Role of Gut Bacteria-Derived Metabolite in Acetaminophen-Induced Liver Injury

Drug-induced liver injury (DILI) refers to the impaired liver function caused by medical treatment and is the leading cause of the withdrawal of approved drugs from the market or black box warning. An increase of evidence has suggested that gut microbiota potentially affect the host's susceptibility to DILI. Gut bacteria produce a large amount of small molecules that enter the systemic circulation via the portal vein and make profound impacts on host physiology and pathology. Using acetaminophen (APAP) as a DILI model drug, this study aims to explore the roles of gut bacteria-derived metabolites in APAP-induced liver injury. Our group previously identified 16 metabolites whose abundance are associated with the different host susceptibility of two vendors of mice. In this work, we screened the effects of these 16 metabolites on APAPinduced cytotoxicity on primary mouse hepatocytes and identified 3,4-hydroxyphenyl-propionic acid (HPPA) and 5-aminovalerate (5-AV) that decreased and increased the cell death, respectively. In mice, administration of HPPA (50 mg/kg) by intra-peritoneal injection significantly attenuated the level of APAP-induced liver injury. To elucidate the underlying mechanism of HPPA’s protective effect, we performed a time profile experiment in which mice were euthanized at multiple different time points from 0.5 to 48 hours after APAP treatment; these time points basically cover all the stages of the progression of APAP hepatotoxicity from onset to recovery. It has been well-established that APAP hepatotoxicity is initiated from the excessive production of a highly-reactive metabolite N-acetyl p-benzoquinone imide (NAPQI) whose amount is decided together by hepatic glucuronidation, sulfation, CYP450-mediated APAP metabolism and detoxification of hepatic glutathione (GSH). Measurements of these events in the time profile experiment samples revealed that HPPA’s protective effect is not attributed to the alteration of NAPQI amount. NAPQI depletes hepatic GSH and triggers oxidative stress and activation of redox-sensitive kinases, leading to c-Jun N-terminal kinase phosphorylation (JNK). The phosphor-JNK (pJNK) is known to play a central role in APAP hepatotoxicity. Our data showed that HPPA significantly decreased APAP-induced JNK phosphorylation, which contributes to alleviated liver injury in HPPA-treated mice. Previously our group found another gut bacterial metabolite phenylpropionic acid (PPA), that protects mice against APAP-induced liver injury. PPA and HPPA are produced by the same bacterial biosynthesis pathway. As gut bacteria determine the host exposure of PPA and HPPA, we aimed to identify the factors that are responsible for PPA and HPPA production in mouse gut microbiota. Using biochemical and bioanalytical approaches, we identified the presence of an amino acid reductive metabolism pathway (called as the Fld pathway) in mouse gut microbiome that metabolize L-phenylalanine and L-tyrosine to PPA and HPPA, respectively. The bacteria species that harbour the Fld pathway remained to be identified in mouse gut microbiome. Inoculation of a PPA and HPPA-producing bacterium Clostridium sporogenes remarkably increased the PPA and HPPA levels in mouse systemic circulation, providing the rationale to develop probiotics as a preventative strategy of DILI. In summary, this work has identified that HPPA alleviates APAP-induced liver injury by inhibiting JNK phosphorylation, which broadened the understanding of the biological functions of gut bacteria-derived metabolites.