Characterizing the Impact of Simvastatin on TCDD-Induced Liver Injury Utilizing Single-Nuclei RNA Sequencing

Objective: 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is associated with metabolic syndrome (MetS) in humans and hepatic effects similar to non-alcoholic fatty liver disease through activation of the aryl hydrocarbon receptor (AHR) pathway. Dysregulation of cholesterol homeostasis, an aspect of MetS, is linked to NAFLD pathogenesis, and TCDD exposure is also linked to the suppression of genes that encode key cholesterol biosynthesis steps and changes in serum cholesterol levels. In a previous experiment, treating mice with TCDD in the presence of simvastatin, a 3-Hydroxy-3-Methylglutaryl-CoA Reductase (HMGCR) competitive inhibitor, increased toxicity in male mice, compared to TCDD alone. The aim of this study was to deduce a possible mechanism(s) for this increased liver injury. Methods: Male C57Bl/6 mice were treated with vehicle control or TCDD via oral gavage and were fed either standard or simvastatin-laced chow. Single nuclei RNA sequencing (snRNASeq) was performed in mouse liver to investigate the impact of treatment on distinct liver cell (sub)types that may explain differences in pathology between treatments. Results: We demonstrated that co-treated mice experienced exacerbated wasting and increased AHR activation compared to TCDD alone. Furthermore, relative proportions of cell (sub)types were different between TCDD alone and co-treated mice including important mediators of NAFLD progression such as hepatocytes and Kupffer cells. Co-treated mice promoted upregulation of lipid metabolism in hepatocytes, which is consistent with decreased fat accumulation seen in previous studies. Interestingly, the proportion of plasmacytoid dendritic cells (pDCs) increased in only co-treated mice, which may play an important role in the differences in immune cell infiltration between treatment groups. Conclusions: Our results suggest simvastatin co-treatment promotes a worse prognosis of TCDD-induced injury in mice and alterations in immune infiltration between treatments may influence liver injury severity. Nuclei were isolated from frozen livers of male C57BL/6 mice either gavaged with sesame oil vehicle or 30 ug/kg TCDD every 4 days for 28 days and fed with normal chow or chow laced with 500 mg/kg simvastatin beginning 3 days prior to first TCDD treatment