Assessment of usnic acid toxicity in rat primary hepatocytes using 13C isotopomer distribution analysis of lactate, glutamate and glucose

The lichen metabolite usnic acid (UA) has been promoted as a dietary supplement for weight loss, although cases of hepatotoxicity have been reported. Here we evaluated UA-associated hepatotoxicity in vitro using isolated rat hepatocytes. We measured cell viability and ATP content to evaluate UA induced cytotoxicity and applied 13C isotopomer distribution measuring techniques to gain a better understanding of glucose metabolism during cytotoxicity. The cells were exposed to 0, 1, 5 or 10μM UA concentrations for 2, 6 or 24h. Aliquots of media were collected at the end of these time periods and the 13C mass isotopomer distribution determined for CO2, lactate, glucose and glutamate.The 1μM UA exposure did not appear to cause significant change in cell viability compared to controls. However, the 5 and 10μM UA concentrations significantly reduced cell viability as exposure time increased. Similar results were obtained for ATP depletion experiments. The 1 and 5μM UA doses suggest increased oxidative phosphorylation. Conversely, oxidative phosphorylation and gluconeogenesis were dramatically inhibited by 10μM UA. Augmented oxidative phosphorylation at the lower UA concentrations may be an adaptive response by the cells to compensate for diminished mitochondrial function.