Preliminary study of the genotoxic potential of homocysteine in human lymphocytes in vitro

Homocysteine (Hcy), an immediate precursor of methionine (Met), is considered a risk factor for cardiovascular disease, Alzheimer's disease and neural tube defects. Hey concentration is also reported to correlate positively with the micronucleus index in lymphocytes in vivo, a marker of chromosome damage. However, it is unclear whether Hey is genotoxic or simply a biomarker of folate deficiency, a known cause of chromosome damage. We investigated whether high concentrations of Hey are genotoxic to human lymphocytes in vitro using the cytokinesis-block micronucleus assay. Eighteen lymphocyte cultures were initiated in Met-free and serum-free RPMI 1640 medium for each of four male volunteers aged 22-23 years. At 0, 24, 44 and 72 h, cultures were spiked with L-Hcy or L-Met to achieve concentrations ranging between 50 and 400 muM. The concentration of Hey at 96 h ranged from 19.45 +/- 2.34 to 149.02 +/- 28.16 muM in Hey cultures and 0.91 +/- 0.17 to 2.15 +/- 0.9 muM in Met cultures spiked with 50 and 400 muM of metabolite, respectively, Forty-four hours after mitogen stimulation, cytokinesis was inhibited with cytochalasin B. After 96 h, cells were transferred to microscope slides and the frequency of micronucleated-binucleate and necrotic cells was scored. Neither Hcy(P = 0.24) nor Met(P = 0.93) had an apparent dose effect on micronucleus frequency. However, when data were pooled, micronucleus frequency was moderately higher (50.1%) in Hcy- than in Met-spiked cultures (P = 0.04; paired t-test). Hey concentration was positively correlated with necrosis (P < 0.0005; r(2) = 0.276), however, when data were pooled, levels of necrosis were higher in Met- than in Hey-spiked cultures (P = 0.01; paired t-test), Further research is required to define more clearly the genotoxic and cytotoxic potential of homocysteine and its metabolites.