Hepatic Effects of Pharmacological Doses of Hydroxy-Cobalamin[c-lactam] in Mice

Abstract

The vitamin B12 analog hydroxy-cobalamin[c-lactam] (HCCL) impairs hepatic mitochondrial protein synthesis and function of the electron transport chain in rats. We aimed to establish an in vivo model for mitochondrial dysfunction in mice, which could be used to investigate hepatotoxicity of mitochondrial toxicants. In a first step, we performed a dose-finding study in mice treated with HCCL 0.4 mg/kg and 4 mg/kg i.p. for two to four weeks. The plasma methylmalonate concentration was strongly increased at 4 mg/kg starting at three weeks of treatment. We subsequently treated mice daily with 4 mg/kg HCCL i.p. for three weeks and characterized liver function and histology as well as liver mitochondrial function. We found an increase in liver weight in HCCL-treated mice, which was paralleled by hepatocellular accumulation of triglycerides. In liver homogenate of HCCL-treated mice, the complex I activity of the electron transport chain was reduced, most likely explaining hepatocellular triglyceride accumulation. The activity of CPT1 was not affected by methylmalonyl-CoA in isolated liver mitochondria. Despite impaired complex I activity, mitochondrial superoxide anion production was not increased and the hepatocellular glutathione (GSH) pool was maintained. Finally, the mitochondrial DNA content was not altered with HCCL treatment. In conclusion, treatment of mice with HCCL is associated with increased liver weight explained by hepatocellular triglyceride accumulation. Hepatocellular fat accumulation is most likely a consequence of impaired activity of the mitochondrial electron transport chain. The impairment of complex I activity is not strong enough to result in ROS accumulation and reduction of the GSH stores.

Fig 1. MMA plasma concentration.

MMA levels were measured with LC-MS/MS analysis using the ClinMass^® Complete Kit after mice were treated with HCCL at 0.4 mg/kg/d or 4 mg/kg/d for two, three or four weeks. Data are expressed as the mean and individual values. **p < 0.01, ***p < 0.001.

Fig 2. Characterization of the animals.

(A) Body weight. (B) Daily food intake. (C) Liver weight. (D) Liver weight/body weight ratio. Data are expressed as mean ± SEM. *p < 0.05.

Fig 3. Cell proliferation and histological stainings.

(A) PAS and oil red stainings of liver sections from HCCL-treated (4 mg/kg/day) and control mice. (B) Histological quantification of PAS staining (reflecting liver glycogen) (C) Histological quantification of oil red staining (reflecting lipids). (D) Ki-67 protein content as a marker for cell proliferation. Data are expressed as mean ± SEM. ***p < 0.001.

Fig 4. CPT1 activity.

Mouse liver mitochondria from control mice were acutely exposed to increased concentrations of MM-CoA. Amiodarone and malonyl-CoA (M-CoA) were used as positive controls. Values represent CPT1 activity relative to the control of at least three independent experiments. Differences between the control and incubations containing toxicants were calculated with a one-way ANOVA followed by a Dunnett’s post test. Statistical significance is indicated as *p < 0.05 or **p < 0.01.

Fig 5. Mitochondrial function.

(A) Mitochondrial membrane potential (Δψ_m) was measured with the [phenyl-³H]-tetra-phenylphosphonium bromide uptake assay in isolated liver mitochondria of HCCL-treated and control mice. (B) Electron transport chain complex activities in mouse liver homogenate of both groups. After addition of substrates and inhibitors of the different complexes, complex activities were determined on an Oxygraph-2k high- resolution respirometer. HCCL: hydroxy-cobalamin[c-lactam]. Data are expressed as mean ± SEM. *p < 0.05.

Fig 6. Evaluation on oxidative stress.

(A) Mitochondrial superoxide production was measured with MitoSOX^™ Red reagent in isolated liver mitochondria in mice treated or not with HCCL. (B) Reduced glutathione (GSH) in the liver of HCCL-treated and control mice, determined with the luminescent GSH-Glo Glutathione assay. (C) Hepatic concentrations of TBARS of mice based on the formation of malondialdehyde. (D) DNA expression of a mitochondrial (ND-1) and a nuclear gene (36B4) determined by qRT-PCR. Data are expressed as mean ± SEM.