Increased lymphocyte micronucleus frequency in early pregnancy is associated prospectively with pre-eclampsia and/or intrauterine growth restriction.

Genome stability is essential for normal foetal growth and development. To date, genome stability in human lymphocytes has not been studied in relation to late pregnancy diseases, such as pre-eclampsia (PE) and intrauterine growth restriction (IUGR), which can be life-threatening to mother and baby and together affect >10% of pregnancies. We performed a prospective cohort study investigating the association of maternal chromosomal damage in mid-pregnancy (20 weeks gestation) with pregnancy outcomes. Chromosome damage was measured using the cytokinesis-block micronucleus cytome (CBMNcyt) assay in peripheral blood lymphocytes. The odds ratio for PE and/or IUGR in a mixed cohort of low- and high-risk pregnancies (N = 136) and a cohort of only high-risk pregnancies (N = 91) was 15.97 (P = 0.001) and 17.85 (P = 0.007), respectively, if the frequency of lymphocytes with micronuclei (MN) at 20 weeks gestation was greater than the mean + 2 SDs of the cohort. These results suggest that the presence of lymphocyte MN is significantly increased in women who develop PE and/or IUGR before the clinical signs or symptoms appear relative to women with normal pregnancy outcomes. The CBMNcyt assay may provide a new approach for the early detection of women at risk of developing these late pregnancy diseases and for biomonitoring the efficacy of interventions to reduce DNA damage, which may in turn ameliorate pregnancy outcome.

The cytokinesis-block micronucleus assay as a biological dosimeter in spleen and peripheral blood lymphocytes of the mouse following acute whole-body irradiation.

To evaluate the application of the cytokinesis-block (CB) micronucleus (MN) assay as a biological dosimeter following in vivo exposure to ionising radiation we determined the micronucleus frequency in spleen and peripheral blood lymphocytes of the mouse, serially, for 14 days following acute whole-body irradiation. The baseline MN frequency of spleen lymphocytes (7.86 +/- 0.68, mean +/- 1 SD) was significantly (p less than 0.001) elevated when compared to that for peripheral blood lymphocytes (4.10 +/- 0.53). Immediately after irradiation there was a substantial dose-related increase in MN, but the MN frequencies in spleen lymphocytes (120.2 +/- 9.4 for 1 Gy; 409.5 +/- 38.4 for 2 Gy) were significantly (p less than 0.009) elevated compared to those in peripheral blood lymphocytes (78.0 +/- 7.0 for 1 Gy; 200.2 +/- 10.9 for 2 Gy). During the 14 days after irradiation, the MN frequency in spleen lymphocytes declined gradually to approximately half of the value observed immediately after irradiation. By contrast the MN frequency in peripheral blood lymphocytes increased during the week after irradiation, but ultimately MN frequencies in blood and spleen became approximately the same by day 14. Study of isolated murine lymphocytes irradiated in vitro showed that the number of MN generated by a given dose of radiation was approximately 2-3 times greater than the number generated by in vivo irradiation. These results suggest that measurement of MN in vivo after irradiation can be used as an in vivo dosimeter. However, precise dosimetry is probably affected by factors such as kinetic changes in different lymphocyte populations and possibly by in vivo factors which influence sensitivity of cells to radiation.

Folate, vitamin B12, homocysteine status and chromosome damage rate in lymphocytes of older men.

Deficient levels of folic acid and vitamin B12 are associated with elevated chromosome damage rate and high concentrations of homocysteine in the blood. We have therefore performed a study to determine the prevalence of folate deficiency, vitamin B12 deficiency and hyperhomocysteinemia in 64 healthy men aged between 50 and 70 years, and evaluate the relationship of these micronutrient levels in the blood with the micronucleus frequency in peripheral blood lymphocytes. We also performed a placebo-controlled, double-blind intervention study to determine whether supplementation of the diet with a daily dose of 0.7 mg (as a supplement in cereal) or 2.0 mg (in a tablet) over a period of 4 months resulted in a significant alteration of folate status, homocysteine status and the micronucleus index. Twenty-three per cent of the men were serum folate deficient (<6.8 nmol/l), 16% were red blood cell folate deficient (<317 nmol/l), 4.7% were vitamin B12 deficient (<150 pmol/l) and 37% has plasma homocysteine levels >10 micromol/l. In total, 56% of the men had one or more abnormal blood values for folate, vitamin B12 or homocysteine. The micronucleus index of these men (n = 34) in cytokinesis-blocked binucleated cells (19.2 +/- 1.1) was significantly elevated (P = 0.02) when compared to the micronucleus index of the rest of the men who had normal levels of folate, vitamin B12 and homocysteine (16.3 +/- 1.3, n = 30). Interestingly, the micronucleus index in men with normal folate and vitamin B12, but homocysteine levels >10 micromol/l (19.4 +/- 1.7, n = 15) was also significantly higher (P = 0.05) when compared to those with normal folate, vitamin B12 and homocysteine. This novel result was also supported by the observation that the micronucleus index and plasma homocysteine were significantly (P = 0.0086) and positively correlated (r2 = 0.172) in those subjects who were not deficient in folate or vitamin B12. The micronucleus index was not significantly correlated with folate indices, but there was a significant (P = 0.013) negative correlation with serum vitamin B12 (r2 = 0.099). Daily supplementation of the diet with 0.7 mg free folic acid in cereal for 2 months followed by 2.0 mg free folic acid via a tablet produced a 4-fold increase in plasma folate, a 2.6-fold increase in red blood cell folate and a 11% reduction in plasma homocysteine; however, these changes were not accompanied by a reduction in the micronucleus index. In conclusion, it is apparent that elevated homocysteine status, in the absence of vitamin deficiency and low, but not deficient, vitamin B12 status are important risk factors for increased chromosome damage in lymphocytes.

Methylenetetrahydrofolate reductase C677T polymorphism does not alter folic acid deficiency-induced uracil incorporation into primary human lymphocyte DNA in vitro.

Methylenetetrahydrofolate reductase (MTHFR) is an enzyme which converts 5,10-methylene tetrahydrofolate (5,10-MnTHF) to 5-methyl tetrahydrofolate. A common C to T transition (C677T) in the MTHFR gene is reported to reduce the risk for colorectal cancer and acute lymphocytic leukemia in homozygotes (TTs). It is hypothesized that because TTs have reduced MTHFR activity, more 5,10-MnTHF is available to provide methyl groups for the conversion of uracil to thymidine. Folic acid deficiency causes the intracellular accumulation of dUMP and the subsequent incorporation of uracil into DNA. The removal of uracil from DNA may result in double-stranded DNA breaks, the accumulation of which is a putative risk factor for cancer. We tested whether human lymphocytes taken from TTs (n = 10) were more able to resist uracil incorporation into DNA than controls (n = 14 CCs and 6 CTs) when cultured in medium containing 12-120 nM folic acid for 9 days. DNA uracil content of these lymphocytes was measured by CG-MS. TTs and controls showed a dose-dependent increase in DNA uracil content during folic acid deficiency (P < 0.0001, R2 = 0.23 for TTs and P < 0.0001, R2 = 0.19 for controls). DNA uracil content was not different between the two groups at any of the folic acid concentrations (two-way ANOVA: media [folic acid], P < 0.0001; genotype, P = 0.4). The results show that, in this in vitro system, the MTHFR C677T polymorphism does not affect the cell's ability to resist uracil incorporation into DNA. Chromosome breakage, as measured by micronuclei, was also shown to correlate with folic acid concentration in a preliminary experiment (P < 0.0001). Although the results appear not to support the hypothesis that a reduced risk for certain cancers in TTs is due to diversion of folic acid to thymidine synthesis, differences between the in vivo and in vitro situation make this conclusion not definitive.