Effects of folic acid deficiency and MTHFRC677T polymorphisms on cytotoxicity in human peripheral blood lymphocytes

Apoptosis (APO) and necrosis (NEC) are two different types of cell death occurring in response to cellular stress factors. Cells with DNA damage may undergo APO or NEC. Folate is an essential micronutrient associated with DNA synthesis, repair and methylation. Methylenetetrahydrofolate reductase (MTHFR) regulates intracellular folate metabolism. Folate deficiency and MTHFR C677T polymorphisms have been shown to be related to DNA damage. To verify the cytotoxic effects of folate deficiency on cells with different MTHFR C677T genotypes, 15 human peripheral lymphocyte cases with different MTHFR C677T genotypes were cultured in folic acid (FA)-deficient and -sufficient media for 9 days. Cytotoxicity was quantified using the frequencies of APO and NEC as endpoints, the nuclear division index (NDI), and the number of viable cells (NVC). These results showed that FA is an important factor in reducing cytotoxicity and increasing cell proliferation. Lymphocytes with the TT genotype proliferated easily under stress and exhibited different responses to FA deficiency than lymphocytes with the CC and CT genotypes. A TT individual may accumulate more cytotoxicity under cytotoxic stress, suggesting that the effects of FA deficiency on cytotoxicity are greater than the effects in individuals with the other MTHFR C677T variants.     

In vitro activity of folic acid on the proliferation dynamics of human bone marrow CFU-GM in propyphenazone-induced granulocytopenia]

The in vitro action of folic acid was tested on the proliferation of bone marrow granulocyte-macrophage progenitor cells from a patient with drug-induced (propyphenazone) neutropenia in remission 20 days after the drug had been suspended. Various bone marrow cultures were prepared with standard stimulant, adding, respectively: folic acid, propyphenazone and both folic acid and propyphenazone together. Growth was tested on day 7, 12 and 19 of incubation. Under baseline culture with standard stimulant, CFU-GM growth was characterized by successive proliferation waves of various entity: the first on day 7 was very high, the second, on day 12 was rather low, and the third, on day 19 was intermediate. This behaviour is different from what is usually observed in normal subjects in steady-state, whose first (AC-A+AC-B) and second (AC-C) proliferation period are of similar entity. The prevalence of the first proliferation period in our case is interpreted as the result of a renewed granulocytopoietic activity after drug-induced bone marrow suppression. This indicates a maintained integrity of the negative-feedback mechanism of homeostatic regulation on granulocytopoietic activity. The sole addition of propyphenazone on the in vitro bone marrow cell cultures of our patient produced a reduction of those CFU-GM that had grown during the first period whereas the growth during the second and third period remained unvaried. Thus the growth peak in cultures treated with propyphenazone occurred on day 19, which seems to correspond with the necessary time for a spontaneous remission from neutropenia, clinically observed to be 20 days after suspension of the propyphenazone-containing drug.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biosynthesis of folic acid

Summary The influence of various vitamins on the biogenesis of folic acid has been studied in microorganisms requiring these as growth factors. In L. arabinosus, the folic acid synthesised was directly proportional to the availability of both riboflavin and pantothenic acid. The influence of cyanocobalamin on folic synthesis varied radically in different organisms. In case of the B₁₂/methionine auxotroph of E. coli there was an inverse relationship of vitamin B₁₂ to folic acid synthesis, while in Euglena the folic acid elaborated was in proportion to cyanocobalamin supplied. Synthesis of both folic acid and vitamin B₁₂ was depressed when thymine supply was adequate in the nutrition of E. coli 15 T ^-, a thymine auxotroph.     

Photogenotoxicity of folic acid

Folic acid (FA), also named vitamin B9, is an essential cofactor for the synthesis of DNA bases and other biomolecules after bioactivation by dihydrofolate reductase (DHFR). FA is photoreactive and has been shown to generate DNA modifications when irradiated with UVA (360 nm) in the presence of DNA under cell-free conditions. To investigate the relevance of this reaction for cells and tissues, we irradiated three different cell lines (KB nasopharyngeal carcinoma cells, HaCaT keratinocytes, and a melanoma cell line) in the presence of FA and quantified cytotoxicity and DNA damage generation. The results indicate that FA is phototoxic and photogenotoxic by two different mechanisms. First, extracellular photodecomposition of FA gives rise to the generation of H₂O₂, which causes mostly DNA strand breaks. If this is prevented, e.g., by the presence of catalase, DNA damage generated by intracellular FA becomes evident. The damage spectrum in this case consists predominantly of oxidatively generated purine modifications sensitive to the repair glycosylase Fpg, as characteristic for type I photoreactions, and is associated with the formation of micronuclei. In KB cells, the DNA damage is strongly enhanced after pretreatment with the DHFR inhibitor methotrexate, which prevents the loss of the chromophore associated with the intracellular reduction of FA by DHFR. The results indicate that FA is photoreactive in cells and gives rise to nuclear DNA damage under irradiation.     

Biosynthesis of folic acid

Summary It has been attempted to isolate and characterize the folate precursors in the culture filtrates of two folate-requiring organisms, Streptococcus faecalis R and Lactobacillus casei. On the basis of paper chromatography, bioautography, ultra violet absorption spectra, chemical reactions, and differential microbiological responses it has been concluded that L. casei cultures contain a compound similar to pteroic acid which can be utilized by S. faecalis R. The S. faecalis R cultures on the other hand appear to accumulate a pteridine derivative active for Crithidia fasciculata It has been confirmed that this pteridine is not derived from the folic acid usually added to the growth medium.Abbreviations PGA Pterolyglutamic acid - PABA p-aminobenzoic acid     

Biosynthesis of folic acid

Summary Biosynthesis of folic acid activity by Bacillus subtilis cell suspensions was studied with respect to substrates utilized as precursors. Among purine bases, adenine was utilized the best and not guanine although guanosine or guanylic acid were utilized efficiently. Among 3-C precursors, glyceraldehyde gave maximum synthesis of folate activity. The cells appeared to utilize p-aminobenzoate preferentially to p-aminobenzoyl-glutamate. Pteroic acid appears to be an intermediate in the synthesis of folate derivatives in this system. Ascorbic acid stimulates the synthesis to a great extent.     

Multispectroscopic studies of the interaction of folic acid with glycated human serum albumin

Abstract

The interaction between glycated human serum albumin (gHSA) and folic acid (FA) was investigated by various spectroscopic techniques, such as fluorescence, circular dichroism, UV–vis absorption spectroscopy and electrophoretic light scattering technique. These methods characterize the binding properties of an albumin–folic acid system. The binding constants values (K_a) at 300 and 310 K are about 10⁴ M^?1. The standard enthalpy change (ΔH) and the standard entropy change (ΔS) were calculated to be ～?20?kJ mol^?1 and ～16 J mol^?1 K^?1, respectively, which indicate characteristic electrostatic interactions between gHSA and folic acid. The CD studies showed that there are no significant conformational changes in the secondary structure of the protein. Moreover, the zeta potential measurements proved that under physiological conditions the gHSA–folic acid complex shows instability. No significant changes in the secondary structure of the protein and reversible drug binding are the desirable effect from pharmacological point of view.

Communicated by Ramaswamy H. Sarma     

Lowering blood homocysteine with folic acid based supplements: meta-analysis of randomised trials

Objective: To determine the size of reduction in homocysteine concentrations produced by dietary supplementation with folic acid and with vitamins B-12 or B-6. Design: Meta-analysis of randomised controlled trials that assessed the effects of folic acid based supplements on blood homocysteine concentrations. Multivariate regression analysis was used to determine the effects on homocysteine concentrations of different doses of folic acid and of the addition of vitamin B-12 or B-6. Subjects: Individual data on 1114 people included in 12 trials. Findings: The proportional and absolute reductions in blood homocysteine produced by folic acid supplements were greater at higher pretreatment blood homocysteine concentrations (P<0.001) and at lower pretreatment blood folate concentrations (P<0.001). After standardisation to pretreatment blood concentrations of homocysteine of 12 μmol/l and of folate of 12 nmol/l (approximate average concentrations for Western populations), dietary folic acid reduced blood homocysteine concentrations by 25% (95% confidence interval 23% to 28%; P<0.001), with similar effects in the range of 0.5-5 mg folic acid daily. Vitamin B-12 (mean 0.5 mg daily) produced an additional 7% (3% to 10%) reduction in blood homocysteine. Vitamin B-6 (mean 16.5 mg daily) did not have a significant additional effect. Conclusions: Typically in Western populations, daily supplementation with both 0.5-5 mg folic acid and about 0.5 mg vitamin B-12 would be expected to reduce blood homocysteine concentrations by about a quarter to a third (for example, from about 12 μmol/l to 8-9 μmol/l). Large scale randomised trials of such regimens in high risk populations are now needed to determine whether lowering blood homocysteine concentrations reduces the risk of vascular disease.

Key messages

• Higher blood homocysteine concentrations seem to be associated with higher risks of occlusive vascular disease and with lower blood concentrations of folate and vitamins B-12 and B-6
• Proportional and absolute reductions in blood homocysteine concentrations with folic acid supplements are greater at higher pretreatment blood homocysteine concentrations and at lower pretreatment blood folate concentrations
• In typical Western populations, supplementation with both 0.5-5 mg daily folic acid and about 0.5 mg daily vitamin B-12 should reduce blood homocysteine concentrations by about a quarter to a third
• Large scale randomised trials of such regimens in people at high risk are now needed to determine whether lowering blood homocysteine concentrations reduces the risk of vascular disease

     

Experiments on the biological activities of various fractions of the folic acid antagonist "X-methyl" folic acid

A crude synthetic preparation called crude "X-methyl" folate has previously been shown to function as a folate antagonist for rats and chicks. This product has been shown to contain two folate antagonists: 9-methyl folate, present as 6% by weight of the product and which has low activity as a folate antagonist for Streptococcus faecalis, and pyrrofolic acid, a compound present in small amounts (0.04%), but having high anti-folate biological activity for S. faecalis. These experiments deal with the antifolate activity of these two fractions for the rat as measured by their effects on histidine oxidation. Rats were fed a purified diet based on 20% vitamin-free casein and containing 1.0% sulfasuxidine. When this diet was supplemented with a marginal amount of folic acid (0.3 mg per kg diet), the addition of 4 g of crude antagonist decreased histidine oxidation and decreased liver folate levels. The addition of 240 mg of pure 9-methyl folic acid (amount of 9-methyl folic acid in 4 g of crude) produced similar decreases in histidine oxidation and liver folate levels. A concentrate of pyrrofolic acid (equivalent to 4 g of crude) free of 9-methyl folic acid produced no decrease in histidine oxidation and minimal changes in liver folate. This indicates that the folate antagonist activity observed previously with animals is probably due to the 9-methyl folic acid component rather than to the pyrrofolic acid activity.