Drug-induced liver injury is not readily detectable using conventional animal studies during pre-clinical drug development. To address this problem, other researchers have proposed the use of co-administration of lipopolysaccharide (LPS), an endotoxin produced by gram-negative bacteria, and a drug. Using this approach, liver injury that is otherwise not detected following drug administration alone can be successfully identified. Previous studies have demonstrated that such injury is suppressed by heparin; therefore, the mechanism may involve coagulation-dependent ischemia. However, it has not been established how LPS-induced ischemia might sensitize hepatocytes to a potentially hepatotoxic drug. In the present study, we aimed to determine the effect of LPS-induced ischemia on liver mitochondrial function and downstream toxicologic responses. Consistent with previous findings, plasma alanine transaminase (ALT) activity was higher in rats co-administered with LPS (1 mg/kg) and diclofenac (100 mg/kg), but reduced by heparin. Liver mRNA expression of Hmox1, encoding heme oxygenase-1, an oxidative stress indicator, was three times higher at 2 hr after LPS administration. Furthermore, respiratory activity via mitochondrial complex II, lipid peroxidation in mitochondria, and the susceptibility to mitochondrial permeability transition pore opening in response to diclofenac administration were significantly increased by LPS administration. The increase in plasma ALT activity and the sensitization to mitochondrial permeability transition pore opening were reduced by the co-administration of heparin. In conclusion, LPS-induced transient ischemia disrupts respiratory chain complex activities, enhances reactive oxygen species production, especially in mitochondria, and sensitizes mitochondria to permeability transition pore opening when testing a potentially hepatotoxic drug in vivo.
Drug-induced mitochondrial dysfunction can lead to severe adverse effects. Accordingly, new in vitro assay systems for assessing mitochondrial-related toxicity are required. Current systems evaluate drug-induced mitochondrial dysfunction based on cell death. However, if mitochondria are damaged without cell death, these methods run the risk of overlooking toxic or dangerous compounds. To solve this problem, we attempted to measure morphological changes semi-quantitatively by transmission electron microscopy and to detect subtle changes in mitochondrial function. To this end, we exposed HepG2 cells cultured in galactose-containing medium to benzbromarone (BBR), which impairs mitochondrial function. After 24 hr of BBR exposure, we compared the rate of cell death between galactose and glucose cultures. Before the onset of cell death, BBR increased the ratio of damaged mitochondria to a greater extent in galactose-cultured than glucose-cultured HepG2 cells. Our results suggested that this new in vitro assay system could detect mitochondrial-related toxicity before the onset of cell death.
Lipopolysaccharide (LPS), a component of the outer membrane of gram-negative bacteria, induces various biological reactions in vivo. Our previous study suggested that LPS administration disrupts respiratory chain complex activities, enhances reactive oxygen species production, especially in the liver mitochondria, and sensitizes mitochondrial permeability transition (MPT) pore opening in rats. However, it is unknown whether LPS-induced MPT pore opening in rats is similarly observed in mice and whether the mechanism is the same. LPS administration to mice increased not only cyclosporin A-sensitive swelling (MPT pore opening) susceptibility, but also induced cyclosporin A-insensitive basal swelling, unlike in rats. In addition, respiratory activity observed after adding ADP was significantly decreased. Based on these results, we further investigated the role of adenine nucleotide translocase (ANT). Carboxyatractyloside (CATR; an ANT inhibitor) treatment decreased respiratory activity after ADP was added in vehicle-treated mitochondria similarly to LPS administration. Additionally, CATR treatment increased MPT pore opening susceptibility in LPS-treated mitochondria compared to that of vehicle-treated mitochondria. Our study shows that ANT maintained a c-state conformation upon LPS administration, which increased MPT pore opening susceptibility in mice. These results suggest that LPS enhances MPT pore opening susceptibility across species, but the mechanism may differ between rat and mouse.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.