RESUMEN
Per- and polyfluoroalkyl substances (PFAS) are pervasive in the environment resulting in nearly universal detection in people. Human serum PFAS concentrations are strongly associated with increased serum low-density lipoprotein cholesterol (LDL-C), and growing evidence suggests an association with serum triacylglycerides (TG). Here, we tested the hypothesis that perfluorooctanoic acid (PFOA) dysregulates liver and serum triacylglycerides in human peroxisome proliferator activated receptor α (hPPARα)-expressing mice fed an American diet. Mice were exposed to PFOA (3.5 mg/L) in drinking water for 6 weeks resulting in a serum concentration of 48 ± 9 µg/ml. In male and female hPPARα mice, PFOA increased total liver TG and TG substituted with saturated and monounsaturated fatty acids. Lack of expression of PPARα alone also increased total liver TG, and PFOA treatment had little effect on liver TG in PPARα null mice. In hPPARα mice, PFOA neither significantly increased nor decreased serum TG; however, there was a modest increase in TG associated with very low-density cholesterol particles in both sexes. Intriguingly, in female PPARα null mice, PFOA significantly increased serum TG, with a similar trend in males. PFOA also modified fatty acid and TG homeostasis-related gene expression in liver, in a hPPARα-dependent manner, but not in adipose. The results of our study and others reveal the importance of context (serum concentration and genotype) in determining the effect of PFOA on lipid homeostasis.
Asunto(s)
Caprilatos/toxicidad , Dieta Occidental , Dislipidemias/inducido químicamente , Fluorocarburos/toxicidad , Hígado/efectos de los fármacos , PPAR alfa/genética , Animales , Peso Corporal/efectos de los fármacos , Dislipidemias/genética , Dislipidemias/metabolismo , Dislipidemias/patología , Femenino , Expresión Génica/efectos de los fármacos , Genotipo , Lipidómica , Hígado/metabolismo , Hígado/patología , Masculino , Ratones Transgénicos , Tamaño de los Órganos/efectos de los fármacos , Triglicéridos/sangre , Triglicéridos/metabolismo , Estados UnidosRESUMEN
Humans are exposed to per- and polyfluoroalkyl substances (PFAS) in their drinking water, food, air, dust, and by direct use of consumer products. Increased concentrations of serum total cholesterol and low density lipoprotein cholesterol are among the endpoints best supported by epidemiology. The objectives of this study were to generate a new model for examining PFAS-induced dyslipidemia and to conduct molecular studies to better define mechanism(s) of action. We tested the hypothesis that perfluorooctanoic acid (PFOA) exposure at a human-relevant level dysregulates expression of genes controlling cholesterol homeostasis in livers of mice expressing human PPARα (hPPARα). Female and male hPPARα and PPARα null mice were fed a diet based on the "What we eat in America" analysis and exposed to PFOA in drinking water (8 µM) for 6 weeks. This resulted in a serum PFOA concentration of 48 µg/ml. PFOA increased liver mass, which was associated with histologically-evident lipid accumulation. Pooled analyses of serum lipoprotein cholesterol suggest that PFOA increased serum cholesterol, particularly in male mice. PFOA induced PPARα and constitutive androstane receptor target gene expression in liver. Expression of genes in four pathways regulating cholesterol homeostasis were also measured. PFOA decreased expression of Hmgcr in a PPARα-dependent manner. PFOA decreased expression of Ldlr and Cyp7a1 in a PPARα-independent manner. Apob expression was not changed. Sex differences were evident. This novel study design (hPPARα mice, American diet, long term exposure) generated new insight on the effects of PFOA on cholesterol regulation in the liver and the role of hPPARα.
Asunto(s)
Caprilatos/toxicidad , Colesterol/sangre , Fluorocarburos/toxicidad , Hígado/efectos de los fármacos , PPAR alfa/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Transcriptoma/efectos de los fármacos , Contaminantes Químicos del Agua/toxicidad , Animales , Peso Corporal/efectos de los fármacos , Peso Corporal/genética , Receptor de Androstano Constitutivo , Dieta Occidental , Femenino , Metabolismo de los Lípidos/efectos de los fármacos , Metabolismo de los Lípidos/genética , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Tamaño de los Órganos/efectos de los fármacos , Tamaño de los Órganos/genética , Receptores Citoplasmáticos y Nucleares/genética , Caracteres SexualesRESUMEN
Tributyltin (TBT) and dioxin-like polychlorinated biphenyls (PCBs) are environmental contaminants that are highly toxic to fish and co-occur in New Bedford Harbor (NBH), an estuarine Superfund site located in Massachusetts, USA. Atlantic killifish (Fundulus heteroclitus) that reside in NBH (and other highly contaminated sites along the east coast of the United States) have developed resistance to activation of the aryl hydrocarbon receptor (AHR) pathway and the toxicity of dioxin-like chemicals, such as 3,3',4,4',5-pentachlorobiphenyl, PCB126. In many biological systems, TBT disregulates adipose and bone development via the PPARγ-RXR pathway; AHR activation also disrupts adipose and bone homeostasis, potentially through molecular crosstalk between AHR and PPARγ. However, little is known about how co-exposure and the interaction of these pathways modulate the toxicological effects of these contaminants. Here, we tested the hypotheses that TBT would induce teratogenesis in killifish via activation of PPARγ and that PCB126 co-exposure would suppress PPARγ pathway activation in PCB-sensitive killifish from a reference site (Scorton Creek, SC, PCB-sensitive) but not in PCB-tolerant NBH killifish. Killifish embryos from both populations exposed to TBT (50 and 100â¯nM) displayed caudal fin deformities. TBT did not change the expression of pparg or its target genes related to adipogenesis (fabp11a and fabp1b) in either population. However, expression of osx/sp7, an osteoblast marker gene, and col2a1b, a chondroblast marker gene, was significantly suppressed by TBT only in SC killifish. An RXR-specific agonist, but not a PPARγ-specific agonist, induced caudal fin deformities like those observed in TBT-treated embryos. PCB126 did not induce caudal fin deformities and did not exacerbate TBT-induced fin deformities. Further, PCB126 increased expression of pparg in SC embryos and not NBH embryos, but did not change the expression of fabp1b. Taken together, these results suggest that in killifish embryos the PPARγ pathway is regulated in part by AHR, but is minimally active at least in this early life stage. In killifish, RXR activation, rather than PPARγ activation, appears to be the mechanism by which TBT induces caudal fin teratogenicity, which is not modulated by AHR responsiveness.
Asunto(s)
Aletas de Animales/efectos de los fármacos , Embrión no Mamífero/efectos de los fármacos , Fundulidae , PPAR gamma/metabolismo , Bifenilos Policlorados/toxicidad , Receptores de Hidrocarburo de Aril/metabolismo , Compuestos de Trialquiltina/toxicidad , Contaminantes Químicos del Agua/toxicidad , Aletas de Animales/anomalías , Animales , Resistencia a Medicamentos/efectos de los fármacos , Sinergismo Farmacológico , Embrión no Mamífero/anomalías , Embrión no Mamífero/metabolismo , Desarrollo Embrionario/efectos de los fármacos , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Massachusetts , PPAR gamma/genética , Receptor Cross-Talk , Receptores de Hidrocarburo de Aril/genética , Transducción de Señal/efectos de los fármacosRESUMEN
Polycyclic aromatic hydrocarbons (PAHs) are toxic chemicals released into the environment by fossil fuel combustion. Moreover, a primary route of human exposure to PAHs is tobacco smoke. Oocyte destruction and ovarian failure occur in PAH-treated mice, and cigarette smoking causes early menopause in women. In many cells, PAHs activate the aromatic hydrocarbon receptor (Ahr), a member of the Per-Arnt-Sim family of transcription factors. The Ahr is also activated by dioxin, one of the most intensively studied environmental contaminants. Here we show that an exposure of mice to PAHs induces the expression of Bax in oocytes, followed by apoptosis. Ovarian damage caused by PAHs is prevented by Ahr or Bax inactivation. Oocytes microinjected with a Bax promoter-reporter construct show Ahr-dependent transcriptional activation after PAH, but not dioxin, treatment, consistent with findings that dioxin is not cytotoxic to oocytes. This difference in the action of PAHs versus dioxin is conveyed by a single base pair flanking each Ahr response element in the Bax promoter. Oocytes in human ovarian biopsies grafted into immunodeficient mice also accumulate Bax and undergo apoptosis after PAH exposure in vivo. Thus, Ahr-driven Bax transcription is a novel and evolutionarily conserved cell-death signaling pathway responsible for environmental toxicant-induced ovarian failure.
Asunto(s)
9,10-Dimetil-1,2-benzantraceno/análogos & derivados , Contaminación Ambiental/efectos adversos , Insuficiencia Ovárica Primaria/genética , Proteínas Proto-Oncogénicas c-bcl-2 , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas/genética , Receptores de Hidrocarburo de Aril/metabolismo , Adulto , Animales , Apoptosis , Femenino , Expresión Génica/efectos de los fármacos , Genes Reporteros , Humanos , Técnicas In Vitro , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones SCID , Microinyecciones , Oocitos/citología , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Ovario/efectos de los fármacos , Ovario/metabolismo , Ovario/trasplante , Insuficiencia Ovárica Primaria/inducido químicamente , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas/deficiencia , Receptores de Hidrocarburo de Aril/deficiencia , Receptores de Hidrocarburo de Aril/genética , Elementos de Respuesta , Transducción de Señal/efectos de los fármacos , Trasplante Heterólogo , Proteína X Asociada a bcl-2RESUMEN
Cytochrome P450 1A (CYP1A) induction is a robust marker for exposure to polynuclear aromatic hydrocarbons and planar halogenated aromatic hydrocarbons that are aryl hydrocarbon receptor agonists. We examined CYP1A expression in mesopelagic fishes from the western North Atlantic. Individuals in 22 species were obtained from slope water and the Sargasso Sea in 1977, 1978, and 1993. Aryl hydrocarbon hydroxylase (AHH), a CYP1A activity, was detected in liver from all species in 1977/78. In some, including Gonostoma elongatum, AHH was inhibited by the CYP1A inhibitor alpha-naphthoflavone. CYP1A-dependent ethoxyresorufin O-deethylase (EROD) was detected in liver microsomes of all species in 1993; rates were highest in G. elongatum and Argyropelecus aculeatus. Immunoblot analysis with the CYP1A-specific monoclonal antibody 1-12-3 detected a single microsomal protein band in most 1993 samples; the highest content was in G. elongatum. Immunohistochemical analysis showed CYP1A staining in gill, heart, kidney, and/or liver of several species. Extracts of the 1993 G. elongatum and A. aculeatus, when applied to fish hepatoma cells (PLHC-1) in culture, elicited a significant induction of EROD in those cells. The capacity of the extracts to induce CYP1A correlated with the content of PCBs measured in the same fish (2-4.6 ng/g total body weight). Mesopelagic fish in the western North Atlantic, which experience no direct exposure to surface waters or sediments, are exposed chronically to inducers of CYP1A at levels that appear to be biochemically active in those fish.
Asunto(s)
Citocromo P-450 CYP1A1/metabolismo , Peces/metabolismo , Contaminantes Químicos del Agua/toxicidad , Animales , Océano Atlántico , Bioensayo , Western Blotting , Citocromo P-450 CYP1A1/biosíntesis , Monitoreo del Ambiente , Inducción Enzimática/efectos de los fármacos , Inmunohistoquímica , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/enzimología , Bifenilos Policlorados/análisis , Bifenilos Policlorados/toxicidad , Agua de Mar , Factores de Tiempo , Distribución Tisular , Contaminantes Químicos del Agua/análisisRESUMEN
Polycyclic aromatic hydrocarbons (PAH) are ubiquitous environmental chemicals that suppress the immune system at multiple levels, including at the level of B cell development in the bone marrow microenvironment. Specifically, PAH induce preB cell apoptosis in primary bone marrow cultures and in cocultures of an early preB cell line (BU-11) and a bone marrow stromal cell line (BMS2). Previous studies focused on the molecular mechanisms through which PAH induce stromal cells to deliver an apoptosis signal to adjacent preB cells. Apoptosis signaling within the preB cell itself was not investigated. Here, the role of NF-kappaB, a lymphocyte survival factor, in PAH-induced preB cell apoptosis was assessed. Analysis of DNA-binding proteins extracted from the nuclei of untreated BU-11 cells indicated DNA-binding complexes comprising NF-kappaB subunits p50, c-Rel, and/or Rel A. NF-kappaB down-regulation with previously described inhibitors induced BU-11 cell apoptosis, demonstrating that the default apoptosis pathway blocked by NF-kappaB is functional at this early stage in B cell development. Similarly, exposure of BU-11/BMS2 cocultures to 7,12-dimethylbenz[a]anthracene (DMBA), a prototypic PAH, down-regulated nuclear Rel A and c-Rel before overt apoptosis. Finally, ectopic expression of Rel A or c-Rel rescued BU-11 cells from DMBA-induced apoptosis. These results extend previous observations by demonstrating that 1) NF-kappaB is a survival factor at an earlier stage of B cell development than previously appreciated and 2) NF-kappaB down-regulation is likely to be part of the molecular mechanism resulting in PAH-induced preB cell apoptosis. These results suggest nonclonally restricted, PAH-mediated suppression of B lymphopoiesis.
Asunto(s)
9,10-Dimetil-1,2-benzantraceno/farmacología , Apoptosis , Linfocitos B/citología , FN-kappa B/fisiología , Animales , Carcinógenos/farmacología , Supervivencia Celular , Regulación hacia Abajo , Contaminantes Ambientales/farmacología , Ratones , FN-kappa B/biosíntesis , FN-kappa B/metabolismo , Proteínas Proto-Oncogénicas c-rel/biosíntesis , Proteínas Proto-Oncogénicas c-rel/fisiología , Factor de Transcripción ReIA , Células Tumorales CultivadasRESUMEN
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.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas , Citocromo P-450 CYP1A1/antagonistas & inhibidores , Citocromo P-450 CYP1A1/biosíntesis , Inhibidores del Citocromo P-450 CYP1A2 , Citocromo P-450 CYP1A2/biosíntesis , Estrés Oxidativo/efectos de los fármacos , Perciformes/metabolismo , Bifenilos Policlorados/farmacología , Animales , Citocromo P-450 CYP2B6 , Citocromo P-450 CYP3A , Sistema Enzimático del Citocromo P-450/metabolismo , Hígado/enzimología , Oxidorreductasas/metabolismo , Oxidorreductasas N-Desmetilantes/metabolismo , ARN Mensajero/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
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.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas , Sistema Enzimático del Citocromo P-450/metabolismo , Delfines/metabolismo , Bifenilos Policlorados/metabolismo , Ballenas/metabolismo , Animales , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP2B6 , Femenino , Masculino , Oxidorreductasas N-Desmetilantes/metabolismoRESUMEN
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.
Asunto(s)
Regulación de la Expresión Génica , Microsomas Hepáticos/enzimología , FN-kappa B/metabolismo , Bifenilos Policlorados/farmacología , Proteínas Proto-Oncogénicas c-rel/genética , Proteínas Proto-Oncogénicas c-rel/metabolismo , Receptores de Hidrocarburo de Aril/agonistas , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Pollos , Secuencia de Consenso , Sistema Enzimático del Citocromo P-450/genética , Peces , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Riñón/metabolismo , Hígado/metabolismo , Ratones , Datos de Secuencia Molecular , Filogenia , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Pavos , Xenobióticos/farmacología , Xenopus laevisRESUMEN
Endothelium is a common site of cytochrome P450 1A (CYP1A) induction in vertebrates, and endothelial CYP1A could affect the distribution and toxicity of CYP1A substrates. We investigated CYP1A induction in organs rich in endothelium, gill, heart, and a microvascular model, the swimbladder rete mirabile, in the eel. Benzo[a]pyrene (BP) and 3, 3',4,4'-tetrachlorobiphenyl (TCB), radiolabeled and injected intraperitoneally, showed similar distribution in eels, with dose-dependent increases in concentration in heart and rete mirabile. BP [given at 0.1, 1, and 10 mg/kg (0.4, 4, and 40 micromol/kg)], TCB [given at 0.1, 1, and 10 mg/kg (0.3, 3, 30, and 60 micromol/kg)], and beta-naphthoflavone (BNF) [given at 0.1, 1, 5, 10, and 100 mg/kg (0.4, 4, 20, 40, and 400 micromol/kg)] induced microsomal CYP1A and ethoxyresorufin O-deethylase in heart and rete mirabile. Immunohistochemical analysis confirmed that induction of CYP1A in heart and rete mirabile occurs in the endothelium. Increasing doses of each compound caused increasing penetration of induction into the vascular bed of the rete, but with BNF and BP induction penetrated further than with TCB. At high doses of BNF there also was induction in epithelial cells adjacent to endothelium in gill and kidney. CYP1A also was induced in heart and rete mirabile of eels from sites heavily contaminated by aryl hydrocarbon receptor (AHR) agonists. The penetration of CYP1A induction into capillaries of the rete mirabile reflects the penetration of the inducer itself, consistent with the idea that endothelial CYP1A can indicate the local distribution of AHR agonists. The microvascular rete mirabile in the eel provides a model system to explore further a hypothesis that endothelial CYP1A participates in removal of some AHR agonists from the circulation and to examine the consequences of CYP1A induction to the vascular system.
Asunto(s)
Citocromo P-450 CYP1A1/biosíntesis , Endotelio Vascular/metabolismo , Branquias/enzimología , Microsomas/enzimología , Animales , Benzo(a)pireno/farmacología , Relación Dosis-Respuesta a Droga , Anguilas , Endotelio Vascular/enzimología , Inducción Enzimática , Branquias/metabolismo , Corazón/fisiología , Inmunohistoquímica , Microcirculación/efectos de los fármacos , Microsomas/metabolismo , Bifenilos Policlorados/farmacología , Receptores de Hidrocarburo de Aril/agonistasRESUMEN
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.
Asunto(s)
Citocromo P-450 CYP1A1/metabolismo , Bifenilos Policlorados/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Acetona/farmacología , Anguilla , Animales , Aves , Pollos , Citocromo P-450 CYP1A1/efectos de los fármacos , Femenino , Lenguado , Fundulidae , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/enzimología , NADP/farmacología , Oxidación-Reducción , Receptores de Hidrocarburo de Aril/agonistas , Receptores de Hidrocarburo de Aril/efectos de los fármacos , Rajidae , Especificidad de la Especie , TortugasRESUMEN
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.
Asunto(s)
Inhibidores Enzimáticos del Citocromo P-450 , Hígado/efectos de los fármacos , Bifenilos Policlorados/farmacología , Especies Reactivas de Oxígeno/metabolismo , Animales , Sitios de Unión , Citocromo P-450 CYP1A1/antagonistas & inhibidores , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1A2/metabolismo , Inhibidores del Citocromo P-450 CYP1A2 , Inhibidores Enzimáticos/farmacología , Peces , Humanos , Hígado/enzimología , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/enzimología , RatasRESUMEN
Cytochrome P450-mediated arachidonic acid (AA) metabolism was investigated in the marine fish scup, Stenotomus chrysops. Liver microsomes incubated with AA and NADPH produced epoxyeicosatrienoic acids (EETs) and their hydration products (dihydroxyeicosatrienoic acids, DHETs), midchain conjugated dienols (midchain HETEs), and C16-through C20-alcohols of AA (omega-terminal HETEs), all identified by HPLC and GC/MS. Gravid females had 4-fold lower AA metabolism rates than males but identical metabolite profiles. The 5,6-EET (inferred from stable metabolites) was most abundant (47% of total EETs) followed by 14,15-, 11,12-, and 8,9-EET (27, 13, and 13%, respectively). The 12-HETE represented 25% of total HETEs followed in abundance by 16-, 15-, 11-, 19-, 20-, 8-, and 9-HETE. Antibodies against scup CYP1A and a scup CYP2B-like protein inhibited liver microsomal AA metabolism by 30 and 46%, respectively. GC/MS analysis revealed EETs and DHETs as endogenous constituents in scup liver; the predominant EETs were 8,9- and 14,15-EET, followed by a lesser amount of 11,12-EET. Chiral analysis showed a preference for the S,R-enantiomers of endogenous 8,9-, 11,12-, and 14,15-EET (optical purities 80, 64, and 64%, respectively). Treatment of scup with the CYP1A inducer benzo(a)pyrene (BP) increased liver microsomal formation of EETs and HETEs by 2.7-fold in spring and 1.7-fold in summer. BP treatment did not affect microsomal EET regioselectivity, but shifted hydroxylation in favor of 19-HETE and induced 17-HETE formation. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) treatment in summer did not induce liver microsomal AA metabolism rates, yet BP and TCDD both increased endogenous EET content of liver (5- and 3-fold, respectively), with a shift to 14,15-EET. BP treatment increased the selectivity for the S,R-enantiomers of endogenous 8,9-, 11,12-, and 14,15-EET (optical purities 91, 84, and 83%, respectively). Kidney, gill, and heart microsomes all metabolized AA, at rates 10- to 30-fold less than liver microsomes. Similar amounts of endogenous 8,9- and 14,15-EET and less 11,12-EET were detected in heart and kidney, and there was a strong enantioselectivity for 8(R),9(S)-EET in heart (optical purity 78%) but not in kidney. BP treatment did not alter the total EET content in these organs but did shift the regiochemical profile in heart to favor 14,15-EET. Thus, scup liver and extrahepatic organs metabolize AA via multiple cytochrome P450 (CYP) forms to eicosanoids in vitro and in vivo. BP or TCDD induced endogenous AA metabolism in liver, altering EET regioselectivity and, with BP, stereoselectivity. While AhR agonists alter metabolism of AA in early diverging vertebrates expressing both CYP1A and AhR, the magnitude of effects may depend upon the type of inducer.