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

Abstract

Homocysteine (Hcy), an immediate precursor of methionine (Met), is considered a risk factor for cardiovascular disease, Alzheimer's disease and neural tube defects. Hcy concentration is also reported to correlate positively with the micro-nucleus index in lymphocytes in vivo, a marker of chromosome damage. However, it is unclear whether Hcy is genotoxic or simply a biomarker of folate deficiency, a known cause of chromosome damage. We investigated whether high concentrations of Hcy are genotoxic to human lymphocytes in vitro using the cytokinesis-block micro-nucleus 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 μM. The concentration of Hcy at 96 h ranged from 19.45 ± 2.34 to 149.02 ± 28.16 μM in Hcy cultures and 0.91 ± 0.17 to 2.15 ± 0.9 μM in Met cultures spiked with 50 and 400 μM 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). Hcy concentration was positively correlated with necrosis (P<0.0005; r2=0.276), however, when data were pooled, levels of necrosis were higher in Met- than in Hcy-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.