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Inhibition of B12‐dependent methionine synthase activity in HepG2 cells over‐expressing CYP2E1

Hyperhomocysteinemia (HHCY) is strongly associated with alcohol abuse and alcoholic liver disease (ALD). However, the molecular mechanisms for the production of elevated homocysteine in alcohol abusers are largely unknown. We hypothesize that HHCY in ALD results from a loss of B12‐dependent methionine synthase (MS). We tested this hypothesis by treating control C34 HepG2 cells and cytochrome P450 2E1 (CYP2E1) over‐expressing cells (E47) with 50 mM ethanol (EtOH) for 0–6 days and measured MS activity and its expression, and intra‐ and extra‐cellular homocysteine (Hcy). Initially, MS activity was 18% lower in E47 cells compared to C34 cells (0.158 ± 0.015 vs 0.192 ± 0.016 nmol/min/mg, p=0.06). However, after 2, 4 and 6 days of EtOH treatment MS activity was further reduced in E47 to 0.133, 0.132 and 0.125 nmol/min/mg protein, respectively (p ≤ 0.05), but there was no change in C34 cells. Using qRT‐PCR, MS mRNA showed 1.5‐fold and 2.0‐fold increases in C34 and E47 cells, respectively, after 2 days of EtOH. The largest increase in mRNA was detected after 6 days of EtOH (E47: 6.2 fold; C34: 4.6 fold). Thus, MS mRNA expression was inversely proportional to its activity. Extracellular Hcy was markedly increased in EtOH treated E47. In summary, MS activity is significantly decreased in response to EtOH in cells over‐expressing CYP2E1, resulting in significant increases in Hcy export and MS mRNA expression. (Supported by NIH AA17671).