Methionine-dependence phenotype in the de novo pathway in BRCA1 and BRCA2 mutation carriers with and without breast cancer.

Methionine-dependence phenotype (MDP) refers to the reduced ability of cells to proliferate when methionine is restricted and/or replaced by its immediate precursor homocysteine. MDP is a characteristic of human tumors in vivo, human tumor cell lines, and normal somatic tissue in some individuals. It was hypothesized that MDP is a risk factor for developing breast cancer in BRCA (BRCA1 and BRCA2) germline mutation carriers. To test the hypothesis, human peripheral blood lymphocytes of BRCA carriers with and without breast cancer and healthy non-carrier relatives (controls) were cultured for 9 days in medium containing either 0.1 mmol/L L-methionine or 0.2 mmol/L D,L-homocysteine, with the ratio of viable cell growth in both types of medium after 9 days used to calculate the methionine-dependence index (MDI), a measure of MDP. We also tested whether MDP was associated with common polymorphisms in methionine metabolism. Viable cell growth, MDI, and polymorphism frequency in MTRR (A66G and C524T) and MTHFR (A1298C and A1793G) did not differ among the study groups; however, MDI tended to be higher in BRCA carriers with breast cancer than those without and was significantly increased in MTHFR 677T allele carriers relative to wild-type carriers (P=0.017). The presence of MTR A2756G mutant allele and MTHFR C677T mutant allele in carriers was associated with increased breast cancer risk [odds ration, 3.2 (P=0.16; 95% confidence interval, 0.76-13.9) and 3.9 (P=0.09; 95% confidence interval, 0.93-16.3), respectively]. The results of this study support the hypothesis that defects in methionine metabolism may be associated with breast cancer risk in BRCA carriers.

Lymphocytes of BRCA1 and BRCA2 germ-line mutation carriers, with or without breast cancer, are not abnormally sensitive to the chromosome damaging effect of moderate folate deficiency.

Mutations in BRCA1 and BRCA2 genes may cause defective DNA repair and increase the risk for breast cancer. Folate deficiency is associated with increased breast cancer risk and induces chromosome abnormalities. We hypothesized that BRCA1 and BRCA2 germline mutation carriers are more sensitive to the genome damaging effect of folate deficiency compared with healthy non-carrier controls and that this sensitivity is further increased in those carriers who develop breast cancer. We tested these hypotheses in lymphocytes cultured in a medium containing 12 or 120 nM folic acid (FA) for 9 days and measured proliferative capacity and chromosomal instability using the cytokinesis-block micronucleus assay. BRCA1 and BRCA2 mutation carriers with or without breast cancer were not abnormally sensitive to FA deficiency-induced chromosome instability; however, BRCA2 mutation carriers had significantly reduced cell proliferation. FA deficiency reduced cell proliferation and increased micronucleus formation significantly, accounting for 45-59% and 70-75% of the variance in these parameters compared with 0.3-8.5% and 0.2-0.3% contributed by BRCA1 or BRCA2 mutation carrier status, respectively. The results of this study suggest that moderate folate deficiency has a stronger effect on chromosomal instability than BRCA1 or BRCA2 mutations found in breast cancer families.

Folic acid deficiency increases chromosomal instability, chromosome 21 aneuploidy and sensitivity to radiation-induced micronuclei.

Folic acid deficiency can lead to uracil incorporation into DNA, hypomethylation of DNA, inefficient DNA repair and increase chromosome malsegregation and breakage. Because ionising radiation increases demand for efficient DNA repair and also causes chromosome breaks we hypothesised that folic acid deficiency may increase sensitivity to radiation-induced chromosome breakage. We tested this hypothesis by using the cytokinesis-block micronucleus assay in 10 day WIL2-NS cell cultures at four different folic acid concentrations (0.2, 2, 20, and 200 nM) that span the "normal" physiological range in humans. The study showed a significant dose-dependent increase in frequency of binucleated cells with micronuclei and/or nucleoplasmic bridges with decreasing folic acid concentration (P<0.0001, P=0.028, respectively). These biomarkers of chromosomal instability were also increased in cells irradiated (1.5 Gy gamma-rays) on day 9 relative to un-irradiated controls (P<0.05). Folic acid deficiency and gamma-irradiation were shown to have a significant interactive effect on frequency of cells containing micronuclei (two-way ANOVA, interaction P=0.0039) such that the frequency of radiation-induced micronucleated cells (i.e. after subtracting base-line frequency of un-irradiated controls) increased with decreasing folic acid concentration (P-trend<0.0001). Aneuploidy of chromosome 21, apoptosis and necrosis were increased by folic acid deficiency but not by ionising radiation. The results of this study show that folate status has an important impact on chromosomal stability and is an important modifying factor of cellular sensitivity to radiation-induced genome damage.

Interactions between flavonoids and proteins: effect on the total antioxidant capacity.

Flavonoids are potent antioxidants. It is also known that flavonoids bind to proteins. The effect of the interaction between tea flavonoids and proteins on the antioxidant capacity was examined. Their separate and combined antioxidant capacities were measured with the Trolox equivalent antioxidant capacity (TEAC) assay. It was observed that the antioxidant capacity of several components of green and black tea with alpha-, beta-, and kappa-casein or albumin is not additive; that is, a part of the total antioxidant capacity is masked by the interaction. This masking depends on both the protein and the flavonoid used. Components in green and black tea, which show the highest masking in combination with beta-casein, are epigallocatechin gallate and gallic acid. The results demonstrate that the matrix influences the efficacy of an antioxidant.