Effect of thermoneutral housing conditions on the hepatotoxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in male C57BL/6 mice

Typical rodent toxicology experiments are performed at human thermoneutral temperatures (thermostandard; TS = 20 – 24° C). Reports have indicated that investigation of metabolic endpoints at mouse thermoneutral temperatures (T­NM = 28 – 30° C) improves translation to humans when they are not chronically cold-stressed. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has been shown to dose-dependently induce metabolic disease in mice at including liver steatosis, steatohepatitis, and periportal fibrosis. To test whether dose-dependent hepatotoxicity caused by TCDD is altered by housing temperature we gavaged male C57BL/6 mice every 4 days for 28 days with TCDD (0.03 – 30 µg/kg) or sesame oil vehicle at TS and TNM. Mice held at TNM exhibited less marked relative liver weight gain and histopathology with milder steatosis, immune cell infiltration, hepatocellular hypertrophy, and an absence of biliary hyperplasia compared to mice held at Ts. Mice housed in both temperature conditions had a similar dose-dependent decrease in total cholesterol, but mice at TS had more elevated serum ALT and decreased glucose and triglycerides at 30 µg/kg TCDD. Mice at TNM had fewer differentially expressed genes (DEGs) than TS, although most of the DEGs at TNM were shared with the DEGs at TS. Benchmark Dose (BMD) analysis performed on the DEGs revealed largely similar trends, however, the most induced DEGs, which constituted some of the AhR-battery genes (e.g. Cyp1a1), exhibited lower BMDs at TNM. Moreover, at 30 µg/kg TCDD both sets of mice had increased pro-inflammatory cytokines. The only cytokine which was clearly different between the two sets of mice irrespective of dose was VEGF-A. Overall, mice held at TNM showed reduced severity at equivalent doses to mice held at TS. Beginning on post-natal day (PND) 30, male C57BL/6 mice were repeatedly gavaged at 28° C every 4 days for 28 days with some dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin (0.03 - 30 µg/kg TCDD) or sesame oil vehicle (n = 5 per dose) and sacrificed on PND 58 with CO2. Gavaging and tissue collection always occurred between ZT0 and ZT3. Tissues were flash frozen in liquid nitrogen and stored at -80°C. For total RNA extraction using trizol, 100 mg was used per liver sample. Only samples which passed Nanodrop and RIN quality control were used. 150 bp paired-end RNA reads were sequenced using a NovoSeq 6000.