4), suggesting protection from cholestasis is a specific physiological function of endogenous serotonin. To investigate potential mechanisms underlying the action of serotonin in cholestasis, we assessed whether the serotonin-dependent protection relates to the elevated plasma bile acids in Tph1−/− mice or is rather due to a more general hepatoprotective role of the neurotransmitter. We first measured ALT levels in WT and Tph1−/− mice following exposure to CCl4.

No differences were observed at 24 and 48 hours after CCl4 treatment (Supporting Fig. 4), suggesting serotonin does not afford general protection from liver injury. To assess whether the serotonergic protection may associate with the bile pool perturbations, we next determined the hepatotoxicity of bile salts by analyzing the composition of plasma

and liver bile salts and adding corresponding Dabrafenib supplier mixtures to hepatocytic cultures. Mass spectrometry (Fig. 3A,B) revealed that about 85% GSI-IX chemical structure of the six analyzed bile salts and acids were taurine-conjugated. Exposure of rat hepatocyte cultures or mouse hepatoma cells to bile acid mixtures demonstrated that the bile acid composition as found in the plasma (about 500 μg/mL) is hepatotoxic in vitro (Fig. 3C,E). The bile acid mix found in the liver was hepatotoxic only in mouse hepatoma cells and at higher doses (Fig. 3D,F). As liver bile salts represent mostly intracellular pools, their extracellular testing may not adequately reveal their toxicity. However, bile salt toxicity was dose-dependent, suggesting the relative increase of bile acids in Tph1−/− mice is augmenting liver injury in vivo. Toxicities of individual bile salts are shown in Supporting Fig. 5. Given the toxicity of bile salts

and their ostensibly reduced clearance in Tph1−/− mice (Supporting Fig. 2), we next examined the expression of genes related to bile salt homeostasis in the liver. Three days of BDL altered the expression of most of the genes examined in the liver. However, no difference was noted between WT and Tph1−/− livers that could explain the increased bile salts and liver injury in Tph1−/− mice (Fig. 4 and Supporting Fig. 6). Notably, the major enzymes related to bile acid production (Fig. 4A), detoxification pheromone (Fig. 4B,C), and transport into plasma (Fig. 4D) were not differentially expressed between WT and Tph1−/− mice. We therefore conclude that serotonin does not affect hepatic bile salt homeostasis in cholestatic mice after 3 days of BDL. Since serotonin does not appear to regulate bile salt homeostasis in the cholestatic liver, we explored whether the kidney may account for the increased bile salt levels in Tph1−/− mice. We tested the expression of renal bile salt transporter genes after 3 days of BDL (Fig. 5).