Cystine/Glutamate Xc- Antiporter Induction Compensates for Transsulfuration Pathway Repression by 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) to Ensure Cysteine for Hepatic Glutathione Biosynthesis.

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 ß-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 (H2S). 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.

Dioxin-elicited decrease in cobalamin redirects propionyl-CoA metabolism to the ß-oxidation-like pathway resulting in acrylyl-CoA conjugate buildup.

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a persistent environmental contaminant that induces diverse biological and toxic effects, including reprogramming intermediate metabolism, mediated by the aryl hydrocarbon receptor. However, the specific reprogramming effects of TCDD are unclear. Here, we performed targeted LC-MS analysis of hepatic extracts from mice gavaged with TCDD. We detected an increase in S-(2-carboxyethyl)-L-cysteine, a conjugate from the spontaneous reaction between the cysteine sulfhydryl group and highly reactive acrylyl-CoA, an intermediate in the cobalamin (Cbl)-independent ß-oxidation-like metabolism of propionyl-CoA. TCDD repressed genes in both the canonical Cbl-dependent carboxylase and the alternate Cbl-independent ß-oxidation-like pathways as well as inhibited methylmalonyl-CoA mutase (MUT) at lower doses. Moreover, TCDD decreased serum Cbl levels and hepatic cobalt levels while eliciting negligible effects on gene expression associated with Cbl absorption, transport, trafficking, or derivatization to 5'-deoxy-adenosylcobalamin (AdoCbl), the required MUT cofactor. Additionally, TCDD induced the gene encoding aconitate decarboxylase 1 (Acod1), the enzyme responsible for decarboxylation of cis-aconitate to itaconate, and dose-dependently increased itaconate levels in hepatic extracts. Our results indicate MUT inhibition is consistent with itaconate activation to itaconyl-CoA, a MUT suicide inactivator that forms an adduct with adenosylcobalamin. This adduct in turn inhibits MUT activity and reduces Cbl levels. Collectively, these results suggest the decrease in MUT activity is due to Cbl depletion following TCDD treatment, which redirects propionyl-CoA metabolism to the alternate Cbl-independent ß-oxidation-like pathway. The resulting hepatic accumulation of acrylyl-CoA likely contributes to TCDD-elicited hepatotoxicity and the multihit progression of steatosis to steatohepatitis with fibrosis.