No abstract

ABSTRACT2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a persistent environmental contaminant, induces steatosis by increasing hepatic uptake of dietary and mobilized peripheral fats, inhibiting lipoprotein export, and repressing β-oxidation. In this study, the mechanism of β-oxidation inhibition was investigated by testing the hypothesis that TCDD dose-dependently repressed straight-chain fatty acid oxidation gene expression in mice following oral gavage every 4 days for 28 days. Untargeted metabolomic analysis revealed a dose-dependent decrease in hepatic acyl-CoA levels, while octenoyl-CoA and dicarboxylic acid levels increased. TCDD also dose-dependently repressed the hepatic gene expression associated with triacylglycerol and cholesterol ester hydrolysis, fatty acid binding proteins, fatty acid activation, and 3-ketoacyl-CoA thiolysis while inducing acyl-CoA hydrolysis. Moreover, octenoyl-CoA blocked the hydration of crotonyl-CoA suggesting short chain enoyl-CoA hydratase (ECHS1) activity was inhibited. Collectively, the integration of metabolomics and RNA-seq data suggested TCDD induced a futile cycle of fatty acid activation and acyl-CoA hydrolysis resulting in incomplete β-oxidation, and the accumulation octenoyl-CoA levels that inhibited the activity of short chain enoyl-CoA hydratase (ECHS1).

2,3,7,8 –Tetrachlorodibenzo –p –dioxin (TCDD) is a persistent environmental contaminant and the prototypical ligand for the aryl hydrocarbon receptor (AhR). AhR mediates the effects of TCDD and related compounds, including the reprograming of intermediate metabolism. Untargeted metabolomics analysis of hepatic extracts prepared from mice orally gavaged with TCDD every 4 days for 28 days identified the dose –dependent induction of acrylyl –CoA, a highly reactive toxic intermediate produced during the metabolism of propionyl –CoA in the cobalamin (Cbl) –independent β –oxidation –like pathway. Acrylyl –CoA is a biomarker of inborn errors of metabolism associated with propionic and methylmalonic acidemia associated with Cbl deficiency, transport and/or defects in Cbl –dependent methylmalonyl –CoA mutase (MUT). Although TCDD repressed gene expression for both the canonical Cbl –dependent carboxylase and the alternate Cbl –independent β –oxidation –like pathways, inhibition occurred only at 30 μg/kg TCDD while acrylyl –CoA levels increased at ~3 μg/kg. In contrast, TCDD decreased serum Cbl and hepatic cobalt levels at ~3 μg/kg TCDD consistent with the dose –dependent increase in acrylyl –CoA levels. TCDD elicited negligible effects on the expression of genes associated with Cbl absorption, transport, trafficking and derivatization to 5 –deoxy –adenosylcobalamin (AdoCbl), the required MUT cofactor. In addition, TCDD induced the decarboxylation of cis –aconitate to itaconate by Acod1. Itaconate can then be activated to itaconyl –CoA, a MUT suicide inactivator that forms an adduct with AdoCbl, blocking MUT activity and reducing Cbl levels. Collectively, these results suggest MUT activity was impaired due to Cbl depletion by TCDD causing propionyl –CoA metabolism to be redirected to the alternate Cbl –independent β –oxidation –like pathway resulting in hepatic acrylyl –CoA accumulation.

Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has been associated with the induction of oxidative stress and the progression of steatosis to steatohepatitis with fibrosis. It also disrupts metabolic pathways including one-carbon metabolism (OCM) and the transsulfuration pathway with possible consequences on glutathione (GSH) levels. In this study, complementary RNAseq and metabolomics data were integrated to examine the hepatic transsulfuration pathway and glutathione biosynthesis in mice following treatment with TCDD every 4 days for 28 days. TCDD dose-dependently repressed hepatic cystathionine β-synthase (CBS) and cystathionine γ-lyase (CTH) mRNA and protein levels. Reduced CBS and CTH levels are also correlated with dose-dependent decreases in hepatic extract hydrogen sulfide (H 2 S). In contrast, cysteine levels increased consistent with the induction of Slc7a11, which encodes for the cystine/glutamate Xc − antiporter. Cotreatment of primary hepatocytes with sulfasalazine, a cystine/glutamate Xc − antiporter inhibitor, decreased labeled cysteine incorporation into GSH with a corresponding increase in TCDD cytotoxicity. Although reduced and oxidized GSH levels were unchanged following treatment due to the induction of GSH/GSSG efflux transporter by TCDD, the GSH:GSSG ratio decreased and global protein S-glutathionylation levels in liver extracts increased in response to oxidative stress along with the induction of glutamate-cysteine ligase catalytic subunit (Gclc), glutathione synthetase (Gss), glutathione disulfide reductase (Gsr), and glutathione transferase π (Gstp). Furthermore, levels of ophthalmic acid, a biomarker of oxidative stress indicating GSH consumption, were also increased. Collectively, the data suggest that increased cystine transport due to cystine/glutamate Xc − antiporter induction compensated for decreased cysteine production following repression of the transsulfuration pathway to support GSH synthesis in response to TCDD-induced oxidative stress.