Abnormal platelet response to PAF and ADP in beta-thalassaemia

1. AGEPC (2 microM) caused a noticeable increment in platelet aggregation, in increasing order, in 9 heterozygous beta-thalassaemic subjects, 18 homozygous beta-thalassaemics and 12 splenectomized homozygous beta-thalassaemics. 2. Recombination experiments with "patient" platelets and "normal" plasma or the reverse, as well as hydrolysis of labelled AGEPC from "normal" and "patient" serum, suggested that the observed abnormalities were due to platelets rather than to the plasma PAF hydrolase. 3. A normal splenectomized subject showed also hyperaggregability and PAF serum levels in a splenectomized patient were found twice as high in a non-splenectomized patient. 4. ADP (5 microM) caused decreased or normal platelet aggregation in the homozygous patients, approximately normal in the heterozygous subjects and increased in the splenectomized patients.     

Sister chromatid exchanges in the peripheral blood of cigarette smokers and in lung cancer patients; and the effect of chemotherapy

Summary Peripheral blood sister chromatid exchange (SCE) rates in chronic cigarette smokers and in subjects with cancer do not differ from those in healthy nonsmokers. SCE patterns were normal in 69 chronic cigarette smokers, including 62 patients with untreated lung cancer. In three chronic smokers with lung cancer, high SCE levels were related to recent intravenous chemotherapy.Presented in part at the 15th Annual Somatic Cell Genetics Conference, Norfolk, Virginia, November 10–12, 1976 (Hollander et al., 1977)     

Sister chromatid exchange in cell lines from malignant lymphomas (lymphoma lines)

Summary The frequency of sister chromatid exchanges (SCEs) was studied in cells from three freshly established lymphoma lines, derived from two patients with Hodgkin's disease and one patient with non-Hodgkin lymphoma. These values were compared to SCE rates found in cells from two long-established lymphoma lines (Raji and BJAB) and to those recorded in control cell lines of normal human donors. The highest SCE levels were demonstrated in the freshly established lymphoma lines, the lowest SCE values separated the lymphoblastoid cell lines from healthy controls, and the older lymphoma lines Raji and BJAB presented rates in between. The influences of BUDR concentration and of the duration of BUDR treatment on the frequency of SCEs were tested. Furthermore, the dependence of the SCE rate on the time interval between establishment of the cell line and its SCE investigation was considered. The connection between elevated SCE rates and the neoplastic nature of lymphoma lines is discussed.     

Dihydrofolate reductase deficiency due to a homozygous DHFR mutation causes megaloblastic anemia and cerebral folate deficiency leading to severe neurologic disease

The importance of intracellular folate metabolism is illustrated by the severity of symptoms and complications caused by inborn disorders of folate metabolism or by folate deficiency. We examined three children of healthy, distantly related parents presenting with megaloblastic anemia and cerebral folate deficiency causing neurologic disease with atypical childhood absence epilepsy. Genome-wide homozygosity mapping revealed a candidate region on chromosome 5 including the dihydrofolate reductase (DHFR) locus. DHFR sequencing revealed a homozygous DHFR mutation, c.458A>T (p.Asp153Val), in all siblings. The patients' folate profile in red blood cells (RBC), plasma, and cerebrospinal fluid (CSF), analyzed by liquid chromatography tandem mass spectrometry, was compatible with DHFR deficiency. DHFR activity and fluorescein-labeled methotrexate (FMTX) binding were severely reduced in EBV-immortalized lymphoblastoid cells of all patients. Heterozygous cells displayed intermediate DHFR activity and FMTX binding. RT-PCR of DHFR mRNA revealed no differences between wild-type and DHFR mutation-carrying cells, whereas protein expression was reduced in cells with the DHFR mutation. Treatment with folinic acid resulted in the resolution of hematological abnormalities, normalization of CSF folate levels, and improvement of neurological symptoms. In conclusion, the homozygous DHFR mutation p.Asp153Val causes DHFR deficiency and leads to a complex hematological and neurological disease that can be successfully treated with folinic acid. DHFR is necessary for maintaining sufficient CSF and RBC folate levels, even in the presence of adequate nutritional folate supply and normal plasma folate.     

Folate Deficiency after Anticonvulsant Drugs: An Effect of Hepatic Enzyme Induction?

Serum and red cell folate levels were reduced in 59% and 58% respectively of 75 children with epilepsy attending a residential school. The degree of folate deficiency was significantly related to increased hepatic microsomal enzyme activity, assessed from increased urinary excretion of D-glucaric acid and also correlated with the daily dose of anticonvulsant taken. Anticonvulsant drugs are known to have inducing properties, and since folate is required as a cofactor in drug hydroxylations it is suggested that folate depletion results from increased demand for the cofactor after induction of drug-metabolizing enzymes. As folate deficiency may ultimately limit drug metabolism this hypothesis would explain why blood phenytoin levels decrease and fit control may worsen after correction of folate deficiency in epileptic patients.     

Malabsorption of Folate Polyglutamates in Tropical Sprue

Malabsorption of folate polyglutamates prepared from yeast has been shown in eight patients with untreated tropical sprue and in three out of six patients receiving therapy for sprue. The absorptive defect for folate polyglutamates among these 14 patients occurred more frequently and in all but one patient more severely than for folic acid.Folate polyglutamates, the principal dietary form of folate, probably require deconjugation by the jejunal enzyme, folate conjugase, before absorption. The mean concentration of jejunal folate conjugase of 21 patients with untreated sprue and of 13 patients with sprue receiving therapy were both significantly less than the mean concentration in a control group. Nevertheless, all but five of the 34 patients had jejunal folate concentrations within the control range. There was no correlation in the individual patients between the jejunal folate conjugase concentration measured in vitro and the ability to absorb folate polyglutamates—nine patients having normal jejunal folate conjugase levels despite showing malabsorption of folate polyglutamates.     

Intermediate hyperhomocysteinemia resulting from compound heterozygosity of methylenetetrahydrofolate reductase mutations.

Four subjects with thermolabile methylenetetrahydrofolate reductase (MTHFR) were discovered among 16 "obligate" heterozygotes for severe MTHFR deficiency and their family members. All four subjects had less than 25% of normal mean MTHFR specific activity in lymphocyte extracts. Three of them with normal serum folate and cyanocobalamin had intermediate hyperhomocysteinemia, and one with high serum folate and cyanocobalamin had no excessive accumulation of serum homocysteine. The biochemical features in these four subjects are distinguishable from subjects homozygous for the thermolabile MTHFR, whose specific activity is approximately 50% of the normal mean, and from heterozygotes for severe MTHFR deficiency, in whom the enzyme is thermostable and has a specific activity of about 50% of the normal mean. We propose that these four subjects are genetic compounds of the allele for the severe mutation and the allele for thermolabile mutation of the MTHFR gene. It is postulated that subjects with this genetic compound are more susceptible to the development of intermediate hyperhomocysteinemia despite normal folate and B12 levels. Nonetheless, hyperhomocysteinemia due to this compound heterozygosity is correctable by oral folic acid therapy.     

Gene-nutrient and gene-gene interactions of controlled folate intake by Japanese women

Elevated serum total homocysteine (tHcy) levels are associated with increased risk for cardiovascular disease and dementia. The prevalence rates of homozygous mutants among Japanese women (n = 300) were 17.3%, 1.3%, 18.6%, and 5.3% for methylenetetrahydrofolate reductase (MTHFR) C677T and A1298C, reduced folate carrier (RFC-1) A80G, and methionine synthase (MS) A2756G, respectively. The tHcy value was significantly lower (p < 0.001) in young women with CC or CT of MTHFR than with TT (10.9+/-4.7 micromol/L) (n =250). Diversities of serum folate and tHcy in women with 23 combinations of different alleles at low folate intake converged to the highest (34.0+/-8.6 nmol/L) and lowest (7.6+/-1.5 micromol/L) levels, respectively, after folic acid (400 microg/day) supplementation. In the regression equation ( y= ax + b) of serum folate ( y nmol/L) plotted against mean folate intake ( x microg/day), the values of "a" were 0.032, 0.037, and 0.045 for individuals with CC, CT, and TT alleles, respectively, of MTHFR.     

The effect of chlorpromazine on SCE frequency in human chromosomes: An in vitro and in vivo study

Schizophrenic patients who were receiving, or who had received chlorpromazine showed SCE levels similar to those in a normal control population. Of 8 normal individuals whose lymphocytes were exposed in vitro to chlorpromazine (0.05–2.00 μg/ml) for two cell cycles, 4 showed a significant increase in SCE, 3 showed no increase and 1 a decrease compared with untreated lymphocytes. Lymphocytes from a further 8 donors treated with 2.0 μg/ml chlorpromazine prior to mitogen stimulation (G₀ lymphocytes) showed a similar SCE response. Only 3 of the 8 donors showed a significant increase in SCEs over the baseline level. When proliferating lymphocytes were exposed to chlorpromazine 38 h after culture initiation and prior to the addition of BrdUrd to the culture medium, metaphase chromosomes from only 3 of the 8 individuals studied showed increased levels of exchange. These results indicate that chlorpromazine can induce SCEs in vitro but that there is considerable variation in SCE response among individuals. Furthermore, our data emphasises the importance of using more than 1 or 2 donors when analysing SCE response in human chromosomes.     

Serum Folate and Vitamine B12 Levels in Acute and Chronic Renal Disease. Effect of Peritoneal Dialysis

Serum folate and vitamin B₁₂ levels have been measured in 32 patients with renal failure. The initial mean serum folate level was raised above normal in seven patients with acute renal failure whereas the mean level in eight patients severely ill from chronic renal failure was significantly lower than normal. Serum folate levels fell during peritoneal dialysis and rose between dialyses in all these patients and also in one patient who was dialysed for acute pancreatitis.The mean serum B₁₂ level was raised in patients with both acute and chronic renal failure, but there was no consistent change in serum B₁₂ level during dialysis.Hypersegmented polymorphs were present in the peripheral blood film of most of the patients with acute or chronic renal failure. Their presence bore no relation to the clinical state, blood urea, serum folate, or serum B₁₂ level of the patients.     

Use of a novel genetic mouse model to investigate the role of folate in colitis-associated colon cancer

Inflammatory bowel disease (IBD) patients are at high risk for developing folate deficiency and colon cancer. Since it is difficult to study the subtle global and gene-specific epigenetic mechanisms involved in folate-mediated tumor initiation and promotion, we have generated genetically modified mouse models by targeting the reduced folate carrier (RFC1) and folate-binding protein (Folbp1) genes. The transgenic mice were fed semi-purified diets for 8 weeks containing either normal (2 mg) or deficient (0.1 mg folate/kg diet) levels of folate. Compound heterozygous mice (Folbp1(+/-); RFC1(+/-)) fed an adequate folate diet exhibited a reduction in plasma folate concentrations compared to heterozygous (Folbp1(+/-)) and littermate wild-type mice (P<.05). In contrast, no differences were observed in colonic mucosa. Consumption of a low folate diet significantly reduced (three- to fourfold) plasma and tissue folate levels in all animal models, although plasma homocysteine levels were not altered. In order to elucidate the relationship between folate status and inflammation-associated colon cancer, animals were injected with azoxymethane followed by dextran sodium sulphate treatment in the drinking water. Mice were fed a normal folate diet and were terminated 5 weeks after carcinogen injection. The number of high multiplicity aberrant crypt foci per centimeter of colon was significantly elevated (P<.05) in compound Folbp1(+/-); RFC1(+/-) (3.5+/-0.4) mice as compared to Folbp1(+/-) (1.9+/-0.3) and wild-type control mice (1.1+/-0.1). These data demonstrate that the ablation of two receptor/carrier-mediated pathways for folate transport increases the risk for developing inflammation-associated colon cancer.     

Excessive chromosome fragility and abundance of sister-chromatid exchanges induced by UV in an Indian muntjac cell line defective in postreplication (daughter strand) repair

Two UV-hypersensitive animal cell mutants defective in postreplication recovery (daughter strand synthesis) display quite different patterns of induced sister-chromatid exchange (SCE). One, an SV40-transformed Indian muntjac cell (SVM), shows extremely high frequencies of SCE after UV; induced exchanges can be measured after UV doses as low as 0.01 J/m2. This cell also displays exaggerated levels of induced and spontaneous chromosome aberrations. By contrast SCE rates in the Chinese hamster cell mutant, UV-1, are essentially normal. In both SVM and UV-1, however, there is a clear correlation between the cell density and spontaneous frequencies of SCE, a feature which could be related to the observed density-dependent rate of DNA maturation.     

A study of sister-chromatid exchange in peripheral lymphocytes of hepatitis B patients with HBsAg positive and their children

Previous studies of sister-chromatid exchange (SCE) in patients with hepatitis B have been reported. But as far as we know, no such work has been done in children born to parents with hepatitis B, either one or both of whom are infected. In the present study, frequencies of SCE in the peripheral lymphocytes of 30 hepatitis B parents with hepatitis B surface antigen (HBsAg) positive and 40 of their children were observed. SCE frequencies of 20 normal adults and 3 normal children were analysed for comparison with the patients and their children. The results obtained from all of the samples were as follows: The hepatitis B patients with HBsAg positive had a significantly higher SCE frequency than the normal adults (P less than 0.01); the children born after their parents contracted hepatitis B had a significantly higher SCE frequency than normal children (P less than 0.01); there was no significant difference in SCE (P greater than 0.05) between children born after their parents contracted hepatitis B, children born after their mothers acquired it and children born after their fathers acquired it. The above results indicate that hepatitis B patients with HBsAg positive and their children born after they contracted hepatitis B had significantly higher frequencies of SCE; these data might throw new light on the study of genetic factors acting on the mechanism of hepatitis B.     

Effect of mouse age and genotype on the frequency of sister chromatid exchange in bone marrow cells

No differences were found in both the baseline and mitomycin C induced levels of sister chromatid exchanges (SCE) between 101/H and C57BL/6J mice differing in chromosome mutability. An increase with the age of the spontaneous and mutagen induced SCE rates was similar in the strains compared, though instability of chromosomes was much higher in old 101/H than in C57BL/6J mice. Thus, no correlation was observed between chromosomal aberration and SCE levels in these strains. As 101/H mice were recently found to be DNA repair-deficient, possible connection of SCE and repair is discussed.     

Effect of high levels of dietary folic acid on folate metabolism in vitamin B12 deficiency

The effect of administering high levels of folic acid to vitamin B12-deficient animals was studied. In B12 deficiency histidine oxidation is decreased. This is the result of both decreased liver folate levels and increases in the proportion of methyltetrahydrofolates. The purpose of this study was to determine if the addition of very high levels of folic acid to B12-deficient diets could increase liver folates and thereby restore histidine oxidation. Rats were fed a soy protein B12-deficient diet containing 10% pectin which has been shown previously to accelerate B12 depletion. When this diet was supplemented with B12 and folic acid, histidine oxidation was 5.4% in 2 h and the livers contained 3.49 micrograms of folate/g. In the absence of B12, the histidine oxidation rate was 0.34% and the liver folate level was 1.33 micrograms/g. When 200 mg/kg of folic acid was added to the B12-deficient diet there was no increase in histidine oxidation (0.35%) but the liver folates were increased to 3.68 micrograms which is about the same as that with B12 supplementation. The percentage tetrahydrofolate of the total liver folates was the same with and without a high level of dietary folic acid. Thus there was an increase in the absolute level of tetrahydrofolate without any increase in folate function as measured by histidine oxidation. Red cell folate levels were the same with and without B12, which is in contrast to the markedly lower liver folate levels in B12 deficiency. These data suggest a difference between B12 regulation of folate metabolism in the liver and in the bone marrow.     

Assessment of Deoxyuridine Suppression Test in Diagnosis of Vitamin B12 or Folate Deficiency

Deoxyuridine (dU) suppression tests have been performed on virtually all marrow samples aspirated at this hospital over the past 12 months. Of the 110 samples studied 26 gave abnormal results, and these 26 samples came from patients deficient in either vitamin B₁₂ or folate. The dU suppression test was found to be of particular value in the diagnosis of vitamin B₁₂ or folate deficiency in non-anaemic patients with macrocytosis and equivocal changes in marrow morphology and in patients in whom the serum vitamin B₁₂ or red cell folate levels were within the normal range.     

Methylenetetrahydrofolate reductase 677C-->T polymorphism affects DNA methylation in response to controlled folate intake in young women

DNA methylation is critical for normal genomic structure and function and is dependent on adequate folate status. A polymorphism (677C-->T) in a key folate enzyme, methylenetetrahydrofolate reductase (MTHFR), may impair DNA methylation when folate intake is inadequate and may increase the risk of reproductive abnormalities. The present study was designed to evaluate the effect of the MTHFR 677C-->T polymorphism on changes in global DNA methylation in young women consuming a low folate diet followed by repletion with the current Recommended Dietary Allowance (RDA). Women (age 20-30 years) with the TT (variant; n = 19) or CC (n = 22) genotype for the MTHFR 677C-->T polymorphism participated in a folate depletion-repletion study (7 weeks, 115 microg DFE/day; 7 weeks, 400 microg DFE/day). DNA methylation was measured at baseline, week 7, and week 14 using a [3H]methyl acceptance assay and a novel liquid chromatography tandem mass spectrometry assay of the DNA bases methylcytosine and cytosine. [3H]Methyl group acceptance tended to increase (P = 0.08) during depletion in all subjects, indicative of a decrease in global DNA methylation. During repletion, the raw change and the percent change in the methylcytosine/total cytosine ratio increased (P = 0.03 and P = 0.04, respectively) only in the subjects with the TT genotype. Moderate folate depletion in young women may cause a decrease in overall DNA methylation. The response to folate repletion suggests that following folate depletion women with the MTHFR 677 TT genotype have a greater increase in DNA methylation with folate repletion than women with the CC genotype.     

Decreased activity of folate transporters in lipid rafts resulted in reduced hepatic folate uptake in chronic alcoholism in rats

Folic acid is an essential nutrient that is required for one-carbon biosynthetic processes and for methylation of biomolecules. Deficiency of this micronutrient leads to disturbances in normal physiology of cell. Chronic alcoholism is well known to be associated with folate deficiency, which is due in part to folate malabsorption. The present study deals with the regulatory mechanisms of folate uptake in liver during chronic alcoholism. Male Wistar rats were fed 1 g/kg body weight/day ethanol (20 % solution) orally for 3 months, and the molecular mechanisms of folate uptake were studied in liver. The characterization of the folate transport system in liver basolateral membrane (BLM) suggested it to be a carrier mediated and acidic pH dependent, with the major involvement of proton coupled folate transporter and folate binding protein in the uptake. The folate transporters were found to be associated with lipid raft microdomain of liver BLM. Moreover, ethanol ingestion decreased the folate transport by altering the Vmax of folate transport process and downregulated the expression of folate transporters in lipid rafts. The decreased transporter levels were associated with reduced protein and mRNA levels of these transporters in liver. The deranged folate uptake together with reduced folate transporter levels in lipid rafts resulted in reduced folate levels in liver and thereby to its reduced levels in serum of ethanol-fed rats. The chronic ethanol ingestion led to decreased folate uptake in liver, which was associated with the decreased number of transporter molecules in the lipid rafts that can be ascribed to the reduced synthesis of these transporters.     

Cell cycle progression and SCE rate of Bloom syndrome cells with/without co-cultivation in the presence/absence of normal cells

The present study was undertaken to examine cell cycle progression and SCE rate in three types of B-lymphoid cell line, viz., normal (KS-86), high-SCE Bloom syndrome (BS (BS2-2) and dimorphic BS (BS-SYW). In order to compare the dimorphic condition (BS-SYW) with artificial dimorphism (co-cultivation of BS2-2 with KS-86) these experiments were designed to test whether the BS B-lymphoid cell line cultures would influence the cell cycle progression and SCE rates of a normal B-lymphoid cell line, and vice versa. The present study resolved the controversy reported in the literature, by finding a definite time period under co-cultivation conditions when the SCE in normal cells was increased after 8 days of co-culture, whereas SCE in the BS cells decreased immediately with co-cultivation. In the dimorphic BS cell line (BS-SYW) the SCE frequency of a high-SCE cell population was also observed to be lower than that of a non-dimorphic BS cell line (BS2-2), thus corroborating the experimental observations under co-cultivation conditions. The decrease in BS SCE and increase in normal SCE (after a particular time period) is attributed to numerous causes discussed in relation to the cell cycle progression.