blood dioxin concentrations and MspI polymorphisms.     

Bioaccumulation of PCBs in Arctic seabirds: influence of dietary exposure and congener biotransformation

Four seabird species and their prey (zooplankton or fish) were collected in the Barents Sea to determine how dietary exposure, cytochrome P450 (CYP) enzyme activities and sex influenced their hepatic PCB concentrations and accumulation patterns. Five males and five females from each seabird species (little auk (Alle alle), Brunnich's guillemot (Uria lomvia), black guillemot (Cepphus grylle) and black-legged kittiwake (Rissa tridactyla)) were analysed. PCB concentrations could not be explained directly by carbon source (delta13C) or trophic position (delta15N), but by a combination of dietary parameters (delta13C, delta15N, migratory pattern, age) and contaminant metabolism. Contrary to previous studies, the PCB pattern differed among seabirds, with a higher proportion of persistent congeners (% of PCB-153, RPCB-153) in black-legged kittiwake than in auks. The PCB pattern also differed among auks, with little auk as the most efficient biotransformer (highest RPCB-153 values of persistent congeners). Based on high RPCB-153 values, Brunnich's guillemot poorly metabolised ortho-meta-unsubstituted congeners, whereas black guillemot poorly metabolised meta-para unsubstituted congeners. Species-specific differences in PCB biotransformation were confirmed by metabolic indices, where PCB patterns in seabirds were adjusted for PCB pattern in prey. The relative contribution of ortho-meta-unsubstituted congeners to SigmaPCBs decreased with increasing EROD activity. There were no differences in PCB concentrations, PCB patterns or cytochrome P450 enzyme activities between males and females. CYP P450 activities (CYP1A- and CYP2B/3A-like: EROD and testosterone 6beta-hydroxylation, respectively) were low and did not correlate with concentrations of non- or mono-ortho Cl-substituted PCBs (NO- and MO-PCBs), or with total toxic equivalent concentrations (TEQs) for dioxin-like effects of NO- and MO-PCBs.     

Hepatic and immune biological effect assays in C57BL/6 mice to measure polycyclic aromatic hydrocarbon bioavailability under laboratory exposures with increasing environmental relevance

Background Monitoring biological responses that are mediated via the aryl-hydrocarbon receptor (AhR) in animals exposed to environmental contaminants can indicate both the presence to chemicals that act through this biochemical pathway and whether these chemicals are bioavailable. Objectives The use of an ex-situ method that incorporated biological responsiveness monitoring in mice for determining the presence of ‘biologically active’ hydrocarbons in contaminated soils was investigated. Methods The use of C57BL/6 as a test organism was validated by determining hepatic and immune responsiveness to two polyaromatic hydrocarbons (PAHs): 3,4 benz[a]pyrene (B[a]P) and 1,2 benz (a)anthracene (BA) administered via intraperitoneal (i.p.) injection. The responsiveness of mice exposed to soils spiked with hydrocarbons or ex situ exposures to soil removed from two contaminated sites was also investigated. Results and Discussion Mice that were exposed to B[a]P via i.p. injections showed a 14-fold increase in liver microsomal ethoxyresorufin O-deethylase (EROD) activity compared to the control group. In contrast EROD activity following BA exposure at the same level was not significantly enhanced. Mouse immune response was significantly inhibited in a dose-dependent manner by i.p. injections of B[a]P. No significant inhibition occurred with the same doses of BA. Following i.p. exposure, the retention of B[a]P in mouse carcasses was greater than BA. Mice exposed to clean soils spiked with environmentally relevant concentrations of B[a]P and BA failed to show any significantly different hepatic or immune responses. Carcass residue data indicated a limited uptake of PAH from the soil. In contrast, EROD activity in mice exposed (ex situ) to hydrocarbon-contaminated soils removed from a fuel-loading depot and decommissioned gas works was significantly enhanced (4- and 2-fold respectively). However, this increase in EROD activity did not appear to correlate with either soil or carcass PAH concentrations. Conclusions and Outlook These results support the assumption that B[a]P has a higher affinity for the aryl hydrocarbon receptor (AhR) compared to BA. Soil parameters such as organic carbon content, structure and particle size distribution can modulate the bioavailability of contaminants to biological receptors. These factors are implicated in the lack of responsiveness demonstrated in the spiked soil experiments. However the responsiveness of EROD activity in mice exposed (ex situ) to soil contaminated with complex mixtures of hydrocarbon compounds confirms the potential usefulness of this model to determine the presence of ‘biologically active’ compounds in aged soils removed from contaminated sites.