Age and hypertension related changes in genotypes of MTHFR 677C>T, 1298A>C and PON1 -108C>T SNPs in men with coronary artery disease (CAD).

The phenotypes of CAD related to arterial hypertension co-occurrence were analysed in 174 male patients and 117 control men for the associations with the polymorphisms of the MTHFR gene (677C>T and 1298A>C) and the PON1 gene (-108C>T) in relation to age at diagnosis (less or equal and more than 50 years). We noted the increased frequency of the three MTHFR genotypes: CC/AC, CT/AA and CC/CC in the CAD group (65.5%) in comparison to the control group (45.3%), corresponding to the 2.3-fold increased risk of CAD for men with these genotypes (95%CI (1.4-3.7); p=0.0005). The higher increase in risk of CAD was noted for the younger men (OR=3.6; 95%CI(1.6-8.3); p=0.002) and lower for the older (OR=1.8; 95%CI(1.0-3.4); p=0.03). In the normotensive men the greater impact on CAD risk had the homozygous genotypes; the 2.3-fold higher risk was associated with MTHFR CC/AC, CC/CC and TT/AA genotypes (95%CI(1.2-4.4); p=0.01). After adjustment for age, the association between CAD and MTHFR was significant only for the younger normotensive men (OR=2.8; 95%CI (1.0-8.0); p=0.04). Additionally, we found that the younger part of the control group was characterized by higher frequency of the low expression PON1 -108T allele and PON1 -108TT genotype (0.54 and 31.9% respectively) in comparison to the older men (0.41 and 17.1% respectively; p=0.03).

[Polymorphism of the methylenetetrahydrofolate reductase gene (MTHFR) and incidence of hyperhomocysteinemia-related diseases]. / Polimorfizm genu reduktazy metylenotetrahydrofolianowej (MTHFR) a wystepowanie chorób zwiazanych z hiperhomocysteinemia.

Methylenetetrahydrofolate reductase (MTHFR), is a cytosolic enzyme, the product of which is N5-metyltetrahydrofolate, the main form of folates in tissues and the carbon donor for methylation of homocysteine to methionine. In MTHFR gene a series of the pathogenic mutations is known which lead to loss of enzymatic activity as well as the two polymorphic alleles (MTHFR 677T and 1298C) with products displaying the lowered enzyme activity resulting in hyperhomocysteinaemia. These polymorphic alleles of MTHFR represent the main genetic factor contributing to hyperhomocysteinaemia. The better known allele MTHFR 677T is found in different populations with frequency between ca. 0.1 and 0.36. In persons inheriting the variant alleles of MTHFR the increase in the level of homocysteine is noted resulting in the increased susceptibility to vascular diseases and the neural tube defects in the progeny. The procedure recommended for the prevention of effects of deficiency of MTHFR activity consists of the supplementation of the diet with 0.4 mg of folic acid daily.

Low activity of the ahr gene in the ahr dd C57BL congenic mice does not prevent the depression of birth rates observed as result of cigarette smoke exposure.

The ahr gene product is a ligand-activated transcription factor which regulates the expression of a number of enzymes involved in the metabolism of aryl hydrocarbons and mediates the effects of dioxins on tumour promotion. We have assessed the cigarette smoke induced depression of the reproductive capacities in the ahr dd C57BL congenic mice and its crosses with C57BL ahr bb mice. The in vivo exposure of animals to toxic concentrations of cigarette smoke during the period of pairing and the early pregnancy was shown by us to affect the reproduction. The transmission of the ahr b and ahr d alleles was assessed by genotyping the STR polymorphism at D12Mit2 locus linked with the ahr gene. Following the exposure to cigarette smoke the changes in the reproductive capacity of the studied mice have been assessed in relation to the presence of the ahr d allele by measurements of the changes in the rates of conceptions, abortions, births and the number of newborns per female after pairings differing in ahr gene alleles and in the expected ahr d allele frequencies in the progeny. The results indicates that the low activity ahr d allele may decrease the abortive effects of cigarette smoke exposure, but does not confer resistance to the depression of births rates following this exposure.

Effect of the route of benzo[a]pyrene administration on sister chromatid exchange and DNA binding in bone marrow of mice differing with respect to cytochrome P450 1A1 induction.

The effects of the route of benzo[a]pyrene administration on sister chromatid exchange (SCE) and B[a]P diol epoxide (B[a]PDE)-DNA adducts formation in bone marrow cells of Ah responsive (C57BL/6; B6) and Ah non-responsive (DBA/2; D2) mice were determined. Animals were treated intraperitoneally (i.p.), intragastrically (i.g.) or topically with two 100 mg/kg doses of benzo[a]pyrene 24 h apart and killed 96 h after the first treatment. Significant increase in the frequencies of SCE and the level of B[a]PDE-DNA adducts as measured by synchronous fluorescence spectrophotometry were detected in D2 mice as compared to B6 mice. The route of administration had little effect on SCE levels in bone marrow cells in D2 mice. In B6 mice higher levels of SCE were observed following i.p. administration as compared to i.g. or topical administration. In both strains the highest level of B[a]PDE-DNA adducts was formed after i.p. administration of B[a]P. We conclude that the i.p. route of B[a]P administration is the most effective in inducing SCE and B[a]P-DNA adducts formation. SCE induction does not correlate linearly with the amount of B[a]PDE-DNA adducts formed in these cells after administration of the above dose of B[a]P.

Ataxia telangiectasia heterozygotes and patients display increased fluidity and decrease in contents of sulfhydryl groups in red blood cell membranes.

In red blood cell membranes of ataxia telangiectasia mutated (ATM) homozygotes and heterozygotes, decreased values of the corrected fluorescence anisotropy and the anisotropy parameter were found, indicating increased fluidity and decreased microviscosity, respectively. These changes in membranes were accompanied by a decrease in SH-groups and an increase in malondialdehyde (MDA) contents. The content of MDA both in homozygotes and in heterozygotes exceeded roughly threefold the respective control values. Decreased content of GSH in red blood cells was found only in ATM homozygotes. The change most specific for the ATM gene appears to be the increase in fluidity, since only this parameter displays the proportionally greater changes in ATM homozygotes compared to ATM heterozygotes. The observations presented here may indicate that the ATM gene is expressed in precursors of red blood cells and deficiency of normal AT gene function may produce the changes which persist in circulating cells.

Unaltered cell proliferation rate of the ataxia telangiectasia lymphocytes dividing in vitro.

Lower cell density was found in cultures of ataxia telangiectasia lymphocytes as compared to control lymphocyte cultures. This fits with the earlier observations of decreased incorporation of 3H-Thymidine into lymphocytes of ataxia telangiectasia (AT) patients. However, the finding that there was no significant difference in proliferation rate index between AT and control cultures was unexpected. This may indicate that both cell count and measurements of thymidine incorporation in lymphocyte cultures characterize cell populations distinct from that of proliferating cells.

Ah locus-associated differences in induction of sister-chromatid exchanges and in DNA adducts by benzo[a]pyrene in mice.

The effect of alleles of the Ah locus on the induction of sister-chromatid exchanges (SCE) was studied in C57Bl/6 and in DBA/2 mice treated twice intragastrically with benzo[a]pyrene (BP, 100 or 10 mg/kg b.w.). To measure the changes in the frequency of SCE, 2 protocols were used: in vivo in bone marrow cells after implantation of 5-bromodeoxyuridine (BrdU) tablets and in vivo/in vitro in spleen lymphocytes cultured with BrdU. On day 5 mice were killed and SCEs estimated in bone marrow cells. BP-DNA adducts in bone marrow and spleen were analyzed on day 5 after the same exposure to BP. In the spleen lymphocytes SCE frequencies were analyzed after an additional 48 h of culture. We found that at both doses of BP, the number of SCEs and BP-DNA adducts in bone marrow and in spleen cells was significantly higher in aryl hydrocarbon hydroxylase (AHH)-non-inducible (DBA/2) mice than in AHH-inducible (C57BL/6) mice. Only marginal induction of SCE was noted after the high dose of BP in C57BL/6 mice in bone marrow in vivo, whereas a highly significant increase in the frequency of SCEs was found in splenocytes in the in vivo/in vitro test. The spleen cells contained larger amounts of BP-DNA adducts and demonstrated higher absolute levels of SCEs than bone marrow cells. The sensitivity of both the in vivo/in vitro and the in vivo SCE test is high enough for assessment of Ah locus-linked differences in BP genotoxicity in mice at the prolonged time between treatment and cell preparation. The present data confirm the influence of inducibility of AHH in the intestine on the genotoxicity of BP to distal tissues after oral exposure to BP.

Increased frequency of chromatid breaks in lymphocytes of heterozygotes of ataxia telangiectasia after in vitro treatment with caffeine.

The frequencies of caffeine-induced chromosomal aberrations (CA), mainly chromatid (CdB) and chromosome (CB) breaks, were studied in lymphocyte cultures derived from 6 obligatory heterozygotes and 1 homozygote of ataxia telangiectasia (AT), and from 4 control adult healthy persons. Caffeine (CF, 1 mM) was added at the beginning of the culture. In control cultures exposed to CF the frequency of CB was 1.9% and of CdB 1.3%. In cells of the AT homozygote, the frequency of CdB was 6.8% in the absence and 8.7% in the presence of caffeine, the frequencies of CB being 3.4 and 10.9%, respectively. In AT heterozygous cells treated with CF, CdB increased 13-fold as compared to a less than 3-fold increase in control cells. Comparing the frequencies of CF-induced chromosomal lesions in control and AT heterozygous cells, potentiation factors (Pf) for the effect of 1 AT gene on cell sensitivity to CF (Pf [AT]) were 3.5 for CB, 6.6 for CdB and 5.5 for CA. These data demonstrate that lymphocytes of AT heterozygotes are significantly more sensitive to caffeine treatment in vitro in terms of increased frequency of CdB than normal cells, which may be useful for the diagnosis of carriers of this defective gene.

Carriers of ataxia-telangiectasia gene display additional protein fraction and changes in the environment of SH groups in erythrocyte membrane.

Additional protein fraction migrating slower than spectrin has been detected in erythrocyte membranes from an ataxia-telangiectasia (A-T) patient and from his mother (A-T heterozygote). In erythrocyte membranes labelled with maleimide spin label changes in signal of the weakly immobilized spin label as related to that of strongly immobilized one (w/s) were noted. In comparison to age-matched control groups the values of w/s were lower in A-T heterozygotes (ten persons) and higher in A-T homozygotes (four persons). In control persons the values of w/s increased with age, whereas in families with A-T no significant differences in this parameter were noted between children and parents. The presence of additional protein fraction in erythrocytes membranes of A-T patient and A-T heterozygote indicates that these phenotypes can be differentiated from the healthy control persons for the first time on the basis of changes detected in the erythrocytes. This change in erythrocyte membrane may explain the decrease in the w/s parameter of electron spin resonance in A-T heterozygotes. On the other hand increased values of w/s in A-T patients may be caused by disease process.

Increase in cooperativity of oxygen binding as a factor related to high maximal oxygen uptake during physical effort.

In a group of trained, nonsmoking men, apparent haemoglobin oxygen affinity was assessed in vitro in the whole blood collected immediately before and after maximal physical effort. Measurements were performed using standard laboratory equipment. Maximal oxygen uptake (V O2 max) has been monitored during maximal physical effort. Increase in app Hb-oxygen affinity after exercise was found by measurements of p25 and by differences between plots of correlations between V O2 max and resting and post-exercise parameters of Hb-oxygen binding. Significant correlations were found between preexercise n50 and VO2 max (r = -0.59) and between post-exercise log P50 and V O2 max (r = 0.6). Exercise-induced increase in Hb-oxygen affinity and in cooperativity of Hb-O2 binding were found to be higher in persons with higher values of VO2 max.

[Use of monoclonal antibodies for studying cytochrome P-450 forms. Use of interspecies homology of amino acid sequence]. / Zastosowanie przeciwcial monoklonalnych do oznaczania odmian cytochromów P-450. Wykorzystanie miedzygatunkowych homologii sekwencji aminokwasów.

The current research on utilization of monoclonal antibodies for isolation of cytochrome P-450 forms, substrate specificity determination, localization in tissues and contribution in DNA adduct formation is reviewed in this paper. Usefulness of monoclonal antibodies directed against rodent cytochrome P-450 isoenzymes to human investigations is emphasized.

The inhibitory effect of theophylline on cell cycle kinetics of human lymphocytes in vitro.

The effect of theophylline (TF) on proliferation of lymphocytes in phytohaemagglutinin (PHA), concanavalin A (Con A) and pokeweed mitogen (PWM) freshly stimulated 3-4 days cultures or of 14-days cultured lymphoblasts was studied. Proliferation was estimated by measurement of incorporation of tritiated thymidine (3H-TdR), and by microscopic analysis of cellular divisions progression up to the 4th generation of cells cultured in presence of 5-bromodeoxyuridine (BrdUrd). The strongest inhibition of proliferation was observed when TF was added at the beginning of the cultures. However, when TF was added for the last 20 h of the cultures, a significant decrease in number of the third (M3) and fourth (M4) generation cells was noted and M3/M4 ratio increased from 6.4 up to 19.1. TF inhibited also incorporation of 3H-TdR into cultured lymphoblasts. The obtained data indicate an inhibitory effect of TF both an activation and cell cycle progression of lymphocytes.

Kinetics of benzo(a)pyrene hydroxylation at varying concentrations of NADPH and NADH in liver microsomes of nontreated and beta-naphthoflavone-treated C57BL/2 and DBA/2 mice.

The biphasic kinetics of hydroxylation of benzo(a)pyrene (B(a)P) dependent on the concentration of either NADPH or NADH has been observed in DBA/2 (AhdAhd) but not in C57BL/6 (AhbAhb) beta-naphthoflavone-treated mice. On the other hand, in nontreated mice, this biphasic kinetics has been observed in both strains of mice. This shows that characteristics of biphasic kinetics differentiate Ahb from Ahd mice only after treatment with methylcholanthrene-type of inducers. The discussed biphasic kinetics was more regular and distinct in the NADH-dependent reaction as compared with NADPH-dependent hydroxylation of B(a)P. It is suggested that the NADH-specific pathway of electron transport to cytochrome P-450 is necessary for the occurrence of this effect both in NADH- and NADPH supported reaction.

Physical effort-induced decrease in apparent whole blood Hb-oxygen affinity normalized for intraerythrocytic pH (pHe) at low initial values of pHe.

Effect of physical effort on haemoglobin-oxygen affinity was studied in whole blood of 28 healthy males. In persons showing decrease in pH gradient between plasma and erythrocytes after physical effort, a parallel decrease in apparent, normalized for intraerythrocytic pH (pHe), Hb-oxygen affinity was noted. An increase in pH gradient after exercise was accompanied by increase in apparent, corrected for pHe, Hb-oxygen affinity and in blood Hb content. Correlation between exercise-induced changes in Hb-oxygen affinity and in pH gradient (-0.66) was highly significant (p less than 0.001). It is suggested that both parameters may be increased by recruitment into circulation of old erythrocytes from spleen. Decrease in both parameters may be related to low values of intraerythrocytic pH before effort. Groups of persons differing in exercise-induced changes of Hb-oxygen affinity did not differ in maximal oxygen utilization.

Exercise-induced increase of methaemoglobin concentration and low cooperativity in haemoglobin-oxygen binding at rest correlate with low oxygen consumption during maximal effort.

Relations between oxygen consumption, physical effort and parameters characterizing oxygen transport by red blood cells [rbc] before and after exercise were assessed in 32 healthy persons. The oxygen affinity of haemoglobin was studied in rbc by several measurements of pO2, pCO2, pH and HbO2 in samples of heparinized venous blood equilibrated with the gas compartment containing decreasing concentrations of O2 and 5% CO2. The actual oxygen affinity significantly decreased after exercise with the values of log p50 [act] equal 1.42 +/- 0.1 at rest and 1.51 +/- 0.12 after the effort; the difference was assessed by the nonparametric test of pairs [p less than 0.05]. No effect of exercise was noted on the mean values of apparent p50, a parameter sensitive to the intraerythrocytic regulators of haemoglobin-oxygen affinity. In subjects with the highest values of efficiency of oxygen utilization [Eff], i.e. the lowest oxygen consumption during maximal effort, the values of Eff were however found to be correlated with the cooperativity in Hb-O2 binding at rest [r = 0.565] and with the exercise-induced changes in metHb concentration [r = 0.258]. The exercise-induced changes in metHb concentration correlated with the exercise-induced change in apparent p50 [r = 0.496].

Low constitutive activity of aryl hydrocarbon hydroxylase in phytohaemoagglutinin-stimulated peripheral blood mononuclear cells of children after treatment for acute lymphoblastic leukemia.

In six children in the first remission after treatment of acute lymphoblastic leukemia (ALL) the constitutive activity of aryl hydrocarbon (benzo-[alpha]pyrene) hydroxylase (AHH) (0.14 +/- 0.04 U, i.e. pmol of 3-OH-benzo[alpha] pyrene x 10(-6) cells x min-1) in phytohaemoagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMC) was significantly lower (P 0.05) than the constitutive activities in the parents of the children (0.25 +/- 0.09 U) and in the control adult group (0.26 +/- 0.09 U). In methylcholanthrene (MC)-induced activities of AHH no significant differences were observed between the above groups.

Reciprocal interactions between oxygen-binding and aquo-ferric chains in partially oxidized human hemoglobin A1.

The concomitant changes in oxygen binding by ferrous hemes and in oxygenation-linked difference in absorbance of ferric hemes at 630 nm were studied at pH 6.8 in human partially oxidized hemoglobin A (Hb++/+++). At concentrations of ferric hemes less than 20% no changes in P50 of oxygenation were noted in comparison to the P50 value of Hb A. An increase of ferric hemes from 20 to 80% resulted in a parallel increase in oxygen affinity of functional hemes. Prevalence of alpha chains among the oxidized chains was confirmed in the studied Hb++/+++ containing less than 20% of ferric hemes. The oxygenation linked increases in absorbance of ferric hemes at 630 nm were related in a linear fashion to the increase in the ferric hemes content fom 0 to 40%. The lack of change in oxygen affinity of HB++/+++ containing less than 20% of ferric hemes, which exhibits the oxygenation-linked changes in absorbance of ferric hemes may be related to the presence and properties of asymmetric tetramers alpha+++ alpha++ beta 2++.