Inhibition of the hepatic vitamin B12-dependent methionine-salvage pathway by trichloroethylene metabolites accounts for the formic aciduria in the rat

The industrial solvent trichloroethylene (TCE) produces a marked formic aciduria in male and female F344 rats and in male C57Bl mice following single or multiple dosing. The two major metabolites of TCE formed by cytochromes P450 metabolism also produce formic aciduria. The quantity of formic acid excreted was about 2-fold higher following trichloroacetic acid (TCA) compared to trichloroethanol (TCE-OH) or TCE, at similar doses of 16mg/kg/day for 3 days. Prior treatment of male F344 rats with 1-aminobenzotriazole a cytochrome P450 inhibitor, followed by TCE, completely prevented the formic aciduria but had no effect on formic acid excretion produced by TCA, suggesting TCA is the proximate metabolite producing this response. Metabolism of formic acid is largely controlled by the vitamin B12 –dependent methionine salvage pathway. Transcriptomic analysis on the liver of rats dosed with 16mg/kg/day TCE for three days when compared to control liver showed nine differentially expressed genes, of particular interest was the down regulation of LMBRD1 involved in the conversion of vitamin B12 into one of two molecules, methylcobalamin (CH3Cbl) or S-adenosylcobalamin (AdoCbl). Administration of CH3Cbl or hydroxocobalamin for 3 days to rats given a single dose of TCE, lead to a reduction in formic acid in their urine. Similarly, rats given TCE followed by L-methionine for 3 days excreted less formic acid in their urine. These findings suggest an effect on the vitamin B12 –dependent methionine salvage pathway. This was supported by the finding that hepatic methionine synthase, which converts homocysteine to methionine, was inhibited following three large daily dose of TCE. We propose that TCE metabolites interact with the vitamin B12 -dependent methionine salvage pathway leading to tetrahydrofolate deficiency and increased excretion of formic acid in rat urine. All animal procedures were performed in accordance with a license issued under the animals (Scientific Procedures) Act, 1986. Wistar-derived rats where bred in the Life Science Support Unit at Liverpool John Moores University. The rats were housed in plastic cages in a room maintained at 20 °C ± 2 °C and a relative humidity of 50% ± 5% with a 12 h light-dark cycle starting at 04.00 h. Rats were allowed food (rat expanded diet), and water ad libitum. Male rats (190–240 g) 7–9 weeks of age were used. Three rats being dosed orally with TCE body weight on in corn oil. Three control animals were dosed orally daily for 3 days with corn oil at. Rats were placed individually in metabolic cages after dosing for the collection of urine, on days 1, 2 and 3 and given food and water ad libitum. Urine was collected from each rat into a container which had 0.1 ml of 10% sodium azide to prevent bacterial growth. Urine volume was determined and the pH measured using a pH meter. Twenty four hours after the last dose all rats were killed by a rising concentration of carbon dioxide. A portion about 50 mg of liver and renal-cortex were quickly harvested and immersed in RNAlater.Rats were dosed orally for 3 days with TCE in corn oil at 16mg/kg/day at 5ml/kg. Controls received corn oil alone.