Search

The most abundant polychlorinated biphenyl (PCB) congeners found in the environment and in humans are neurotoxic. This is of particular concern for early life stages because the exposure of the more vulnerable developing nervous system to neurotoxic chemicals can result in neurobehavioral disorders. In this study, we uncover currently unknown links between PCB target mechanisms and neurobehavioral deficits using zebrafish as a vertebrate model. We investigated the effects of the abundant non-dioxin-like (NDL) congener PCB153 on neuronal morphology and synaptic transmission linked to the proper execution of a sensorimotor response. Zebrafish that were exposed during development to concentrations similar to those found in human cord blood and PCB contaminated sites showed a delay in startle response. Morphological and biochemical data demonstrate that even though PCB153-induced swelling of afferent sensory neurons, the disruption of dopaminergic and GABAergic signaling appears to contribute to PCB-induced motor deficits. A similar delay was observed for other NDL congeners but not for the potent dioxin-like congener PCB126. The effects on important and broadly conserved signaling mechanisms in vertebrates suggest that NDL PCBs may contribute to neurodevelopmental abnormalities in humans and increased selection pressures in vertebrate wildlife., (© 2021. The Author(s).)

Previous reports suggested that non-dioxin-like (NDL) PCB153 effects on cytochrome P450 3A (Cyp3a) expression in Atlantic killifish (Fundulus heteroclitus) gills differed between F0 generation fish from a PCB site (New Bedford Harbor; NBH) and a reference site (Scorton Creek; SC). Here, we examined effects of PCB153, dioxin-like (DL) PCB126, or a mixture of both, on Cyp3a56 mRNA in killifish generations removed from the wild, without environmental PCB exposures. PCB126 effects in liver and gills differed between populations, as expected. Gill Cyp3a56 was not affected by either congener in NBH F2 generation fish, but was induced by PCB153 in SC F1 fish, with females showing a greater response. PCB153 did not affect Cyp3a56 in liver of either population. Results suggest a heritable resistance to NDL-PCBs in killifish from NBH, in addition to that reported for DL PCBs. Induction of Cyp3a56 in gills may be a biomarker of exposure to NDL PCBs in fish populations that are not resistant to PCBs., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Atlantic killifish (Fundulus heteroclitus) thrive in New Bedford Harbor (NBH), MA, highly contaminated with polychlorinated biphenyls (PCBs). Resident killifish have evolved tolerance to dioxin-like (DL) PCBs, whose toxic effects through the aryl hydrocarbon receptor (AhR) are well studied. In NBH, non-dioxin like PCBs (NDL PCBs), which lack activity toward the AhR, vastly exceed levels of DL congeners yet how killifish counter NDL toxic effects has not been explored. In mammals and fish, NDL PCBs are potent activators of ryanodine receptors (RyR), Ca(2+) release channels necessary for a vast array of physiological processes. In the current study we compared the expression and function of RyR related pathways in NBH killifish with killifish from the reference site at Scorton Creek (SC, MA). Relative to the SC fish, adults from NBH displayed increased levels of skeletal muscle RyR1 protein, and increased levels of FK506-binding protein 12 kDa (FKBP12) an accessory protein essential for NDL PCB-triggered changes in RyR channel function. In accordance with increased RyR1 levels, NBH killifish displayed increased maximal ligand binding, increased maximal response to Ca(2+) activation and increased maximal response to activation by the NDL PCB congener PCB 95. Compared to SC, NBH embryos and larvae had increased levels of mtor and ryr2 transcripts at multiple stages of development, and generations, while levels of serca2 were decreased at 9 days post-fertilization in the F1 and F2 generations. These findings suggest that there are compensatory and heritable changes in RyR mediated Ca(2+) signaling proteins or potential signaling partners in NBH killifish., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Killifish survive and reproduce in the New Bedford Harbor (NBH) in Massachusetts (MA), USA, a site severely contaminated with polychlorinated biphenyls (PCBs) for decades. Levels of 22 different PCB congeners were analyzed in liver from killifish collected in 2008. Concentrations of dioxin-like PCBs in liver of NBH killifish were ∼400 times higher, and the levels of non-dioxin-like PCBs ∼3000 times higher than in killifish from a reference site, Scorton Creek (SC), MA. The NBH killifish are known to be resistant to the toxicity of dioxin-like compounds and to have a reduced aryl hydrocarbon receptor (AhR) signaling response. Little is known about the responses of these fish to non-dioxin-like PCBs, which are at extraordinarily high levels in NBH fish. In mammals, some non-dioxin-like PCB congeners act through nuclear receptor 1I2, the pregnane-X-receptor (PXR). To explore this pathway in killifish, a PXR cDNA was sequenced and its molecular phylogenetic relationship to other vertebrate PXRs was determined. Killifish were also collected in 2009 from NBH and SC, and after four months in the laboratory they were injected with a single dose of either the dioxin-like PCB 126 (an AhR agonist) or the non-dioxin-like PCB 153 (a mammalian PXR agonist). Gills and liver were sampled three days after injection and transcript levels of genes encoding PXR, cytochrome P450 3A (CYP3A), P-glycoprotein (Pgp), AhR2 and cytochrome P450 1A (CYP1A) were measured by quantitative PCR. As expected, there was little effect of PCB exposure on mRNA expression of AhR2 or CYP1A in liver and gills of NBH fish. In NBH fish, but not in SC fish, there was increased mRNA expression of hepatic PXR, CYP3A and Pgp upon exposure to either of the two PCB congeners. However, basal PXR and Pgp mRNA levels in liver of NBH fish were significantly lower than in SC fish. A different pattern was seen in gills, where there were no differences in basal mRNA expression of these genes between the two populations. In SC fish, but not in NBH fish, there was increased mRNA expression of branchial PXR and CYP3A upon exposure to PCB126 and of CYP3A upon exposure to PCB153. The results suggest a difference between the two populations in non-AhR transcription factor signaling in liver and gills, and that this could involve killifish PXR. It also implies possible cross-regulatory interactions between that factor (presumably PXR) and AhR2 in liver of these fish., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The teleost swim bladder is assumed a homolog of the tetrapod lung. Both swim bladder and lung are developmental targets of persistent aryl hydrocarbon receptor (AHR(2)) agonists; in zebrafish (Danio rerio) the swim bladder fails to inflate with exposure to 3,3',4,4',5-pentachlorobiphenyl (PCB126). The mechanism for this effect is unknown, but studies have suggested roles of cytochrome P450 1 (CYP1) and cyclooxygenase 2 (Cox-2) in some Ahr-mediated developmental effects in zebrafish. We determined relationships between swim bladder inflation and CYP1 and Cox-2 mRNA expression in PCB126-exposed zebrafish embryos. We also examined effects on β-catenin dependent transcription, histological effects, and Ahr2 dependence of the effect of PCB126 on swim bladder using morpholinos targeting ahr2. One-day-old embryos were exposed to waterborne PCB126 or carrier (DMSO) for 24h and then held in clean water until day 4, a normal time for swim bladder inflation. The effects of PCB126 were concentration-dependent with EC(50) values of 1.4 to 2.0 nM for induction of the CYP1s, 3.7 and 5.1 nM (or higher) for cox-2a and cox-2b induction, and 2.5 nM for inhibition of swim bladder inflation. Histological defects included a compaction of the developing bladder. Ahr2-morpholino treatment rescued the effect of PCB126 (5 nM) on swim bladder inflation and blocked induction of CYP1A, cox-2a, and cox-2b. With 2nM PCB126 approximately 30% of eleutheroembryos(3) failed to inflate the swim bladder, but there was no difference in CYP1 or cox-2 mRNA expression between those embryos and embryos showing inflated swim bladder. Our results indicate that PCB126 blocks swim bladder inflation via an Ahr2-mediated mechanism. This mechanism seems independent of CYP1 or cox-2 mRNA induction but may involve abnormal development of swim bladder cells., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The Xenopus tropicalis genome shows a single gene in each of the four cytochrome P450 1 (CYP1) subfamilies that occur in vertebrates, designated as CYP1A, CYP1B1, CYP1C1, and CYP1D1. We cloned the cDNAs of these genes and examined their expression in untreated tadpoles and in tadpoles exposed to waterborne aryl hydrocarbon receptor agonists, 3,3',4,4',5-pentachlorobiphenyl (PCB126), β-naphthoflavone (βNF), or indigo. We also examined the effects of PCB126 on expression of genes involved in stress response, cell proliferation, thyroid homeostasis, and prostaglandin synthesis. PCB126 induced CYP1A, CYP1B1, and CYP1C1 but had little effect on CYP1D1 (77-, 1.7-, 4.6- and 1.4-fold induction versus the control, respectively). βNF induced CYP1A and CYP1C1 (26- and 2.5-fold), while, under conditions used, indigo tended to induce only CYP1A (1.9-fold). The extent of CYP1 induction by PCB126 and βNF was positively correlated to the number of putative dioxin response elements 0-20 kb upstream of the start codons. No morphological effect was observed in tadpoles exposed to 1 nM-10 μM PCB126 at two days post-fertilization (dpf) and screened 20 days later. However, in 14-dpf tadpoles a slight up-regulation of the genes for PCNA, transthyretin, HSC70, Cu-Zn SOD, and Cox-2 was observed two days after exposure to 1 μM PCB126. This study of the full suite of CYP1 genes in an amphibian species reveals gene- and AHR agonist-specific differences in response, as well as a much lower sensitivity to CYP1 induction and short-term toxicity by PCB126 compared with in fish larvae. The single genes in each CYP1 subfamily may make X. tropicalis a useful model for mechanistic studies of CYP1 functions., (© 2010 Elsevier Inc. All rights reserved.)

Knowledge of the complement of cytochrome P450 (CYP) genes is essential to understanding detoxification and bioactivation mechanisms for organic contaminants. We cloned three new CYP1 genes, CYP1B1, CYP1C2 and CYP1D1, from the killifish Fundulus heteroclitus, an important model in environmental toxicology. Expression of the new CYP1s along with previously known CYP1A and CYP1C1 was measured by qPCR in eight different organs. Organ distribution was similar for the two CYP1Cs, but otherwise patterns and extent of expression differed among the genes. The AHR agonist 3,3',4,4',5-pentachlorobiphenyl (PCB126) (31 pmol/g fish) induced expression of CYP1A and CYP1B1 in all organs examined, while CYP1C1 was induced in all organs except testis. The largest changes in response to PCB126 were induction of CYP1A in testis (approximately 700-fold) and induction of CYP1C1 in liver (approximately 500-fold). CYP1B1 in liver and gut, CYP1A in brain and CYP1C1 in gill also were induced strongly by PCB126 (> 100-fold). CYP1C1 expression levels were higher than CYP1C2 in almost all tissues and CYP1C2 was much less responsive to PCB126. In contrast to the other genes, CYP1D1 was not induced by PCB126 in any of the organs. The organ-specific response of CYP1s to PCB126 implies differential involvement in effects of halogenated aromatic hydrocarbons in different organs. The suite of inducible CYP1s could enhance the use of F. heteroclitus in assessing aquatic contamination by AHR agonists. Determining basal and induced levels of protein and the substrate specificity for all five CYP1s will be necessary to better understand their roles in chemical effects and physiology.

Enzymes in the cytochrome P450 1 family oxidize many common environmental toxicants. We identified a new CYP1, termed CYP1D1, in zebrafish. Phylogenetically, CYP1D1 is paralogous to CYP1A and the two share 45% amino acid identity and similar gene structure. In adult zebrafish, CYP1D1 is most highly expressed in liver and is relatively highly expressed in brain. CYP1D1 transcript levels were higher at 9h post-fertilization than at later developmental times. Treatment of zebrafish with potent aryl hydrocarbon receptor (AHR) agonists (3,3',4,4',5-pentachlorobiphenyl or 2,3,7,8-tetrachlorodibenzo-p-dioxin) did not induce CYP1D1 transcript expression. Morpholino oligonucleotide knockdown of AHR2, which mediates induction of other CYP1s, did not affect CYP1D1 expression. Zebrafish CYP1D1 heterologously expressed in yeast exhibited ethoxyresorufin- and methoxyresorufin-O-dealkylase activities. Antibodies against a CYP1D1 peptide specifically detected a single electrophoretically-resolved protein band in zebrafish liver microsomes, distinct from CYP1A. CYP1D1 in zebrafish is a CYP1A-like gene that could have metabolic functions targeting endogenous compounds.

Persistent organic pollutants (POPs) bioaccumulate in blubber of marine mammals. Therefore, it is important to understand the structure and dynamics of blubber layers and how they affect the accumulation of POPs and subsequent biochemical responses. We used established histological and immunohistochemical methods to document the structure of bottlenose dolphin (Tursiops truncatus) blubber and to assess the expression of cytochrome P4501A1 (CYP1A1) in skin-blubber biopsies of dolphins sampled in the waters off Charleston, SC (CHS) (N=38), and Indian River Lagoon, FL (IRL) (N=36). CYP1A1 expression was strongest and most frequent in capillary endothelial cells and was stratified in blubber; the greatest CYP1A1 staining was in the deepest layer. CYP1A1 expression in deep blubber and 2,3,7,8-TCDD Toxic Equivalents measured in the entire blubber were significantly higher in dolphins from CHS as compared to those from IRL. Adipocyte size was associated with the extent of CYP1A1 expression. Male dolphins with smaller adipocytes from CHS and IRL had higher levels of CYP1A1 expression in deep blubber. In CHS females, CYP1A1 expression in vascular endothelial cells varied with reproductive status. CYP1A1 expression in the deep layer was highest in simultaneously pregnant-lactating dolphins, and these dolphins had the smallest adipocytes in deep blubber. In all dolphins, CYP1A1 expression in the deep blubber layer was positively related to concentrations of hydroxylated PCBs (OH-PCBs) in plasma. In summary, redistribution of AHR agonists from blubber into the circulatory system may enhance PCB metabolism and production of OH-PCBs by induction of CYP1A1 in hepatocytes and, possibly, by induction of CYP1A1 in endothelial cells of the deep blubber. The OH-PCBs thus formed have the potential to interfere with thyroid hormone homeostasis.

Halogenated agonists for the aryl hydrocarbon receptor (AHR), such as 3,3',4,4',5-pentachlorobiphenyl (PCB126) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), cause developmental toxicity in fish. AHR dependence of these effects is known for TCDD but only presumed for PCB126, and the AHR-regulated genes involved are known only in part. We defined the role of AHR in regulation of four cytochrome P450 1 (CYP1) genes and the effect of PCB126 on cell cycle genes (i.e., PCNA and cyclin E) in zebra fish (Danio rerio) embryos. Basal and PCB126-induced expression of CYP1A, CYP1B1, CYP1C1, and CYP1C2 was examined over time as well as in relation to cell cycle gene expression and morphological effects of PCB126 in developing zebra fish. The four CYP1 genes differed in the time for maximal basal and induced expression, i.e., CYP1B1 peaked within 2 days postfertilization (dpf), the CYP1Cs around hatching (3 dpf), and CYP1A after hatching (14-21 dpf). These results indicate developmental periods when the CYP1s may play physiological roles. PCB126 (0.3-100nM) caused concentration-dependent CYP1 gene induction (EC50: 1.4-2.7nM, Lowest observed effect concentration [LOEC]: 0.3-1nM) and pericardial edema (EC50: 4.4nM, LOEC: 3nM) in 3-dpf embryos. Blockage of AHR2 translation significantly inhibited these effects of PCB126 and TCDD. PCNA gene expression was reduced by PCB126 in a concentration-dependent manner, suggesting that PCB126 could suppress cell proliferation. Our results indicate that the four CYP1 genes examined are regulated by AHR2 and that the effect of PCB126 on morphology in zebra fish embryos is AHR2 dependent. Moreover, the developmental patterns of expression and induction suggest that CYP1 enzymes could function in normal development and in developmental toxicity of PCB126 in fish embryos.

The cytochrome P4501C (CYP1C) gene subfamily was recently discovered in fish, and zebrafish (Danio rerio) CYP1C1 transcript has been cloned. Here we cloned the paralogous CYP1C2, showing that the amino acid sequence is 78% identical to CYP1C1, and examined gene structure and expression of CYP1A, CYP1B1, CYP1C1, and CYP1C2. Xenobiotic response elements were observed upstream of the coding regions in all four genes. Zebrafish adults and embryos were exposed (24 h) to 100 nM 3,3',4,4',5-polychlorinated biphenyl (PCB126) or 20 ppm acetone and subsequently held in clean water for 24 h (adults) or 48 h (embryos). All adult organs examined (eye, gill, heart, liver, kidney, brain, gut, and gonads) and embryos showed basal expression of the four genes. CYP1A was most strongly expressed in liver, whereas CYP1B1, CYP1C1, and CYP1C2 were most strongly expressed in heart and eye. CYP1B1 and the CYP1C genes showed an expression pattern similar to one another and to mammalian CYP1B1. In embryos CYP1C1 and CYP1C2 tended to have a higher basal expression than CYP1A and CYP1B1. PCB126 induced CYP1A in all organs, and CYP1B1 and CYP1C1 in all organs except gonads, or gonads and brain, respectively. CYP1C2 induction was significant only in the liver. However, in embryos all four genes were induced strongly by PCB126. The results are consistent with CYP1C1 and CYP1C2, as well as CYP1A and CYP1B1, being regulated by the aryl hydrocarbon receptor. While CYP1A may have a protective role against AHR agonists in liver and gut, CYP1B1, CYP1C1, and CYP1C2 may also play endogenous roles in eye and heart and possibly other organs, as well as during development.

Integument biopsy is a nondestructive method for sampling free-ranging cetaceans, which allows for the determination of both contaminant concentrations and biomarker responses. Cytochrome P450 1A1 (CYP1A1) expression is induced by polycyclic aromatic hydrocarbons and planar halogenated aromatic hydrocarbons such as the non-ortho and mono-ortho polychlorinated biphenyls (PCBs). CYP1A induction has been used extensively as a biomarker of exposure to such compounds in vertebrates. We measured PCB concentrations and CYP1A1 expression in integument biopsies from bottlenose dolphins (Tursiops truncatus) resident in Sarasota Bay, FL. This population of dolphins has been the subject of long-term population and health assessment, affording the opportunity to evaluate the influence of age, sex, and reproductive status on CYP1A1 expression. CYP1A1 expression was seen in endothelial cells, vascular smooth muscle, and nerve cells in the dermis, similar to what has been observed in other cetacean species. Endothelial CYP1A1 expression varied along the length of the biopsy, which could be related to differences in the structure and functionality of the blubber in different parts of the integument. Neither age nor sex was related to CYP1A1 expression in these biopsies, and reproductive status did not relate to levels of CYP1A1 in females. Total PCB and toxic equivalent quotient concentrations in blubber were positively correlated with dermal endothelial CYP1A1 expression, although Sigmamono-ortho PCBs concentrations did not show this relationship. Contaminant concentrations appear to be stronger determinants of CYP1A1 expression in integument of these dolphins, than are age, sex, or reproductive status.

The present study characterized cytochrome P4501A (CYP1A) isoforms from common cormorant (Phalacrocorax carbo) with regard to their evolutionary relationships and their roles in disposition of dioxin and related compounds (DRCs). Two clones isolated from a cormorant liver cDNA library were named CYP1A4 and CYP1A5 on the basis of greatest overall amino acid identity shared with chicken (Gallus gallus) CYP1A4 (78%) and CYP1A5 (78%), respectively. Spatial heterogeneity in phylogenetic signal along the sequences strongly indicated that cormorant CYP1A4 and CYP1A5 have undergone partial interparalog gene conversion, similar to chicken and mammalian CYP1As. Phylogenetic analysis of a putatively unconverted region produced a tree topology consistent with the orthology of avian CYP1A5s with mammalian CYP1A2s and avian CYP1A4s with mammalian CYP1A1s. Hepatic CYP1A4 and CYP1A5 mRNA levels in wild cormorants from Lake Biwa, Japan, were quantified to examine the effects of DRCs on isoform-specific expression and to evaluate the toxicokinetics of DRCs in which CYP1A expression is involved. Both CYP1A4 and CYP1A5 mRNA levels were positively correlated with total tetrachlorodibenzo-p-dioxin toxic equivalents and concentrations of each congener in most cases in the liver, suggesting the induction of both enzymes through a shared transcriptional mechanism. The lack of correlation of 2,3,7,8-tetrachlorodibenzofuran and 3,3',4,4'-tetrachlorobiphenyl (PCB77) to CYP1A gene expression is likely due to the rapid metabolism of these two congeners. Liver-to-muscle concentration ratios for most DRC congeners except PCB77 and mono-ortho coplanar polychlorinated biphenyls significantly increased with an elevation of CYP1A4 and CYP1A5 mRNA levels. The present data suggest that hepatic sequestration of some DRCs occurs in cormorant via binding to either CYP1A5 or both CYP1A4 and CYP1A5.

The non-ortho-polychlorinated biphenyl (PCB) congener 3,3'4,4'-tetrachlorobiphenyl (PCB 77) can uncouple the catalytic cycle of fish (scup) cytochrome P4501A (CYP1A) and mammalian (rat, human) CYP1A1, stimulating release of reactive oxygen species (ROS). PCB 77 also inactivates CYP1A in an NADPH-, oxygen-, and time-dependent process, linked to uncoupling. We addressed a hypothesis that planar halogenated hydrocarbons generally will uncouple CYP1A. Thus, additional PCB congeners including non-ortho-3,3',4,4',5'-pentachlorobiphenyl (PCB 126) and 3,3',4,4',5,5'-hexachlorobiphenyl (PCB 169), mono-ortho-2,3,3',4,4'-pentachlorobiphenyl (PCB 105) and di-ortho-2,2',5,5'-tetrachlorobiphenyl (PCB 52), as well as the polycyclic aromatic hydrocarbon benzo[a]pyrene (B[a]P), were examined for their ability to stimulate microsomal ROS production and to inactivate CYP1A. Incubated without NADPH, non-ortho-PCB 126 and -PCB 169 both inhibited microsomal CYP1A activity (ethoxyresorufin O-deethylase; EROD). When NADPH was included, these congeners caused a progressive inactivation of CYP1A, in addition to the inhibition. The determined K(Inact) values for inactivation were 0.14 and 0.08 microM, respectively, for PCB 126 and PCB 169, similar to the 0.05 microM for PCB 77 previously reported. The mono-ortho-PCB 105 weakly inhibited and weakly inactivated CYP1A. The di-ortho-PCB 52 neither inhibited nor inactivated CYP1A. Alone, B[a]P strongly inhibited CYP1A, but when NADPH was added that inhibition was reversed, apparently by metabolic depletion of the substrate, and there was no inactivation. PCB 126 and PCB 169 stimulated release of ROS from induced liver microsomes, while B[a]P, PCB 52 and PCB 105 did not. ROS release and CYP1A inactivation stimulated by the non-ortho-PCB 126 and PCB 169 indicate an uncoupling of CYP1A like that previously shown with PCB 77. The uncoupling and release of ROS further suggest a participation of CYP1A in the oxidative stress associated with some planar halogenated aryl hydrocarbon receptor agonists.

The planar polychlorinated biphenyl (PCB) 3,3',4,4'-tetrachlorobiphenyl (TCB) causes dose-dependent induction and post-transcriptional suppression of hepatic cytochrome P450 1A (CYP1A) in the marine teleost scup (Stenotomus chrysops). That suppression is linked to inhibition and oxidative inactivation of CYP1A by TCB. Other planar PCBs, including 3,3',4,4',5-pentachlorobiphenyl (PeCB), inactivate scup CYP1A in vitro leading us to hypothesize that PeCB also will suppress CYP1A in vivo. We examined induction and suppression of CYP1A by PeCB in scup, as related to oxidative stress. PeCB at a low dose (0.01 mg/kg) induced hepatic microsomal spectral P450 and CYP1A protein and catalytic activities (ethoxyresorufin o-deethylase (EROD) and methoxyresorufin o-demethylase (MROD)) over an 18 day period. A high dose (1 mg PeCB/kg) only minimally induced hepatic spectral P450 and CYP1A content, and EROD and MROD rates remained at control levels at all sampling times, while CYP1A mRNA expression was induced strongly (up to 35-fold) at both doses. High dose PeCB had minimal effects on content of P450A (a CYP3A protein), P450B (a CYP2B-like protein) and cytochrome b5 in scup liver, suggesting that the suppression was specific for CYP1A. High dose PeCB suppressed EROD but not CYP1A protein in the kidney but did not strongly suppress either CYP1A or EROD in the heart or gill. PeCB stimulated ROS production (oxidation of dihydroethidium) by liver microsomes from the low dose but not the high dose fish, and the rate of PeCB-stimulated ROS production was correlated with EROD activity (r(2)=0.641, P<0.0005). Oxidative stress, indicated by increased levels of catalase, glutathione peroxidase, glutathione reductase and superoxide dismutase activities, was stimulated in the liver by low dose but not high dose PeCB. The results support a hypothesis that many PHAH can inactivate teleost CYP1A in vivo, and that CYP1A is a source of ROS. However, there appears to be a complex balance between the effects of PeCB on the levels of active CYP1A, ROS release and oxidative stress.

Monoclonal antibody 1-12-3 (MAb 1-12-3) recognizes an epitope exclusive to cytochrome P450s in subfamily 1A (CYP1A) from all vertebrates tested so far, including one amphibian species. In this study, we first tested the utility of MAb 1-12-3 for detection of presumed CYP1A proteins in hepatic microsomes of northern leopard frogs treated without or with 3,3',4,4',5-pentachlorobiphenyl (PCB 126). Statistical analysis showed that ethoxyresorufin-O-deethylase (EROD) activities and CYP1A equivalents in treated groups were significantly increased at doses > or = 2.3 mg/kg compared with the control groups (p < 0.05), and the increases were maintained for at least four weeks. This result confirmed that MAb 1-12-3 can be used for detection of CYP1A in northern leopard frogs and indicated that CYP1A is the primary catalyst for EROD in this species. In a subsequent experiment, sections of organs of PCB 126-treated frogs were immunohistochemically stained with MAb 1-12-3 to identify localization of the CYP1A in different cell types. The CYP1A staining was seen prominently in hepatocytes and epithelium of nephronic duct, while capillaries close to gastric epithelium and submucosal vascular epithelium in both stomach and intestine exhibited moderate to strong staining. The CYP1A was immunodetected in coronary endothelium and the vascular endothelium of lung and gonad. In skin, mild staining was seen in epithelial cells of mucous glands and serous glands and in vascular endothelium, demonstrating induction of CYP1A in the dermal layer.

Models of receptor action are valuable for describing properties of ligand-receptor interactions and thereby contribute to mechanism-based risk assessment of receptor-mediated toxic effects. In order to build such a model for the aryl hydrocarbon receptor (AHR), binding affinities and CYP1A induction potencies were measured in PLHC-1 cells and were used to determine intrinsic efficacies for 10 halogenated aromatic hydrocarbons (HAH): 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,7, 8-tetrachlorodibenzofuran (TCDF), and eight polychlorinated biphenyls (PCB). TCDD, TCDF, and non-ortho-substituted PCBs 77, 81, 126, and 169 behaved as full agonists and displayed high-intrinsic efficacy. In contrast, the mono- and di-ortho-substituted PCBs bound to the AHR but displayed lower or no intrinsic efficacy. PCB 156 was a full agonist, but with an intrinsic efficacy 10- to 50-fold lower than non-ortho-substituted PCBs. PCB 118 was a very weak partial agonist. PCBs 105 and 128 were shown to be competitive antagonists in this system. The model was then used to predict CYP1A induction by binary mixtures. These predictions were tested with binary mixtures of PCB 126, 128, or 156 with TCDD. Both PCB 156 (a low-intrinsic efficacy agonist) and PCB 128 (a competitive antagonist) inhibited the response to TCDD, while the response to TCDD and PCB126 was additive. These data support the following conclusions: 1) only 1-2% of the receptors in the cell need be occupied to achieve 50% of maximal CYP1A induction by one of the high-intrinsic efficacy agonists, demonstrating the existence of "spare" receptors in this system; 2) the insensitivity of fish to ortho-substituted PCBs is due to both reduced affinity and reduced intrinsic efficacy compared to non-ortho-substituted PCBs; 3) PCB congeners exhibit distinct structure-affinity and structure-efficacy relationships. Separation of AHR ligand action into the properties of affinity and intrinsic efficacy allows for improved prediction of the behavior of complex mixtures of ligands, as well as mechanistic comparisons across species and toxic endpoints., (Copyright 2000 Academic Press.)

We measured rates of oxidative metabolism of two tetrachlorobiphenyl (TCB) congeners by hepatic microsomes of two marine mammal species, beluga whale and pilot whale, as related to content of selected cytochrome P450 (CYP) forms. Beluga liver microsomes oxidized 3,3',4,4'-TCB at rates averaging 21 and 5 pmol/min per mg for males and females, respectively, while pilot whale samples oxidized this congener at 0.3 pmol/min per mg or less. However, rates of 3,3',4,4'-TCB metabolism correlated with immunodetected CYP1A1 protein content in liver microsomes of both species. The CYP1A inhibitor alpha-naphthoflavone inhibited 3,3',4,4'-TCB metabolism by 40% in beluga, supporting a role for a cetacean CYP1A as a catalyst of this activity. Major metabolites of 3,3',4,4'-TCB generated by beluga liver microsomes were 4-OH-3,3',4',5-TCB and 5-OH-3,3',4,4'-TCB (98% of total), similar to metabolites formed by other species CYP1A1, and suggesting a 4,5-epoxide-TCB intermediate. Liver microsomes of both species metabolized 2,2',5,5'-TCB at rates of 0.2-1.5 pmol/min per mg. Both species also expressed microsomal proteins cross-reactive with antibodies raised against some mammalian CYP2Bs (rabbit; dog), but not others (rat; scup). Whether CYP2B homologues occur and function in cetaceans is uncertain. This study demonstrates that PCBs are metabolized to aqueous-soluble products by cetacean liver enzymes, and that in beluga, rates of metabolism of 3,3',4,4'-TCB are substantially greater than those of 2,2',5,5'-TCB. These directly measured rates generally support the view that PCB metabolism plays a role in shaping the distribution patterns of PCB residues found in cetacean tissue.

Members of the Rel family of proteins have been identified in Drosophila, an echinoderm, Xenopus, birds and mammals. Dimers of Rel proteins form the transcription factor nuclear factor kappaB (NF-kappaB) that rapidly activates genes encoding cytokines, cell surface receptors, cell adhesion molecules and acute phase proteins. Evidence suggests that xenobiotic compounds also may alter the activation of NF-kappaB. This study had a dual objective of identifying members of the Rel family and examining their activation by xenobiotic compounds in a marine fish model, scup (Stenotomus chrysops). A DNA-protein crosslinking technique demonstrated that liver, kidney and heart each had at least three nuclear proteins that showed specific binding to an NF-kappaB consensus sequence, with molecular weights suggesting that the proteins potentially corresponded to mouse p50, p65 (RelA) and c-rel. In addition, an approximately 35kD NF-kappaB binding protein was evident in liver and kidney. The 50 kD protein was immunoprecipitated by mammalian p50-specific antibodies. The presence of Rel members in fish implied by those results was confirmed by RT-PCR cloning of a Rel homology domain (an apparent c-rel) from scup liver. NF-kappaB activation occurred in vehicle-treated fish, but this appeared to decrease over time. In fish treated with 0.01 or 1 mg 3,3',4,4', 5-pentachlorobiphenyl per kg, NF-kappaB activation in liver did not decrease, and there was a 6-8-fold increase in activation 16-18 days following treatment. Treatment with 10 mg benzo[a]pyrene/kg had no effect on NF-kappaB-DNA binding, either at 3 or 6 days following treatment. The data show that the Rel family of proteins is present in fish, represented at least by a p50/105 homologue, and support a hypothesis that some aryl hydrocarbon receptor agonists can activate NF-kappaB in vivo.

Previously we showed that the polychlorinated biphenyl 3,3',4,4'-tetrachlorobiphenyl (TCB) caused a release of reactive oxygen species (ROS) from cytochrome P450 1A (CYP1A) of the fish scup (Stenotomus chrysops), and from rat and human CYP1A1. This was linked to a TCB- and NADPH-dependent oxidative inactivation of the enzyme, which in scup and rat was inversely related to the rates of TCB oxidation. We examined the relationship between rates of TCB oxidation, CYP1A inactivation and ROS production in liver microsomes from additional vertebrate species, including skate (Raja erinacea), eel (Anguilla rostrata), killifish (Fundulus heteroclitus), winter flounder (Pleuronectes americanus), chicken (Gallus domesticus), cormorant (Phalacrocorax auritus), gull (Larus argentatus), and turtle (Chrysemys picta picta). TCB oxidation rates were induced in all fish and birds treated with aryl hydrocarbon receptor agonists. Induced rates of TCB oxidation were <1 pmol/min/mg microsomal protein in all fish, and 6-14 pmol/min/mg in the birds. In all species but one, TCB oxidation rates correlated positively with EROD rates, indicating likely involvement of CYP1A in TCB oxidation. Incubation of liver microsomes of most species with TCB+NADPH resulted in an immediate (TCB-dependent) inhibition of EROD, and a progressive loss of EROD capacity, indicating an oxidative inactivation of CYP1A like that in scup. NADPH stimulated production of ROS (H(2)O(2) and/or O(2)(-*)) by liver microsomes, slightly in some species (eel) and greatly in others (chicken, turtle). Among the birds and the fish, NADPH-stimulated ROS production correlated positively with EROD activity. TCB caused a significant stimulation of ROS production by liver microsomes of flounder, killifish, cormorant and gull, as well as scup. The stimulation of CYP1A inactivation and ROS generation indicates an uncoupling of CYP1A by TCB in many species, and when compared between species, the rates of CYP1A inactivation correlated inversely with rates of TCB oxidation. Some feature(s) of binding/active site topology may hinder TCB oxidation, enhancing the likelihood for attack of an oxidizing species in the active site.

The effects of many chemicals on cellular processes are governed by their ability to enter the cell, which is in turn a function of the composition of the cell's external environment. To examine this relationship, the effect of serum in cell culture medium on the bioavailability of cytochrome P450 1A (CYP1A)-inducing compounds was determined in PLHC-1 (Poeciliopsis lucida hepatocellular carcinoma) cells. The presence of 10% calf serum in the medium increased the EC50 (effective concentration to achieve 50% maximal response) for induction of ethoxyresorufin O-deethylase (EROD) activity by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) 20-fold as compared to treatment in serum-free medium. Measurement of [3H]TCDD uptake and Ah receptor binding indicated that the apparent difference in potencies was a result of decreased bioavailability in the presence of serum, effectively reducing the concentration of TCDD within the cells. Induction of EROD and CYP1A protein in response to treatment with each of three coplanar polychlorinated biphenyls (PCB congeners 77, 126, and 169) was similarly affected by serum, although the magnitude varied among inducers and assays. Relative potencies (calculated as EC50TCDD / EC50PCB) for EROD induction by the three PCBs were significantly higher in the absence of serum. However, serum showed no significant effect on the relative potencies for CYP1A protein induction. These results demonstrate that measured inducing potencies, and relative potencies for EROD induction, by halogenated aromatic hydrocarbons are strongly dependent on the composition of culture medium, which can lead to artificial differences in comparisons among cell types.

Microsomal cytochrome P-450 1A (CYP1A) in a vertebrate model (the teleost fish scup) is inactivated by the aryl hydrocarbon receptor agonist 3,3',4,4'-tetrachlorobiphenyl (TCB). Here, the mechanism of CYP1A inactivation and its relationship to reactive oxygen species (ROS) formation were examined by using liver microsomes from scup and rat and expressed human CYP1As. In vitro inactivation of scup CYP1A activity 7-ethoxyresorufin O-deethylation by TCB was time dependent, NADPH dependent, oxygen dependent, and irreversible. TCB increased microsomal NADPH oxidation rates, and CYP1A inactivation was lessened by adding cytochrome c. CYP1A inactivation was accompanied by loss of spectral P-450, a variable loss of heme and a variable appearance of P-420. Rates of scup liver microsomal metabolism of TCB were < 0.5 pmol/min/mg, 25-fold less than the rate of P-450 loss. Non-heme iron chelators, antioxidant enzymes, and ROS scavengers had no influence on inactivation. Inactivation was accelerated by H(2)O(2) and azide but not by hydroxylamine or aminotriazole. TCB also inactivated rat liver microsomal CYP1A, apparently CYP1A1. Adding TCB to scup or rat liver microsomes containing induced levels of CYP1A, but not control microsomes, stimulated formation of ROS; formation rates correlated with native CYP1A1 content. TCB stimulated ROS formation by baculovirus-expressed human CYP1A1 but not CYP1A2. The results indicate that TCB uncouples the catalytic cycle of CYP1A, ostensibly CYP1A1, resulting in formation of ROS within the active site. These ROS may inactivate CYP1A or escape from the enzyme. ROS formed by CYP1A1 may contribute to the toxicity of planar halogenated aromatic hydrocarbons.

The metabolism of the polychlorinated biphenyl congener 3,3',4,4'-tetrachlorobiphenyl (TCB) was examined in vitro and in vivo in the marine fish scup (Stenotomus chrysops). Untreated scup liver microsomes catalyzed metabolism of TCB with an estimated KM of 0.7 microM, at a rate < or = 0.13 pmol/min/mg. Metabolism was NADPH-dependent and inhibited by cytochrome c and CO, indicating cytochrome P450 (CYP) involvement. alpha-Naphthoflavone strongly inhibited microsomal TCB metabolism, and treatment of fish with CYP1A inducers increased the rates by approximately 2-fold, suggesting involvement of CYP1A. Scup were injected intraperitoneally with 0.1 or 5 mg TCB/kg and sampled on days 1-16 after treatment (after 3 days without food at each sampling). Concentrations of unmetabolized TCB in liver peaked on day 5 in low dose fish and on day 12 in high dose fish. In both groups the TCB content in the liver had declined 60% or more by day 16, suggesting depuration or redistribution from the liver. GC and MS revealed TCB and TCB metabolites in bile within 24 hr of treatment. The concentrations of TCB and metabolites in bile peaked at the same time that TCB concentrations peaked in the liver. The major metabolites were 5-hydroxy-3,3'4,4'-TCB (5-OH-TCB) and 4-hydroxy-3,3',5,4'-TCB (4-OH-TCB); 2-hydroxy-3,3',4,4'-TCB and 6-hydroxy-3,3',4,4'-TCB were minor metabolites. Animals given the high dose had much less 5-OH-TCB and much more parent TCB in bile than did fish given the low dose. Amounts of 4-OH-TCB in bile did not differ between doses. The reduced excretion of 5-OH-TCB coincided with a suppression of CYP1A in fish given the high dose, that did not occur in low dose fish, consistent with an involvement of CYP1A in TCB metabolism and particularly in formation of 5-OH-TCB. This study provides the first direct demonstration of 3,3',4,4'-TCB metabolism by fish. Data also indicate that these fish are able to eliminate TCB both as parent compound and as metabolites, despite a very slow rate of metabolism in vitro.

3,3',4,4'-Tetrachlorobiphenyl (TCB) can induce and inhibit cytochrome P450 1A1 (CYP1A1) in vertebrates. TCB may also suppress CYP1A1 protein levels, but the mechanism is unknown. This study examined transcriptional and translational aspects of hepatic CYP1A1 regulation in the fish scup (Stenotomus chrysops) given single intraperitoneal injections of low (0.1 mg/kg) or high (5 mg/kg) doses of TCB, and sampled over 16 days. The low dose strongly induced hepatic CYP1A1 mRNA (25-fold), protein (12-fold), and activity [ethoxyresorufin O-deethylase (EROD)] (15-fold). The high dose also strongly induced CYP1A1 mRNA (29-fold), in a pattern like that at the low dose, but microsomal CYP1A1 protein content was induced only 4-fold and EROD rates were near control levels. Both TCB doses caused similar increases in microsomal cytochrome b5 content, and in rates of NADPH-cytochrome c (P450) reductase and UDP-glucuronosyltransferase (with p-nitrophenol). The contents of CYP forms other than CYP1A1 (putative CYP2B or CYP3A) were only weakly affected by TCB at either dose. The strong and largely specific post-transcriptional suppression of CYP1A1 content was associated with high concentrations of TCB measured in the liver. Incubation of scup hepatic microsomes with TCB plus NADPH led to a time-dependent inactivation of CYP1A1 that was distinct from catalytic inhibition, and appeared not to involve reactive metabolites of TCB. This in vitro result suggests that TCB may inactivate CYP1A1 in vivo, which could account for the apparent antagonistic effect of TCB on CYP1A1 induction.

The regulation of different cytochrome P-450 forms and their functions in different organs and cell types could determine the susceptibility of those cells and organs to toxic effects of xenobiotics, including chemical carcinogenesis. Here we describe the cellular localization of cytochrome P-450E (P-450IA1) induced in 10 major organs or organ systems of a marine vertebrate species, the fish, Stenotomus chrysops (scup). Scup were injected ip with 3,3',4,4'-tetrachlorobiphenyl (TCB) at 1 mg/kg, or with 2,3,7,8-tetrachlorodibenzofuran (TCDF) at 3 micrograms/kg. Induction was verified by Western blot analysis of microsomes from selected organs (liver, kidney, and gill) using monoclonal antibody (MAb) 1-12-3 to scup P-450IA1. The localization of P-450IA1 was subsequently determined in sections prepared by standard histological methods (10% buffered formalin fixation, paraffin embedding), and stained with MAb 1-12-3 and peroxidase-labeled second antibody. P-450IA1 was induced in epithelial and endothelial cells in liver (including pancreatic tissue), kidney, gill, gut, spleen, testis, and ovary. Induction also was detected in endothelial cells, but not other types, in heart, brain, and red muscle. In heart, the staining was present in the endocardium as well as in the endothelium of the coronary vasculature and great vessels. Although TCDF and TCB both induced P-450IA1 in various cells of all organs examined, the effect of TCB was in most cases greater than that of TCDF. This may be due to a relatively higher TCB dosage. A wider staining distribution was seen in gut, gill, kidney, and gonad of TCB-treated fish, which might be explained by a greater penetration, or by excretion of parent TCB, as opposed to TCDF. In any case, the results show that these important environmental agents induce P-450IA1 in generally similar patterns in all organs examined. The common finding of a strong induction of P-450IA1 in endothelial cells in all organs examined supports the suggestion that the endothelium may be a primary site of P-450IA1 induction.

Polychlorinated biphenyl congeners that are abundant in environmental samples, and known to induce hepatic monooxygenase isozymes in the P450IA gene subfamily in mammals, were examined for their ability to induce hepatic monooxygenase activity in scup, a marine teleost. Scup were dosed ip with 3,3',4,4'-tetrachlorobiphenyl (congener 77), 2,3,3',4,4'-pentachlorobiphenyl (congener 105), 2,3',4,4',5-pentachlorobiphenyl (congener 118), 2,2',3,4,4',5'-hexachlorobiphenyl (congener 138), 2,2',3,3',4,4'-hexachlorobiphenyl (congener 128), or beta-naphthoflavone and examined for increases in ethoxyresorufin O-deethylase (EROD) activity, immunodetectable cytochrome P450E (the EROD catalyst in scup), and in vitro translatable mRNA for P450E. Monooxygenase parameters were significantly induced only by 3,3',4,4'-tetrachlorobiphenyl (TCB). However, while translatable mRNA for P450E was induced at all doses (1, 5, and 10 mg/kg), EROD activity and P450E were decreased at the 5 and 10 mg/kg doses, relative to the response at 1 mg/kg. A strong relationship between residual TCB concentration in the liver and the decreased EROD activity was evident at the higher doses of TCB. Aminopyrine N-demethylase, a monooxygenase activity not catalyzed by P450E, was unaffected by TCB treatment, indicating a specificity in the TCB effect. Analysis in vitro revealed that TCB was a potent competitive inhibitor of EROD activity, with half-maximal inhibition at 0.3 microM, near the Km for ethoxyresorufin, suggesting one mechanism for the in vivo effect of TCB. These results demonstrate that PCB congeners with ortho-chlorine substitution, and which are effective inducers of AHH and EROD activity in mammals, are ineffective, at the doses tested, as inducers in the teleost scup.