H&E re Table S2

This study was performed under a licence by the UK Home Office with local Animal Welfare and Ethical Review Body (Newcastle University) approval. Adult C57Bl6 male mice (5 months of age) were purchased from Charles River and housed in the Comparative Biology Centre at Newcastle University. Mice (up to five per cage) were housed in Maxiseal 420 cm² mouse cages (Arrowmight, Hereford, UK) in an enriched environment (nesting material, chew sticks and cardboard tubes) and were provided with food (RM3 Special Diet Services, UK) and water ad libitum in an air-conditioned environment on a 12 h light/dark cycle with regulated humidity (50% ± 10%) and temperature (23^oC ± 1^oC). Cages were randomly assigned to 3 groups and provided with drinking water ad libitum (control, 5 animals) or either drinking water containing BMI (10 animals) or M8OI (10 animals) at a concentration of 440 mg/L (prepared fresh every 4 weeks). Aliquots were tested by HPLC and both remained stable in drinking water for at least 4 weeks. No vehicle was required in these studies. One control mouse was culled during the study due to a handling error. There are no data available on potential exposure levels in man. A single dose was therefore chosen to reduce the number of animals used prior to any future dose-response study to determine a threshold for any effects. The dose chosen for this study was based on acute exposure via i.p. administration, as a dose likely to give no more than mild renal and hepatic effects. This route was chosen because the oral route is the most relevant route of exposure in man and relevant to test potential direct effects on the microbiota and liver. Based on a default factor for sub-chronic studies (0.15), it can be estimated that mice were exposed to 66 mg of BMI or M8OI/kg bw. Mice were exposed for 18 weeks prior to cervical dislocation and removal of tissues, blood, and urine (by direct extraction from the bladder) for analyses. Tissues were fixed in 4% formalin in 1xPBS, processed, embedded in wax and 4μm sections stained with haematoxylin and eosin (H&E) essentially as previously outlined (Marek CJ, Tucker SJ, Konstantinou DK, Elrick LJ, Haefner D, Sigalas C, Murray GI, Goodwin B, Wright MC. Pregnenolone-16alpha-carbonitrile inhibits rodent liver fibrogenesis via PXR (pregnane X receptor)-dependent and PXR-independent mechanisms. Biochem J. 2005; 387: 601-8). Each section is representative of a view from a different animal.