Involvement of Cytochrome P450 1A in the toxicity of aryl hydrocarbon receptor agonists : alteration arachidonic acid metabolism and production of reactive oxygen species

Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at the Massachusetts Institute of Technology and the Woods Hole Oceanographic Institution August 1998Two cytochrome P4501A-dependent mechanisms of aryl hydrocarbon receptor (AhR) agonist toxicity were examined in the marine teleost scup (Stenotomus chrysops), alteration of arachidonic acid (AA) metabolism and production of reactive oxygen species (ROS). In scup hepatic microsomes, cytochrome P450s including CYP1A and CYP2B-like proteins catalyzed regioselective metabolism of AA to eicosatrienoic and hydroxyeicosatetraenoic acids. Benzo[a]pyrene (BP) treatment induced liver microsomal AA metabolism, but that effect varied with season. Endogenous AA epoxides were recovered from scup liver, heart, and kidney, and their composition in the liver was altered by treatment with BP or 2,3,7,8-tetrachlorodibenzo-p-dioxin. In scup and mammals, the formation of ROS was stimulated by binding of 3,3',4,4-tetrachlorobiphenyl (TCB) to CYP1A, apparently CYP1Al. Attack of that ROS inactivated scup CYP1A. ROS release and inactivation of CYP1A were stimulated only by substrates of CYP1A that are slowly metabolized. In vivo, 3,3',4,4',5- pentachlorobiphenyl (PeCB) potently induced CYP1A mRNA, protein and catalytic activity at low doses (0.01-0.1 mg/kg), suppressed induction of CYP1A protein and catalytic activity at a high dose (1 mg/kg) and transiently induced oxidative stress in scup liver. The suppression of CYP1A induction was organ-dependent, with hepatic CYP1A being most susceptible to inactivation. The results suggest that ROS could be involved in the in vivo suppression of scup liver CYP1A by planar halogenated aromatic hydrocarbons. The reactive oxygen sensitive transcription factor, nuclear factor-KB (NF-KB), was characterized in scup. An NF-KB consensus binding sequence bound specifically to 3 proteins in scup liver, heart and kidney. One protein was recognized by an antibody to mammalian p50. Injection alone appeared to activate NF-KB. BP did not increase the activation ofNF-KB, and PeCB activated NF-KB in only 1 of 2 experiments. Last, CYP1A induction in endothelial cells of the American eel (Anguilla rostrata), a site which may be particularly susceptible to alterations in AA metabolism and ROS production, was described. Eel liver CYP1A responded to BP, 13-naphthoflavone and TCB in a dose-dependent fashion, and induction was correlated with hepatic inducer concentration. Endothelial CYP1A was inducible in a number of organs and was metabolically active. In the rete mirabile, penetration of endothelial CYP1A induction increased with increasing dose of AhR agonists, corresponding with an increase in inducer concentration. A transition from endothelial to epithelial staining occurred in the gill, heart and kidney at high inducer doses.My research was supported in part by the Lyons Fellowship, the MIT!WHOI Joint program, the Rinehart Coastal Research Center, NIH grant P42-ES07381, EPA grant R823890 and the Air Force Office of Scientific Research grant F40620-94-1039

Direct Assessment of Cumulative Aryl Hydrocarbon Receptor Agonist Activity in Sera from Experimentally Exposed Mice and Environmentally Exposed Humans

Background: Aryl hydrocarbon receptor (AhR) ligands adversely affect many biological processes. However, assessment of the significance of human exposures is hampered by an incomplete understanding of how complex mixtures affect AhR activation/inactivation. Objectives: These studies used biological readouts to provide a broader context for estimating human risk than that obtained with serum extraction and gas chromatography/mass spectroscopy (GC/MS)-based assays alone. Methods: AhR agonist activity was quantified in sera from dioxin-treated mice, commercial human sources, and polychlorinated biphenyl (PCB)–exposed Faroe Islanders using an AhR-driven reporter cell line. To validate relationships between serum AhR agonist levels and biological outcomes, AhR agonist activity in mouse sera correlated with toxic end points. AhR agonist activity in unmanipulated (“neat”) human sera was compared with these biologically relevant doses and with GC/MS-assayed PCB levels. Results: Mouse serum AhR agonist activity correlated with injected dioxin dose, thymic atrophy, and heptomegaly, validating the use of neat serum to assess AhR agonist activity. AhR agonist activity in sera from Faroe Islanders varied widely, was associated with the frequency of recent pilot whale dinners, but did not correlate with levels of PCBs quantified by GC/MS. Surprisingly, significant “baseline” AhR activity was found in commercial human sera. Conclusions: An AhR reporter assay revealed cumulative levels of AhR activation potential in neat serum, whereas extraction may preclude detection of important non-dioxin-like biological activity. Significant levels of AhR agonist activity in commercial sera and in Faroe Islander sera, compared with that from experimentally exposed mice, suggest human exposures that are biologically relevant in both populations

Cytochrome P450 induced differentially in endothelial cells cultured from different organs of Anguilla rostrata

Author Posting. © The Authors, 2004. This is the author's version of the work. It is posted here by permission of Society for In Vitro Biology for personal use, not for redistribution. The definitive version was published in In Vitro Cellular & Developmental Biology - Animal 41 (2005): 57-63, doi:10.1290/0409063.1.Endothelial cells are a structural barrier and an active regulator of many bodily processes. CYP1A activity is induced in the endothelium of teleosts and mammals exposed to lipophilic xenobiotics, such as polycyclic aromatic hydrocarbons, and can have significant consequences for endothelial functions. We exposed cultures of characterized endothelial cells from the heart, kidney and rete mirabile of the eel, Anguilla rostrata, to AhR agonists. In heart endothelial cells the maximum response (based on EROD activity) to TCDD, 113 pmol/mg-min, was at 1 nM TCDD and the peak response to βNF, 135 pmol/mg-min, was at 3 μM βNF. The maximum response to TCDD in the kidney endothelial cells is 12 pmol/mg-min at 0.3 nM TCDD. The rete mirabile capillary endothelial cells responded minimally or not at all to exposure to TCDD and βNF. Both the heart and kidney endothelial cells (but not the rete mirabile capillary cells) have a low level of EROD activity (12.7 and 5.2 pmol/mg-min respectively) in untreated or DMSO-treated cells. The robust response of the heart endothelial cells to induction and the lack of response in the rete mirabile capillary endothelial cells indicate that these cells are a good resource to use to investigate the physiological consequences of AhR agonist exposure and CYP1A induction in different areas of the vasculature.The Faculty Research Council of Fordham University provided partial support for RAG. This research was supported by NIH grant 5-P42-ES07381 and by U.S.EPA grant R827102-01-0

Exposure to environmental contaminants is associated with altered hepatic lipid metabolism in non-alcoholic fatty liver disease

Background & aims: Recent experimental models and epidemiological studies suggest that specific environmental contaminants (ECs) contribute to the initiation and pathology of nonalcoholic fatty liver disease (NAFLD). However, the underlying mechanisms linking EC exposure with NAFLD remain poorly understood and there is no data on their impact on the human liver metabolome. Herein, we hypothesized that exposure to ECs, particularly perfluorinated alkyl substances (PFAS), impacts liver metabolism, specifically bile acid metabolism. Methods: In a well-characterized human NAFLD cohort of 105 individuals, we investigated the effects of EC exposure on liver metabolism. We characterized the liver (via biopsy) and circulating metabolomes using 4 mass spectrometry-based analytical platforms, and measured PFAS and other ECs in serum. We subsequently compared these results with an exposure study in a PPARa-humanized mouse model. Results: PFAS exposure appears associated with perturbation of key hepatic metabolic pathways previously found altered in NAFLD, particularly those related to bile acid and lipid metabolism. We identified stronger associations between the liver metabolome, chemical exposure and NAFLD-associated clinical variables (liver fat content, HOMA-IR), in females than males. Specifically, we observed PFAS-associated upregulation of bile acids, triacylglycerols and ceramides, and association between chemical exposure and dysregulated glucose metabolism in females. The murine exposure study further corroborated our findings, vis-a-vis a sex-specific association between PFAS exposure and NAFLD-associated lipid changes. Conclusions: Females may be more sensitive to the harmful impacts of PFAS. Lipid-related changes subsequent to PFAS exposure may be secondary to the interplay between PFAS and bile acid metabolism. Lay summary: There is increasing evidence that specific environmental contaminants, such as perfluorinated alkyl substances (PFAS), contribute to the progression of non-alcoholic fatty liver disease (NAFLD). However, it is poorly understood how these chemicals impact human liver metabolism. Here we show that human exposure to PFAS impacts metabolic processes associated with NAFLD, and that the effect is different in females and males. (C) 2021 The Author(s). Published by Elsevier B.V. on behalf of European Association for the Study of the Liver.Peer reviewe

Environmental chemical-induced bone marrow B cell apoptosis: Death receptor-independent activation of a caspase-3 to caspase-8 pathway

ABSTRACT Programmed cell death is a critical process in B lymphocyte development. Premature apoptosis in developing B cells could affect the repertoire and number of mature B cells produced. Of particular concern is the ability of environmentally ubiquitous polycyclic aromatic hydrocarbons (PAH) to induce B cell apoptosis within the bone marrow microenvironment in a clonally nonspecific way. Here, models of bone marrow B cell development were used to assess the role of the "extrinsic" apoptosis pathway in PAH-induced apoptosis and to compare PAH-induced apoptosis with that induced during clonal deletion. A

Myasthenia gravis and pregnancy: clinical implications and neonatal outcome

BACKGROUND: The myasthenia gravis is twice as common in women as in men and frequently affects young women in the second and third decades of life, overlapping with the childbearing years. Generally, during pregnancy in one third of patients the disease exacerbates, whereas in two thirds it remains clinically unchanged. Complete remission can occur in some patients. METHODS: To describe the clinical course, delivery and neonatal outcome of 18 pregnant women with the diagnosis of myasthenia gravis. Retrospective chart review of pregnant patients with myasthenia gravis, followed at the National Institute of Perinatology in Mexico City over an 8-year period. Data was abstracted from the medical records on the clinical course during pregnancy, delivery and neonatal outcome. RESULTS: From January 1, 1996 to December 31, 2003 18 patients with myasthenia gravis were identified and included in the study. The mean ± SD maternal age was 27.4 ± 4.0 years. During pregnancy 2 women (11%) had an improvement in the clinical symptoms of myasthenia gravis, 7 women (39%) had clinical worsening of the condition of 9 other patients (50%) remained clinically unchanged. Nine patients delivered vaginally, 8 delivered by cesarean section and 1 pregnancy ended in fetal loss. Seventeen infants were born at mean ± SD gestational age of 37.5 ± 3.0 weeks and a mean birth weight of 2710 ± 73 g. Only one infant presented with transient neonatal myasthenia gravis. No congenital anomalies were identified in any of the newborns. CONCLUSIONS: The clinical course of myasthenia gravis during pregnancy is variable, with a significant proportion of patients experiencing worsening of the clinical symptoms. However, neonatal transient myasthenia was uncommon in our patient population

Exposure to environmental contaminants is associated with altered hepatic lipid metabolism in non-alcoholic fatty liver disease

Background & aimsRecent experimental models and epidemiological studies suggest that specific environmental contaminants (ECs) contribute to the initiation and pathology of NAFLD. However, the underlying mechanisms linking EC exposure with NAFLD remain poorly understood and there is no data on their impact on the human liver metabolome. Herein, we hypothesized that exposure to ECs, particularly perfluorinated alkyl substances (PFAS), impacts liver metabolism, specifically bile acid metabolism.MethodsIn a well-characterized human NAFLD cohort of 105 individuals, we investigated the effects of EC exposure on liver metabolism. We characterized the liver (via biopsy) and circulating metabolomes using four mass spectrometry-based analytical platforms, and measured PFAS and other ECs in serum. We subsequently compared these results with an exposure study in a PPARα-humanized mouse model.ResultsPFAS exposure appears associated with perturbation of key hepatic metabolic pathways previously found altered in NAFLD, particularly as regards bile acid and lipid metabolism. We identified stronger associations between the liver metabolome, chemical exposure and NAFLD-associated clinical variables (liver fat content, HOMA-IR), in female subjects versus males. Specifically, we observed PFAS-associated up-regulation of bile acids, triacylglycerols and ceramides, and association between chemical exposure and dysregulated glucose metabolism in females. The murine exposure study further corroborated our findings, vis-à-vis a sex-specific association between PFAS exposure and NAFLD-associated lipid changes.ConclusionsFemales may be more sensitive to the harmful impacts of PFAS. Lipid-related changes subsequent to PFAS exposure may be secondary to the interplay between PFAS and bile acid metabolism.</p

Reproducibility of adipogenic responses to metabolism disrupting chemicals in the 3T3-L1 pre-adipocyte model system: An interlaboratory study

The 3T3-L1 murine pre-adipocyte line is an established cell culture model for screening Metabolism Disrupting Chemicals (MDCs). Despite a need to accurately identify MDCs for further evaluation, relatively little research has been performed to comprehensively evaluate reproducibility across laboratories, assess factors that might contribute to varying degrees of differentiation between laboratories (media additives, plastics, cell source, etc.), or to standardize protocols. As such, the goals of this study were to assess interlaboratory variability of efficacy and potency outcomes for triglyceride accumulation and pre-adipocyte proliferation using the mouse 3T3-L1 pre-adipocyte cell assay to test chemicals. Ten laboratories from five different countries participated. Each laboratory evaluated one reference chemical (rosiglitazone) and three blinded test chemicals (tributyltin chloride, pyraclostrobin, and bisphenol A) using: 1) their Laboratory-specific 3T3-L1 Cells (LC) and their Laboratory-specific differentiation Protocol (LP), 2) Shared 3T3-L1 Cells (SC) with LP, 3) LC with a Shared differentiation Protocol (SP), and 4) SC with SP. Blinded test chemical responses were analyzed by the coordinating laboratory. The magnitude and range of bioactivities reported varied considerably across laboratories and test conditions, though the presence or absence of activity for each tested chemical was more consistent. Triglyceride accumulation activity determinations for rosiglitazone ranged from 90 to 100% across test conditions, but 30–70 % for pre-adipocyte proliferation; this was 40–80 % for triglyceride accumulation induced by pyraclostrobin, 80–100 % for tributyltin, and 80–100 % for bisphenol A. Consistency was much lower for pre-adipocyte proliferation, with 30–70 % active determinations for pyraclostrobin, 30–50 % for tributyltin, and 20–40 % for bisphenol A. Greater consistency was observed for the SC/SP assessment. As such, working to develop a standardized adipogenic differentiation protocol represents the best strategy for improving consistency of adipogenic responses using the 3T3-L1 model to reproducibly identify MDCs and increase confidence in reported outcomes.Over-arching project supported by grants [R01 ES016099 to HMS; R00 ES030405 to CDK] from the National Institute of Environmental Health Sciences (NIEHS); University of Turin; European Union's Horizon 2020 research and innovation program under grant agreement GOLIATH No. 825489; Brunel University London; NIEHS (1K22ES026208 and R01ES027863); NIEHS (Z0ES102785); Spanish Institute of Health Carlos III (grant FIS-PI16/01812)