Organohalogens and their hydroxylated metabolites in the blood of pigs from an open waste dumping site in south India: association with hepatic cytochrome P450.

The concentrations of polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers (PBDEs), and their hydroxylated metabolites (OH-PCBs and OH-PBDEs) were measured in the blood of Eurasian wild pigs (Sus scrofa) from a municipal waste open dumping site (DS) and a reference site (RS) in South India. We showed that contamination with OH-PCBs was higher in female pigs from the DS than in all other adult pigs. The highest OH-PCB concentrations were found in piglets from the DS. Moreover, the hepatic expression levels of CYP1A and CYP2B were higher in piglets than in their dam, implying metabolism of PCBs by cytochrome P450 (CYP) enzymes. The OH-PCB congener profiles differed according to sex and collection sites, possibly because of variations in the expression levels of phase I and phase II enzymes among individual pigs, differences in the exposure sources, and maternal transfer of parent PCBs. The hepatic CYP1A expression levels were positively correlated with the blood concentrations of 4OH-CB107, 4OH-CB162, and 4OH-CB187, implying CYP1A-dependent formation of these OH-PCBs in the pig liver. We found no significant correlations between the blood concentrations of OH-PCBs and thyroid hormones (THs); however, the thyroxin (T4) levels were lower in pigs from the DS than in pigs from the RS. Our limited dataset suggest that induced CYP enzymes accelerate the metabolism of xenobiotics and endogenous molecules in pigs. Thus, besides parental compounds, the risk of hydroxylated metabolites entering wildlife and humans living in and around municipal open waste dumping sites should be considered.

Effects of persistent organochlorine exposure on the liver transcriptome of the common minke whale (Balaenoptera acutorostrata) from the North Pacific.

Hepatic concentrations of persistent organochlorines (OCs) were determined in the common minke whale (Balaenoptera acutorostrata) from the North Pacific. To investigate the effects of OCs on the transcriptome in the minke whale, the present study constructed a hepatic oligo array of this species where 985 unique oligonucleotides were spotted and further analyzed the relationship between the OC levels and gene expression profiles of liver tissues. The stepwise multiple linear regression analysis identified 32 genes that correlated with hepatic OC levels. The mRNA expression levels of seven cytochrome P450 (CYP) genes, CYP1A1, 1A2, 2C78, 2E1, 3A72, 4A35, and 4V6 showed no clear correlations with the concentration of each OC, suggesting that the accumulated OCs in the liver did not reach levels that could alter CYP expression. Among the genes screened by the custom oligo array analysis, hepatic mRNA expression levels of 16 genes were further measured using quantitative real-time reverse transcription polymerase chain reaction. The mRNA levels of vitamin D-binding protein (DBP) were negatively correlated with non-ortho coplanar polychlorinated biphenyl (PCB) levels. Androgen receptor-associated coregulator 70 (ARA70) expression levels showed a significant positive correlation with concentrations of non-ortho coplanar PCB169. These correlations suggest that coplanar PCB-reduced DBP expression could suppress vitamin D receptor-mediated signaling cascades in peripheral tissues. Alternatively, the suppression of vitamin D receptor signaling cascade could be enhanced through competition with the androgen receptor signaling pathway for ARA70. In addition, a negative correlation between kynureninase and PCB169 levels was also observed, which suggest an enhanced accumulation of an endogenous aryl hydrocarbon receptor agonist, kynurenine in the minke whale population. Further studies are necessary to translate the changes in the transcriptome to toxicological outcomes including the disruption of the nervous and immune systems.

Toxicokinetics of dioxins and other organochlorine compounds in Japanese people: association with hepatic CYP1A2 expression levels.

Concentrations of persistent organochlorine compounds (OCs) including polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), and polychlorinated biphenyls (PCBs) in the liver and adipose tissue of Japanese cadavers were measured, and their toxicokinetics were examined in association with hepatic cytochrome P450 (CYP) 1A protein expression levels. Total 2,3,7,8-tetrachlorodibenzo-p-dioxin toxic equivalents (TEQs) were 66±74 and 65±57 pg/g lipid weight (mean±S.D.) in the liver and adipose tissue, respectively. Total PCBs (sum of 62 congeners targeted), p,p'-dichlorodiphenyl-dichloroethylene (p,p'-DDE) and ß-hexachlorocyclohexane (ß-HCH) were detected at concentrations over 1 µg/g lipid in both tissues of some specimens. For most of the dioxin-like congeners, total PCBs, p,p'-DDE, oxychlordane, α- and ß-HCH, hexachlorobenzene (HCB), and tris(4-chlorophenyl)methane (TCPMe), age-dependent increases in concentrations were found in the adipose tissue of males. No such age-dependent trend was observed in the liver, suggesting that there are different mechanisms underlying the hepatic concentrations of OCs. Immunoblot analyses indicated detectable expression of hepatic CYP1A2 protein, whereas no CYP1A1 protein was detected. The CYP1A2 expression levels were positively correlated with concentrations (on wet weight basis) of 2,3,4,7,8-P5CDF, the dominant TEQ-contributed congeners in the liver, indicating the induction of this CYP. Hepatic CYP1A2 protein levels were strongly correlated with the liver to adipose concentration (L/A) ratios of PCDD/F congeners with more than 5 chlorine atoms. Together with higher concentrations of the congeners in the liver than in the adipose tissue, the observation on L/A ratios of highly chlorinated PCDD/Fs suggests that induced hepatic CYP1A2 protein is involved in their sequestration in this human population, as observed in model animals (rodents). Nonetheless, the magnitude of hepatic sequestration (L/A ratio) of PCDD/Fs in this human population was lower than in other mammals and birds, reported previously. This study emphasizes the fact that toxicokinetics of some OCs can be affected at least partly by CYP1A2 protein levels in humans. For the extrapolation of their toxicokinetics from model animals to humans, knowledge on the induction and sequestration potencies of CYP1A is necessary.

Accumulation of dioxins and induction of cytochrome P450 1A4/1A5 enzyme activities in common cormorants from Lake Biwa, Japan: temporal trends and validation of national regulation on dioxins emission.

To validate the outcome of the national regulation on dioxins emission implemented in 1999, this study investigated temporal trends of chlorinated dioxins and related compounds (DRCs) in liver of common cormorants (Phalacrocorax carbo) collected from Lake Biwa, Japan between 2001 and 2008, as a part of the "Survey on the State of Dioxins Accumulation in Wildlife" conducted by the Ministry of the Environment, Japan. We also measured a biomarker of DRCs exposure, the cytochrome P450 1A (CYP1A)-dependent O-dealkylation activity of alkoxyresorufins (AROD), including methoxy-, ethoxy-, pentoxy- and benzyloxy-resorufins in the samples over 2001-2007. Neither TEQ nor AROD activity showed any clear declining trend over the time period, although the emission of DRCs during the corresponding period was estimated to be apparently decreasing. Our data indicate that the concentration of recalcitrant DRCs in the cormorant during 2001-2008 was scarcely affected by the national regulation on dioxins emission.

Dioxin-like and perfluorinated compounds in pigs in an Indian open waste dumping site: toxicokinetics and effects on hepatic cytochrome P450 and blood plasma hormones.

Dioxins and related compounds (DRCs) and perfluorinated compounds were measured in the livers of pigs (Sus scrofa) collected from an open waste dumping site in South India. Hepatic concentrations of DRCs and perfluorooctanesulfonate (PFOS; up to 200 ng/g wet wt) were significantly higher in male and female pigs, respectively, collected from the dumping site than in those from a reference site. Results suggest that dumping sites are a source of DRCs and PFOS. Hepatic concentrations of DRCs in piglets were higher than in mothers, especially for the congeners with molecular weights in the range of 360 to 400, implying congener-specific maternal transfer of DRCs in swine. Concentrations of polychlorinated dibenzo-p-dioxins and dibenzofurans and some non-ortho dioxin-like polychlorinated biphenyls (PCBs) in the liver of pigs were higher than those in the adipose fat and muscle of the same specimens. In addition, the liver-to-adipose concentration ratios for each congener had a significant positive correlation with the levels of hepatic cytochrome P450 (CYP)1A-like protein, suggesting congener-specific and CYP1A-dependent hepatic sequestration of DRCs in the swine. Total hepatic 2,3,7,8-tetrachlorodibenzo-p-dioxin toxic equivalents (TEQs; 8.9-350 pg/g fat wt) had a significant positive correlation with CYP1A-like protein expression (r=0.56, p=0.012), suggesting the induction of CYP1A by DRCs. However, the total TEQs had a significant negative correlation with CYP4A-like protein (r=-0.49, p=0.029), suggesting repression of peroxisome proliferator-activated receptor-alpha (PPARalpha)-mediated signaling pathway by DRCs. Decreases in plasma total thyroxine (T4), free T4, and immunoglobulin (Ig) G were also found in pigs from the dumping site compared with those from the reference site. This study provides insight into the toxicological impacts of DRCs and perfluorinated compounds in wild animals from open waste dumping sites.

Effects of in ovo exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin on hepatic AHR/ARNT-CYP1A signaling pathways in common cormorants (Phalacrocorax carbo).

Our previous studies have isolated multiple isoforms of aryl hydrocarbon receptors (AHRs) and AHR nuclear translocators (ARNTs) in avian species. However, roles of such genes on cytochrome P450 1A (CYP1A) expression are not fully understood. To investigate the effects of dioxins on the hepatic expression profiles of AHR1, AHR2, ARNT1 and ARNT2 in avian species, and whether the expression levels of AHRs and ARNTs affect the transcriptions of CYP1A4 and CYP1A5 genes, the eggs of common (great) cormorants (Phalacrocorax carbo) collected from Lake Biwa, Japan, were in ovo administrated with 0, 1500 and 4500pg/g egg of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and the hepatic expression levels of AHRs, ARNTs and CYP1As in embryos were monitored with two-step real-time RT-PCR. In young and adult cormorants collected from the same location, the hepatic expressions of these genes were also measured to understand the effects of growth stage. The residue levels of TCDD and other chlorinated dioxin-like congeners (DLCs) in the body of cormorants were quantified with high-resolution gas chromatography equipped with mass-spectrometry. There was no observable effect of in ovo TCDD treatment even at the highest dosage on mortality, body weight and morphology of the liver, heart, spleen, kidney and lung in the embryos. The mRNA expression of ARNT2 was slightly suppressed by the treatment with TCDD, while no alteration was observed for the expression of AHR1, AHR2 and ARNT1. Expressions of CYP1A4 and CYP1A5 were dose-dependently enhanced by TCDD, but CYP1A4 mRNA level increased more prominently than CYP1A5, indicating the difference in induction efficiency between the CYP1A isozymes. Comparison of hepatic mRNA levels of these genes among embryonic, young and adult cormorants revealed that young and adult cormorants had greater CYP1A5 expression levels than embryos, independently of the accumulation levels of DLCs. These results suggest that the hepatic induction of each CYP1A by DLCs in cormorants occurs in an isoform-specific manner and CYP1A5 expression, at least partially, depends on the factors related to the growth of cormorants, but the transcriptional processes of CYP1As are not related to the expression levels of AHRs and ARNTs. This study yielded results supporting our previous observations that in reality, high accumulation of DLCs induces hepatic CYP1A4 and 1A5 expressions in the wild cormorant population.

Effects of co-exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin and perfluorooctane sulfonate or perfluorooctanoic acid on expression of cytochrome P450 isoforms in chicken (Gallus gallus) embryo hepatocyte cultures.

In this study we investigated the effect of a single-compound exposure or two compound co-exposure to tetrachlorodibenzo-p-dioxin (TCDD) plus perfluorooctane sulfonate (PFOS) or perfluorooctanoic acid (PFOA) on the mRNA expression of cytochromes P450 (CYP) 1A4, 4V2 and 3A37, ethoxyresorufin-O-deethylase (EROD) activity and cell viability in chicken (Gallus gallus domesticus) embryo primary hepatocyte cultures. Cell viability after 24 h of incubation was significantly decreased in cells exposed to PFOS at concentrations between 30 microM and 60 microM with or without co-exposure to TCDD (0.3 nM at maximum). PFOA did not decrease cell viability even at maximum concentrations of 60 microM. TCDD induced CYP1A4 mRNA and EROD activity substantially as reported previously. PFOS also increased CYP1A4 mRNA in a concentration-dependent manner. Co-exposure of cells to PFOS plus TCDD did not change CYP1A4 mRNA levels compared to cells treated with TCDD alone. PFOS alone did not induce CYP4V2 mRNA, however 40-50 microM PFOS plus TCDD (0.3 nM) induced CYP4V2 mRNA compared to TCDD alone (P<0.05). This trend was similar to that observed with co-exposure to TCDD plus PFOA, suggesting that PFOA alone did not induce CYP4V2 mRNA, whereas co-exposure to TCDD plus PFOA induced the expression levels. PFOS alone decreased CYP3A37 mRNA by a maximum of 45%, however after co-exposure to TCDD, recovery of mRNA expression to levels measured in DMSO-treated cells was observed. Our data suggest a complex gene response to mixtures of dioxin-like and perfluorinated compounds.

Analysis of biomagnification of persistent organic pollutants in the aquatic food web of the Mekong Delta, South Vietnam using stable carbon and nitrogen isotopes.

The present study elucidated the biomagnification profiles of persistent organic pollutants (POPs) through a tropical aquatic food web of Vietnam based on trophic characterization using stable nitrogen analysis. Various biological samples collected from the main stream of the Mekong Delta were provided for the analysis for both POPs, and stable nitrogen and carbon isotope ratios. Of the POPs analyzed, dichlorodiphenyltrichloroethane and its metabolites (DDTs) were the predominant contaminants with concentrations ranging from 0.058 to 12 ng/g wet weight, followed by polychlorinated biphenyls (PCBs) at 0.017-8.9 ng/g, chlordane compounds (CHLs) at 0.0043-0.76 ng/g, tris-4-chlorophenyl methane (TCPMe) at N.D.-0.26 ng/g, hexachlorocyclohexane isomers (HCHs) at N.D.-0.20 ng/g and hexachlorobenzene (HCB) at 0.0021-0.096 ng/g. Significant positive increases of concentrations in DDTs, CHLs, and TCPMe against the stable nitrogen ratio (delta(15)N) were detected, while, concentrations of HCHs and HCB showed no significant increase. The slopes of the regression equations between the log-transformed concentrations of these POPs and delta(15)N were used as indices of biomagnification. The slopes of the POPs for which positive biomagnification was detected ranged from 0.149 to 0.177 on a wet weight basis. The slopes of DDTs and CHLs were less than those reported for a marine food web of the Arctic Ocean, indicating that less biomagnification had occurred in the tropical food web. Of the isomers of CHLs, unlike the studies of the Arctic Ocean, oxychlordane did not undergo significant biomagnification through the food web of the Mekong Delta. This difference is considered to be due to a lack of marine mammals, which might metabolize cis- and trans-chlordane to oxychlordane, in the Mekong Delta ecosystem. The biomagnification profile of TCPMe is reported for the first time in the present study.

Identification and hepatic expression profiles of cytochrome P450 1-4 isozymes in common minke whales (Balaenoptera acutorostrata).

Full-length cDNA sequences of cytochrome P450 (CYP) 2C78, 2E1, 3A72, 4A35 and 4V6 isozymes were isolated from a hepatic cDNA library of common minke whale (Balaenoptera acutorostrata). The deduced amino acid sequences of minke whale CYP2C78, 2E1, 3A72, 4A35 and 4V6 showed high identities with cattle CYP2C86 (83%), pig CYP2E1 (85%), sheep CYP3A24 (82%), pig CYP4A21 (80%), and human CYP4V2 (76%), respectively. To investigate whether or not these CYP expression levels are altered by contamination of organochlorine contaminants (OCs), mRNA levels of these CYPs in the liver of common minke whale were measured using a quantitative real-time RT-PCR method, and the quantified mRNA levels were employed for the statistical analysis with the residue levels of OCs including PCBs, DDTs (p,p'-DDT, p,p'-DDD and p,p'-DDE), chlordanes (cis-chlordane, trans-chlordane, cis-nonachlor, trans-nonachlor and oxychlordane), HCHs (alpha-, beta- and gamma-isomers) and hexachlorobenzene that have already been reported elsewhere. Spearman's rank correlation analyses showed no significant correlation between CYP expression levels and each OC level in the common minke whale liver, implying that these environmental chemicals have no potential to alter the expression levels of these CYPs or the residue levels encountered in the whale livers may not reach their transcriptional regulation levels. This suggests that the expression of individual CYPs in the whale liver may be at basal level. Relationships among hepatic mRNA expression levels of these CYP2-4 isozymes together with CYP1A1 and CYP1A2 were also examined. Significant positive correlations were detected among mRNA expression levels of individual CYP isozymes in most cases. These associations indicate that the transcriptional regulation of these CYPs examined in this study may be reciprocally related. CYP1A1 levels showed a positive correlation with CYP1A2 levels (r=0.64, p<0.01) indicating that both CYP isozymes were regulated by aryl hydrocarbon receptor activated by endogenous ligands. A strong positive correlation between CYP2C78 and 3A72 (r=0.90, p<0.001) suggests that expression of these CYP isozymes may be under a regulation mechanism of cross-talk in which specific nuclear receptors such as constitutive androstane receptor and pregnane X receptor are involved. The present study indicates that minke whale from the North Pacific may be a model species to investigate the mechanism of basal regulation of these CYPs.

PCDDs, PCDFs, and coplanar PCBs in wild terrestrial mammals from Japan: congener specific accumulation and hepatic sequestration.

The present study determined the contamination levels and congener-specific accumulation features of dioxins and related compounds (DRCs) in wild terrestrial mammals such as large Japanese field mice (LJFM), lesser Japanese moles (LJMs), and raccoon dogs (RDs) collected from Kanto region in Japan during 2001. The toxic equivalent quantity (TEQ) levels in the carcasses or adipose tissues were in the order of RDs > or = LJMs > LJFM. Comparison of DRC congener profiles in the three species and principal component analysis (PCA) demonstrated a higher contribution of OCDD, T4CB77, and P5CB118 in LJMs. Analysis of liver-adipose distribution of DRC congeners in RDs showed that livers contained significantly higher TEQs than adipose tissues, indicating that liver is a depository organ and critical for determining the toxicokinetics of DRCs. As for most T4, P5, H6CDD/DFs and for P5CB126, H6CB169 and mono-ortho PCB congeners, their liver/adipose concentration ratios in RDs revealed a tendency to increase with hepatic TEQ levels, suggesting TEQ-dependent hepatic sequestration.

Induction of cytochrome P450 1A5 mRNA, protein and enzymatic activities by dioxin-like compounds, and congener-specific metabolism and sequestration in the liver of wild jungle crow (Corvus macrorhynchos) from Tokyo, Japan.

This study presents concentrations of polychlorinated dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs), and dioxin-like coplanar PCBs (Co-PCBs) in the liver and breast muscle of jungle crows (JCs; Corvus macrorhynchos) collected from Tokyo, Japan. 2,3,7,8-Tetrachlorodibenzo-p-dioxin toxic equivalents (TEQs) derived by WHO bird-TEF were in the range of 23 to 280 pg/g (lipid) in the liver, which are lower or comparable to the lowest-observed-effect-level of CYP induction in chicken, and 5.6-78 pg/g (lipid) in the pectoral muscle. Cytochrome P450 (CYP) 1A-, 2B-, 2C-, and 3A-like proteins were detected using anti-rat CYP polyclonal antibodies in hepatic microsomal fractions. Significant (p < 0.05) positive correlations between hepatic TEQs and CYP1A or CYP3A-like protein expression levels were noticed, implying induction of these CYP isozymes by TEQs. On the other hand, there was no significant positive correlation between muscle TEQ and any one of analyzed CYP isozyme expression levels. CYP1A- and CYP3A-like protein expression levels represented better correlations with pentoxy- and benzyloxyresorufin-O-dealkylase activities rather than methoxy- and ethoxyresorufin-O-dealkylase activities, indicating unique catalytic functions of these CYPs in JCs. Furthermore, we succeeded in isolating CYP1A5 cDNA from the liver of JC, having an open reading frame of 531 amino acid residues with a predicted molecular mass of 60.3 kDa. JC CYP1A5 mRNA expression measured by real-time RT-PCR had a significant positive correlation with hepatic TEQs, suggesting induction of CYP1A5 at the transcriptional level. Ratios of several Co-PCB congeners to CB-169 in the liver of JCs revealed significant negative correlations with CYP1A protein or CYP1A5 mRNA expression levels, implying metabolism of these congeners by the induced CYP1A. The liver/breast muscle concentration (L/M) ratios of PCDDs/DFs and CB-169 increased with an increase in hepatic CYP1A protein or CYP1A5 mRNA expression levels, suggesting congener-specific hepatic sequestrations by the induced CYP1A. The present study provides insights into the propensity of CYP1A induction to the exposure of dioxin-like chemicals, and unique metabolic and sequestration capacities of CYP1A in JC.

Molecular cloning and mRNA expression of cytochrome P4501A1 and 1A2 in the liver of common minke whales (Balaenoptera acutorostrata).

This study presents full-length cDNA sequences of CYP1A1 and 1A2, in common minke whale (Balaenoptera acutorostrata) from the North Pacific. Both CYP1A1 and CYP1A2 cDNAs had an open reading frame of 516 amino acid residues, and predicted molecular masses were 58.3 kDa and 58.1 kDa, respectively. The deduced full-length amino acid sequence of CYP1A1 revealed higher identities with those of sheep (86%) and pig (87%), and that of CYP1A2 was most closely related to human (82%) and monkey CYP1A2 (82%) among species from which CYP1A2 has been isolated so far. Differences in certain conserved and functional amino acid residues of CYP1A1 and 1A2 between common minke whale and other mammalian species indicate the possibility of their specific metabolic function. Concentrations of organochlorine compounds (OCs) including PCBs and DDTs analyzed in common minke whale liver showed no significant correlation with hepatic mRNA expression levels of CYP1A1 and CYP1A2, indicating no induction of these enzymes by such OCs.

Bioaccumulation of organochlorines in crows from an indian open waste dumping site: evidence for direct transfer of dioxin-like congeners from the contaminated soil.

To assess the significance of waste dumping sites as a source of chemical contamination to ecosystems, we analyzed the residue levels of polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), polychlorinated biphenyls (PCBs), and other organochlorines in the breast muscle of crows from a dumping site in the south of Chennai city, South India. Crows from the dumping site contained significantly higher total TEQs (60 +/- 27 pg/g lipid wt) than those from the reference sites (26 +/- 18 pg/g lipid wt). Especially, certain dioxin-like coplanar PCB congeners (Co-PCBs), such as CB-77 and CB-105, whose source is commercial PCBs,were significantly higher in crows from the dumping site than those from the reference sites. Profiles of PCDDs/DFs and Co-PCBs in crows from the dumping site were similar to those of soil at the same site, which was confirmed by principal component analysis. Furthermore, significant positive correlations were obtained between the congener-specific bioconcentration factors (BCFs) of PCDDs/DFs estimated from concentrations in crows and soil from the dumping site and the theoretical BCFs calculated from water-particle and lipid-water partitioning coefficients. On the other hand, the estimated BCFs had significant negative correlations with the molecular weight of PCDDs/DFs, indicating that molecular size limits their bioaccumulation. These results suggest that dioxin-like congeners in the soil of the dumping site were transferred directly to the crows through the ingestion of on-site garbage contaminated with soil, rather than through trophic transfer in the ecosystem. The present study provides insight into the ecological impacts of dumping sites.