In-vitro and in-vivo functional observation studies to establish therapeutic potential of alpha-ketoglutarate against methotrexate induced liver injury

BACKGROUND: Methotrexate (MTX) is widely used in chemotherapy but its associated hepatotoxicity is a major complication, limiting its use. This study evaluates possible therapeutic effect of oral alpha-ketoglutarate (AKG) supplementation against MTX-induced hepatotoxicity. METHODS: HepG2 cells were used to evaluate in-vitro cyto-protection conferred by AKG against MTX induced cytotoxicity. For in-vivo animal study, rats were divided into three groups. Group-I served as control. Group-II animals were administered single intraperitoneal injection of MTX (20 mg/kg/body weight), while Group-III received MTX as in group-II followed by oral AKG (2 gm/kg body weight) for 5 days. (99m)Tc-Mebrofenin hepatobiliary study was performed under a gamma camera to determine real time functional status of rats’ livers. Multiple parameters concerning hepatic mebrofenin uptake and excretion, including T(peak) and T(1/2 peak) in control and treated animals were determined. Biochemical analysis of the liver homogenate in terms of hepatic enzyme activities in serum, antioxidant status, tissue factor activity, tissue collagen content and histological analysis of the liver tissue were also done. RESULTS: AKG supplementation significantly reversed MTX induced derangement in activities of serum liver enzymes [ALT and ALP (p = 0.003); AST (p = 0.005)], antioxidant status [LPO and GSH (p = 0.005); CAT (p = 0.004)], tissue factor activity (p = 0.005) and tissue collagen content (p = 0.005). Functional imaging confirmed that hepatic retention and fractional biliary excretion were significantly abnormal in MTX treated group (T(peak): 234 s ± 40 s; T(1/2 peak): 846sec ± 32sec) as compared to AKG supplemented group (T(peak): 144 s ± 35sec; T(1/2peak): 468sec ± 27sec). Hepatic extraction fraction (HEF) was 92.2 ± 1.8%, 48.7 ± 2.6% and 69.8 ± 4.3% in control, MTX and AKG supplemented rats respectively. CONCLUSION: (99m)Tc-mebrofenin imaging strongly suggests therapeutic action of AKG in protecting liver damage by MTX in rats. Functional imaging parameters correlated well with biochemical and histopathological findings.