N-acetyl cysteine reduces chromosomal DNA damage in circulating lymphocytes during cardiac catheterization procedures: a pilot study.

BACKGROUND: N-acetylcysteine (NAC) is considered a promising radio-protector for its antioxidant and anticarcinogenic properties. We examined the ability of NAC to confer protection against radiation-induced chromosomal DNA damage during cardiac catheterization procedures. METHODS: Sixty-five patients (52 males, age 64.4 ± 11.9 years) undergoing invasive cardiovascular procedures (peripheral transluminal angioplasty, n=45; cardiac resynchronization therapy, n=15 and ablation therapy n=5) were enrolled: 35 patients (26 males, age 63.4 ± 11.1 years) received the standard hydration protocol consisting of intravenous isotonic saline for 12h after catheterization (Group I), and 30 patients (26 males, age 65.5 ± 12.9 years) received a clinically driven double intravenous dose of NAC (6 mg/kg/h diluted in 250 mL of NaCl 0.9%) for 1h before and a standard dose (6 mg/kg/h diluted in 500 mL of NaCl 0.9%) for 12h following catheterization (Group II). Micronucleus assay (MN) was performed as biomarker of chromosomal DNA damage before, 2 and 24h after the radiation exposure. Dose-area product (DAP; Gy cm(2)) was assessed as physical measure of radiation load. RESULTS: DAP was higher in NAC-treated patients (I=54.7 ± 23.6 vs II=126.2 ± 79.2 Gy cm(2), p=0.0001). MN frequency was 13.7 ± 4.7 ‰ at baseline and showed a significant rise at 2h (18.0 ± 6.8 p=0.01) and 24h (17.6 ± 5.9, p=0.03) in the Group I. There was no significant increase of MN in the Group II (13.7 ± 7.0, 15.5 ± 6.0 and 14.9 ± 6.3 for baseline, 2h and 24h respectively, p=0.4). CONCLUSION: NAC treatment given to prevent contrast-induced nephropathy may also reduce DNA damage induced by ionizing radiation exposure during cardiac catheterization procedures.

Maternal and sex dependency of insulin resistance: longitudinal PET and echocardiography study from the healthy fetus to the adult minipig.

UNLABELLED: Cardiovascular and metabolic vulnerability have an early developmental origin. We evaluated the potential influence of innate life factors, including the metabolism of the mother and the sex of the offspring, on cardiometabolic risk, including organ-specific insulin resistance, subclinical cardiac dysfunction, and DNA oxidative damage throughout the lifespan. METHODS: Two female minipigs were studied during late pregnancy, and their offspring were restudied at the ages of 1 mo (n = 11), 6 mo (n = 9), and 9 mo (n = 10, 6 offspring and 4 age-matched animals). We measured insulin-mediated glucose disposal in skeletal muscle, adipose tissue, liver, and myocardium using (18)F-FDG PET; cardiac function using 2-dimensional strain echocardiography; and DNA damage using the comet assay. RESULTS: Glucose metabolism showed the 2 sows to have differences similar to those in their respective 1-mo-old offspring. Over time, compared with female animals, male animals developed myocardial insulin resistance (male animals vs. female animals: 34 ± 5 vs. 58 ± 8 µmol/min/kg at 6 mo, P = 0.03; 29 ± 8 vs. 60 ± 7 µmol/min/kg at 9 mo, P = 0.02). Cardiac function progressively deteriorated in male animals from 1 mo (radial strain, -60% ± 7%; strain rate, -5.4 ± 0.9 s(-1)) to 6 mo (radial strain, -41% ± 5%; strain rate, -2.5 ± 0.2 s(-1), P < 0.05 vs. 1 mo) and 9 mo (radial strain, -32% ± 5%; strain rate, -1.6 ± 0.2 s(-1), P < 0.01 vs. 1 mo) and was significantly different from that in female animals (radial strain, -48% ± 4%; strain rate, -3.1 ± 0.2 s(-1), P < 0.05 and P < 0.01, respectively). Oxidative damage was reduced in female animals and increased in male animals across age categories (P < 0.05). CONCLUSION: The metabolism of minipig offspring is influenced by maternal insulin sensitivity during early life stages. Sex-related effects prevail thereafter in healthy minipigs, documenting a precocious onset of cardiometabolic vulnerability in male offspring.

Genetic polymorphisms in XRCC1, OGG1, APE1 and XRCC3 DNA repair genes, ionizing radiation exposure and chromosomal DNA damage in interventional cardiologists.

Interventional cardiologists working in high-volume cardiac catheterization laboratory are exposed to significant occupational radiation risks. Common single-nucleotide polymorphisms (SNPs) in DNA repair genes are thought to modify the effects of low-dose radiation exposure on DNA damage, the main initiating event in the development of cancer and hereditary disease. The aim of this study was to determine the relationship between XRCC1 (Arg194Trp and Arg399Gln), OGG1 (Ser326Cys), APE1 (Asp148Glu) and XRCC3 (Thr241Met) SNPs and chromosomal DNA damage. We enrolled 77 subjects: 40 interventional cardiologists (27 male, 41.3+/-9.4 years and 13 female, 37.8+/-8.4 years) and 37 clinical cardiologists (26 male, 39.4+/-9.5 years and 11 female, 35.0+/-9.8 years) without radiation exposure as the control group. Micronucleus (MN) assay was performed as biomarker of chromosomal DNA damage and an early predictor of cancer. MN frequency was significantly higher in interventional cardiologists than in clinical physicians (19.7+/-7.8 per thousand vs. 13.5+/-6.3 per thousand, p=0.0003). Within the exposed group, individuals carrying a XRCC3 Met241 allele had higher frequency than homozygous XRCC3 Thr241 (21.2+/-7.8 per thousand vs. 16.6+/-7.1 per thousand, p=0.03). Individuals with two or more risk alleles showed a higher MN frequency when compared to subjects with one or no risk allele (18.4+/-6.6 per thousand vs. 14.4+/-6.1 per thousand, p=0.02). An interactive effect was found between smoking, exposure >10 years and the presence of the two or more risk alleles on the MN frequency (F=6.3, p=0.02). XRCC3 241Met alleles, particularly in combination with multiple risk alleles of DNA repair genes, contribute to chromosomal DNA damage levels in interventional cardiologists.

Glutathione S-transferase T1- and M1-null genotypes and coronary artery disease risk in patients with Type 2 diabetes mellitus.

INTRODUCTION: Since long-term exposure to oxidative stress is strongly implicated in the pathogenesis of diabetic complications, polymorphic genes of detoxifying enzymes must be involved in the development of coronary artery disease (CAD). We assessed the potential glutathione S-transferase (GST) gene-gene (GSTM1(null)-GSTT1(null)) and gene-smoking interactions on the development of CAD in patients with Type 2 diabetes. MATERIALS & METHODS: In a case-only design, we enrolled 231 patients with Type 2 diabetes (147 male, 66.1 +/- 9.7 years) referred to our institute for coronary angiography investigation. CAD was diagnosed if there was over 50% obstruction of one or more major vessels. RESULTS: Coronary angiography revealed significant CAD in 184 patients (80%). Male gender (p < 0.001), smoking habits (p = 0.003) and GSTT1(null) genotype (p = 0.003) were significantly correlated with the increasing extent of the coronary atherosclerosis. Case-only analysis revealed that patients with both M(null)-T(null) genotypes had the highest risk for 3-vessel CAD compared with patients who express both GST genes (odds ratio: 3.1; 95% confidence interval: 1.0-10.3, p = 0.04). A nearly threefold interaction existed between cigarette smoking and M(null)-T(null) genotypes (odds ratio: 2.9, 95% confidence interval: 1.7-7.8, p = 0.03). A significant interaction between M(null)-T(null) genotypes and smoking was also observed on the increasing number of coronary vessels that were diseased (chi(2) = 14.0; p = 0.03). CONCLUSION: These data suggest that polymorphisms in GSTM1 and GSTT1 genes are risk factors for CAD in Type 2 diabetic patients, especially among smokers. These genetic markers may permit the targeting of preventive and early intervention on high-risk patients to reduce their cardiovascular risk.

Relation of increased chromosomal damage to future adverse cardiac events in patients with known coronary artery disease.

Somatic deoxyribonucleic acid (DNA) damage has been associated with early-phase and/or acute complications of atherosclerosis. However, it remains unclear whether circulating levels of DNA damage have prognostic value in patients with coronary artery disease (CAD). The aim of this study was to assess the prognostic significance of chromosomal DNA damage in human lymphocytes on the rate of major adverse cardiovascular events in patients with CAD. A follow-up prospective cohort study was carried out of 178 patients (153 men, mean age 61.9 +/- 9.7 years) with angiographically proved CAD who underwent micronucleus assay, a sensitive biomarker of chromosomal damage and genetic instability, from March 1999 and June 2001. During a mean follow-up period of 51.4 +/- 23.8 months, 58 patients had major adverse cardiovascular events (cardiac death, myocardial infarction, stroke, congestive heart failure, unstable angina, or coronary and peripheral revascularization). The overall event-free survival rates were 77.5%, 70.4%, and 49.0% in patients in the lower, middle, and upper tertiles of micronucleus level, respectively (log rank = 11.5, p = 0.003). In a multivariate Cox regression model, only the upper tertiles were significantly associated with a higher risk for major adverse cardiovascular events (hazard ratio 2.2, 95% confidence interval 1.1 to 4.7, p = 0.03). In conclusion, levels of peripheral chromosomal DNA damage may be a new sensitive biomarker of prognostic stratification in patients with known CAD.

A case report of myocardial infarction in young patient with a parental history of premature cardiovascular death: combination of prothrombotic gene mutations.

We report a case of myocardial infarction at a young age in a subject heterozygous for the G20210A prothrombin gene variant and homozygous for the C677T MTHFR polymorphism, who presented a strong family history of atherothrombosis. Genetic screening for inherited thrombophilia, especially in the presence of a strong familiarity, may be a critical information for secondary prevention of arterial thrombosis.

Acute chromosomal DNA damage in human lymphocytes after radiation exposure in invasive cardiovascular procedures.

AIMS: We evaluated whether radiation exposure during interventional cardiovascular procedures can induce damage to deoxyribonucleic acid (DNA). METHODS AND RESULTS: Micronucleus assay (MN) was performed as biomarker of chromosomal damage and intermediate endpoint in carcinogenesis. Seventy-two patients (54 males, age = 63.8 +/- 10.5 years) undergoing a wide range of radiation exposure during invasive cardiovascular procedures (coronary angiography, n = 9; percutaneous coronary intervention, n = 9; peripheral transluminal angioplasty, n = 37; and cardiac resynchronization therapy, n = 17) were enrolled. MN frequency was evaluated before, 2, and 24 h after the radiation exposure. Dose-area product (DAP; Gy cm(2)) was assessed as physical measure of radiation load. DAP value was 96.0 +/- 63.9 Gy cm(2). MN frequency was 15.1 +/- 7.1 per thousand at baseline and showed a significant rise at 2 h (17.5 +/- 6.5 per thousand, P = 0.03) and 24 h (18.5 +/- 7.3 per thousand, P = 0.004) after procedures. CONCLUSION: Our results corroborate the current radioprotection assumption that even modest radiation load can damage the DNA of the cell and induce chromosome alterations which are early predictors of increased cancer risk.

GSTM1, GSTT1 and CYP1A1 detoxification gene polymorphisms and susceptibility to smoking-related coronary artery disease: a case-only study.

Cigarette smoking is a powerful risk factor for coronary artery disease (CAD), leading to the formation of DNA alterations within blood vessels and heart. However, the degree of smoking-related atherosclerosis varies from individual to individual. Genetic polymorphisms of relevant xenobiotic metabolising enzymes may determine the susceptibility of an individual response to environmental toxicants. The purpose of this study was to test the hypothesis that the inheritance of polymorphic genes encoding cytochrome P450 1A1 (CYP1A1 MspI) and glutathione S-transferases (GSTM1(null) and GSTT1(null)) may be causally associated with the presence and severity of smoking-induced CAD. In a case-only design, 222 (179 male, 57.8+/-10.3 years) consecutive smoker patients who had undergone elective and diagnostic coronary angiography were recruited. We found a group (n=169) of smoker patients with significant CAD, defined as>50% reduction in diameter of at least one major vessel, and a group without obstructive CAD (n=53). No significant differences were observed in CYP1A1 genotypes frequencies between CAD and non-CAD smokers (p=0.1). Homozygous deletion of GSTM1 had a frequency of 58.6% among patients with CAD and 45.3% among those without CAD (p=0.08). The frequency of the GSTT1(null) genotype was 43.8% among the patients with CAD and 24.5% among CAD-free subjects (p=0.01). After adjustment for traditional risk factors, the presence of combined GSTM1(null)GSTT1(null) genotypes was significantly associated with an increased risk of CAD (OR=3.9; 95% CI: 1.3-11.4, p=0.01). Moreover, smokers with combined GSTM1(null)GSTT1(null) genotypes had significantly higher number of stenosed vessels than those with the positive genotype (2.3+/-0.9 versus 1.7+/-0.8, p=0.03). Our findings showed that smokers carrying GST deleted genotypes have an increased susceptibility to the smoking related coronary artery disease. Exploring gene-smoking effect provides an excellent model in order to understand gene-environment toxicants interaction and its implications to cardiovascular disease.

Cardiac catheterization and long-term chromosomal damage in children with congenital heart disease.

AIMS: Medical radiological exposure is associated with an additional risk of cancer. Children with repaired congenital heart disease (CHD) are theoretically at a relatively greater cancer risk as the radiological exposure can be intensive in these patients. Chromosomal aberrations test (CA) and micronucleus assay (MN) in peripheral blood lymphocytes are biomarkers of chromosomal damage and intermediate endpoints in carcinogenesis. METHODS AND RESULTS: The frequency of CA and MN was assessed in three groups of patients: Group I, 32 exposed patients (17 males, age=15.5+/-8.3 years) who underwent cardiac procedures employing ionizing radiation (mostly cardiac catheterization) for CHD between 1965 and 2000; Group II, 32 healthy age- and sex-matched subjects (17 males, age=14.1+/-12.3 years), and Group III, 10 newborn non-exposed patients (7 males) with CHD. Exposed patients of Group I had a mean value of 2.9+/-1.4 cardiac catheterization (range 1-5) procedures per person. The mean frequency of CA was higher in the exposed patients (Group I=2.8+/-1.9% vs. Group II=0.7+/-0.7%; vs. Group III=0.8+/-0.8%; P<0.0001). Similarly, the mean values of MN were higher in the exposed patients (Group I =12.3+/-5.1 per thousand vs. Group II=6.0+/-3.8 per thousand; vs. Group III=4.4+/-1.4 per thousand; P<0.0001). CONCLUSION: Cardiac ionizing procedures are associated with a long-lasting mark in the chromosomal damage of exposed children with CHD.

Hormone replacement therapy: one-year follow up of DNA damage.

Although hormone replacement therapy (HRT) may offer considerable benefits for menopausal women, the potential cancer risk may limit its use. This work aimed at assessing whether HRT is able to induce DNA damage in postmenopausal women monitored by the micronucleus (MN) test, which provides a reliable biomarker of genotoxicity and cancer risk assessment. A group of 16 healthy women (non-smokers) in spontaneous menopause were given oral estradiol (2 mg oral micronized 17-beta estradiol daily) for 1 month, followed by a 30-day wash-out period and a transdermal treatment with 17-beta estradiol (1.5 mg gel daily) during 1 month. Oral intake of dihydrogesterone (10 mg/day for 12 days/month) was cyclically combined with oral or transdermal estradiol during the next 9 months. Venous blood samples were collected before the treatment, and after 1, 3, 6 and 12 months of therapy. Slides were scored blind and MN frequency was evaluated as number of micronuclei per 1000 binucleated cells. The baseline plasma levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH) and estradiol (E2) were simultaneously measured. The means of MN frequency were 18.2+/-1.6, 18.6+/-2.1, 14.8+/-1.5, 15.9+/-1.0 and 17.7+/-1.3 for samples collected before and at 1, 3, 6 and 12 months, respectively. The MN frequencies at every sampling time did not statistically differ from the basal values. In addition, no statistically significant associations between MN values and hormone levels of E2 and FSH were observed throughout the entire study. This study shows the absence of any significant increase of MN frequencies in women undergoing oral and/or transdermal HRT, sequentially monitored for up to 12 months of therapy.

AMPD1 (C34T) polymorphism and clinical outcomes in patients undergoing myocardial revascularization.

BACKGROUND: C34T variant of adenosine monophosphate deaminase 1 (AMPD1) gene has been associated with a prolonged survival in heart failure and coronary artery disease, hypothetically linked to an enhanced production of adenosine. DESIGN: Since adenosine administration is a promising approach for the prevention of the ischemia-reperfusion in myocardial revascularization, the aim of this study was to investigate whether the AMPD1 (-) allele is associated with a favorable prognosis after coronary revascularization. In addition, we assessed the association between AMPD1 polymorphism and plasma adenosine levels. METHODS: We investigated a total of 161 patients receiving coronary revascularization (70 percutaneous transluminal coronary angioplasty and 91 coronary artery bypass graft). They were investigated for a composite endpoint including recurrent angina, non-fatal MI, target vessel revascularization, heart failure and cardiac death. Plasma adenosine was also measured by high-performance liquid chromatography methods on a subset of 25 patients. RESULTS: During the follow-up period (7.0+/-0.3 months), the overall combined endpoint accounted for 17 events (10 cardiac-related deaths, 6 revascularization procedures and 1 congestive heart failure). The composite endpoint was 9.8% for AMPD1 (-) allele carriers vs. 11.5% for non-carriers (log-rank statistic, p=n.s.). In the logistic analysis only low (

Somatic DNA damage in interventional cardiologists: a case-control study.

Interventional cardiologists who work in cardiac catheterization laboratories are exposed to low doses of ionizing radiation that could pose a health hazard. DNA damage is considered to be the main initiating event by which radiation damage to cells results in development of cancer and hereditary disease. The aim of the present study was to assess the effects of chronic low-dose X-ray radiation exposure on somatic DNA damage of interventional cardiologists working in high-volume cardiac catheterization laboratories. For this analysis, we used peripheral lymphocytes and the assay for micronuclei (MNs), which is considered to be a reliable biological dosimeter for radiation exposure. We obtained peripheral blood from 62 physicians (mean age+/-se = 40.6+/-1.5 years): 31 interventional cardiologists (group I, exposed) and 31 age- and sex-matched clinical cardiologists (group II, nonexposed). Interventional cardiologists showed higher MN values (group I=20.5+/-1.6 vs. group II=12.8+/-1.3, P=0.001), although some overlap was apparent in the individual subject analysis. A correlation between years of professional activity and MN frequency value was detectable for interventional cardiologists (r=0.428, P=0.02) but not for clinical cardiologists (r=0.253, P=0.17). The results indicated that, overall, interventional cardiologists working in a high-volume catheterization laboratory have higher levels of somatic DNA damage when compared with clinical cardiologists working outside the catheterization laboratory. The amount of this damage varies and is only weakly related to the duration of professional exposure, which suggests that a dominant modulation of the underlying genetic substrate by environmental factors has a role in determining the harm in individual physicians.

Diabetes and chronic nitrate therapy as co-determinants of somatic DNA damage in patients with coronary artery disease.

Somatic DNA damage has been linked to coronary artery disease (CAD). However, whether genetic instability is linked to CAD per se or to concomitant potentially genotoxic metabolic and pharmacological factors remains still unclear. The aim of this study was to evaluate the determinants of somatic DNA damage in a large population of patients undergoing coronary angiography. A total of 278 in-hospital patients (215 men, age 61.8+/-0.7 years) were studied by using micronucleus assay (MN) in human lymphocytes, which is one of the most commonly used biomarker for somatic DNA damage. Significant CAD (>50% diameter stenosis) was present in 210 patients (179 men, age 62.3+/-0.7 years). Normal coronary arteries were observed in 68 patients (35 men, age 60.2+/-1.7 years). There were no significant differences between patients with and without CAD, but patients with multivessel disease had the highest MN levels (P=0.01). MN frequency was also found significantly higher in presence of type 2 diabetes (P<0.0001), dyslipidemia (P=0.048) and nitrate therapy (P=0.0002). A significant additive effect was also observed between diabetes and nitrate therapy (P=0.02). On multivariate logistic regression analysis, diabetes [odds ratio=6.8 (95% confidence interval, 3.2-14.5), P<0.0001] and nitrate therapy [odds ratio=2.4 (95% confidence interval, 1.3-4.7), P=0.01] remained the only significant determinants for the 50th percentile of MN (>12 per thousand). These results indicated that diabetes and, to a lesser extent, chronic nitrate therapy are major determinants of somatic DNA instability in patients with CAD. DNA damage might represent an additional pathogenetic dimension and a possible therapeutic target in the still challenging management of coronary artery disease concerning diabetics.

Detection of mtDNA with 4977 bp deletion in blood cells and atherosclerotic lesions of patients with coronary artery disease.

Recent evidence suggests that somatic mutations in nuclear and mitochondrial DNA accumulated during aging, may significantly contribute to the pathogenesis of chronic-degenerative illness such as coronary artery disease (CAD). Mitochondrial DNA with 4977 bp deletion mutation (mtDNA4977) is a common type of mtDNA alteration in humans. However, little attempt has been made to detect the presence of mtDNA4977 deletion in cells and tissues of cardiovascular patients. This study investigated the presence of mtDNA4977 in blood samples of 65 cardiovascular patients and 23 atherosclerotic plaques of human coronaries with severe atherosclerosis. Moreover, the presence of the deletion has been investigated in blood cells from 22 healthy age-matched subjects. The detection of mtDNA4977 has been performed by using a nested polymerase chain reaction (PCR) protocol and normalized to wild-type mtDNA. A significant higher incidence of mtDNA4977 was observed in CAD patients with respect to healthy subjects (26.2% versus 4.5%; P=0.03). Furthermore, the relative amount of the deletion was significantly higher in the patients compared to the control group (P=0.02). The mtDNA4977 was detected in 17 of the 65 patients blood samples (26.2%) and deletion levels ranged from 0.18 to 0.46% of the total mtDNA (mean: 0.34+/-0.02%). For what concerns atherosclerotic lesions, 5 patients (21.7%) showed the deletion ranging from 0.13 to 0.45% of the total mtDNA (mean: 0.35+/-0.06%). In both samples from patients, the incidence and the relative amount of mtDNA4977 was not significantly influenced by atherogenic risk factors and clinical parameters. The obtained results may suggest that the increase of oxidative stress in cardiovascular disease may be responsible for the accumulation of mtDNA damage in coronary artery disease patients.

Genetic polymorphisms in folate and homocysteine metabolism as risk factors for DNA damage.

Epidemiological studies indicated a role for polymorphisms in genes of folate and homocysteine (Hcy) metabolism in the etiology of neurodegenerative disease, congenital defects and coronary artery disease (CAD). This study investigated the effect of several polymorphisms [C677 T, A1298C of methylenetetrahydrofolate reductase (MTHFR) and A66G of methionine synthase reductase (MTRR) genes] on Hcy levels and DNA damage in 68 patients who underwent coronary angiography. Plasma Hcy concentrations were higher in patients with multivessel disease with respect to monovessel disease and no-CAD patients (19.4+/-2.6 vs 11.6+/-1.2 and 13.7+/-1.4 micromol/l, respectively; P=0.03). 677TT patients had higher Hcy levels than those with 677CC or 677CT genotypes (26.2+/-4.3 vs 13.1+/-1.4 and 13.0+/-1.4 micromol/l, respectively; P=0.0006). No significant associations were found between A1298C and A66G polymorphisms and plasma Hcy levels. Among patients with 677CC genotype, 66GG individuals tended to have higher levels of Hcy than 66AA homozygotes (14.5+/-1.9 vs 8.9+/-0.7 micromol/l, P=0.06). Multivessel disease patients showed an increased frequency of DNA damage, measured by the micronucleus (MN) frequency, as compared to monovessel disease and no-CAD subjects (12.5+/-1.1 vs 8.5+/-0.8 and 8.2+/-0.9, respectively; P=0.006). The MN were positively correlated with Hcy levels (r=0.33, P=0.006) and were significantly higher in subjects with the 677TT genotype compared with the 677CC or 677CT genotypes (14.4+/-2.0 vs 8.8+/-1.2 and 9.5+/-0.7, respectively; P=0.006). A1298C and A66G polymorphisms had no effect on MN frequency. However, among 677TT patients, 66GG subjects tended to have higher levels of MN than those 66AG and 66AA (18.2+/-3.6 vs 13.8+/-4.0 and 10.3+/-1.7, respectively; P=NS). Our results indicate that genetic instability may be associated with increased risk for multiple Hcy-related diseases.

Interactive effect of the glutathione S-transferase genes and cigarette smoking on occurrence and severity of coronary artery risk.

Cardiovascular diseases and cancer are the main causes of death in developed countries. Mortality trends for these diseases suggest that they share common pathogenetic mechanisms. Glutathione S-transferase (GST) is a family of enzymes that detoxify reactive electrophiles, particularly present in tobacco smoke. Glutathione S-transferase null M1 and T1 (GSTM1 and GSTT1) genotypes have often been associated with increased risk of developing cancer. Our hypothesis was that the polymorphic GSTM1 and GSTT1 genes modulate the risk of smoking-coronary artery disease (CAD). We evaluated the distribution of GST genotypes in 430 angiographically defined patients (308 CAD and 122 non-CAD). The frequencies of GST null genotypes did not differ significantly between patients with CAD and without CAD. However, smokers with GSTM1 and GSTT1 null genotypes had a significantly higher risk of CAD than never-smokers with these genotypes present (OR 2.2 and 3.4 for smokers with null GSTM1 and GSTT1 genes, respectively). There was also evidence of multiple interaction between GSTM1 and GSTT1 deleted genotypes and smoking. In nonsmokers carrying both null genotypes the risk of CAD was 0.66. In smokers with both present genotypes the OR was 1.5 and was significantly increased in smokers with concurrent lack for GSTM1 and GSTT1 genes (OR=4.0). Moreover, smokers lacking GST genes had both more stenosed vessels and a higher Duke score than smokers expressing the genes. We also examined the levels of DNA damage in 66 men patients using the micronucleus test, a sensitive assay for evaluating chromosome damage. Micronucleus levels were higher in smokers with null genes than in smokers with present genes. These observations suggest that GST-null genotypes strengthen the effect of smoking on CAD risk by modulating the detoxification of genotoxic atherogens.

Endothelial nitric oxide synthase gene polymorphisms and risk of coronary artery disease.

BACKGROUND: Endothelial nitric oxide synthase (eNOS) could be a candidate gene for coronary artery disease (CAD). This study investigated the relationship of the eNOS Glu(298)-->Asp and T(786)-->C polymorphisms with the presence and severity of CAD in the Italian population. METHODS: We enrolled 415 unrelated individuals who underwent coronary angiography. The severity of CAD was expressed by means of the Duke score. The eNOS Glu(298)-->Asp and T(786)-->C variants were analyzed by PCR. RESULTS: There was significant linkage disequilibrium between the two eNOS polymorphisms (P <0.0001). Both variants were significantly associated with the occurrence and severity of CAD (P = 0.01 and 0.004 for Glu(298)-->Asp and T(786)-->C, respectively). The risk of CAD was increased among individuals homozygous for the C allele of the T(786)-->C polymorphism compared with individuals homozygous for the T allele (odds ratio = 2.5; P <0.01) and was independent of the other common risk factors (P = 0.04). Moreover, individuals with both the Asp/Asp genotype of the Glu(298)-->Asp polymorphism and at least one C allele of the T(786)-->C variant in the promoter region of the eNOS gene had an increased risk of CAD (odds ratio = 4.0; P <0.001) and a significantly higher mean Duke score (26.2 +/- 2.9 vs 45.2 +/- 3.7; P = 0.002) compared with individuals with the TT genotype and the Glu allele. CONCLUSIONS: The present study provides evidence that the Glu(298)-->Asp and T(786)-->C polymorphisms of the eNOS gene are associated with the presence and severity of angiographically defined CAD in the Italian population and that those individuals carrying both eNOS variants simultaneously might have a higher risk of developing CAD.

Methylenetetrahydrofolate reductase gene C677T polymorphism, homocysteine, vitamin B12, and DNA damage in coronary artery disease.

Elevated levels of plasma homocysteine (Hcy), a risk factor for coronary artery disease (CAD), can result from genetic errors, e.g., the methylenetetrahydrofolate reductase (MTHFR) polymorphism, or nutritional deficiencies, e.g., in vitamin B12 and folate. The mechanism by which Hcy induces atherosclerosis is not fully understood. Recently, Hcy has also been observed to induce DNA damage. In this study, we have investigated whether DNA damage is related to the C677T variant in the MTHFR gene and to plasma levels of Hcy, B12, and folate in patients with CAD. Patients ( n=46) with angiographically proven CAD were studied by using the micronucleus (MN) test, an accepted method for evaluating genetic instability. TT patients had plasma Hcy levels higher than those with the CT or CC genotypes (27.8+/-5.2 vs 13.7+/-2.2 and 12.9+/-1.9 micro mol/l, respectively; P=0.02). Patients with multi-vessel disease had higher plasma Hcy levels (11.6+/-1.2, 22.0+/-4.7, 19.3+/-3.9 micromol/l for one-, two- and three-vessel disease, respectively; P=0.05). The MN index increased with the number of affected vessels (8.4+/-0.7, 11.1+/-2.0, 14.2+/-1.7 for one-, two-, and three-vessels disease, respectively; P=0.02) and was significantly higher in subjects with the TT genotype compared with the CC or CT genotypes (15.7+/-2.4 vs 8.9+/-1.7 and 9.9+/-0.8; P=0.02). The MN index was also correlated negatively with plasma B12 concentration ( r=-0.343; P=0.019) and positively with plasma Hcy ( r=0.429, P=0.005). These data indicate that the MN index is associated with the severity of CAD and is related to the MTHFR polymorphism, suggesting an interesting link between coronary atherosclerosis and genetic instability in humans.

Elevated levels of oxidative DNA damage in patients with coronary artery disease.

BACKGROUND: Somatic DNA damage has been suggested to contribute to the pathogenesis of atherosclerosis. However, little is known about the role of oxidative DNA damage in patients with coronary artery disease (CAD). METHODS: In this study, we used the comet assay to measure oxidative DNA damage (DNA strand breaks and enzyme-sensitive sites) in peripheral blood lymphocytes from 13 patients with angiographically documented CAD and 11 age- and sex-matched control participants. RESULTS: Mean values of DNA strand breaks, oxidized pyrimidines and altered purines were significantly higher in CAD patients than in the control group (11.9 +/- 1.4, 18.0 +/- 2.7 and 18.1 +/- 3.1 compared with 3.3 +/- 0.2, 2.7 +/- 0.5 and 4.5 +/- 1.1; P < 0.0001, P < 0.0001 and P = 0.0009, respectively). Moreover, oxidized purines (for example, 8-oxo-guanine) increased with the number of affected vessels and positively correlated with the extent of CAD measured by means of the number of the coronary lesions (P = 0.76, P = 0.003) and the Duke scoring system (P = 0.66, P = 0.01). Diabetic patients showed higher levels of oxidized pyrimidines (31.3 +/- 5.5 compared with 14.1 +/- 2.7; P = 0.013), while patients with dyslipidemia had elevated altered purines compared with normal patients (20.4 +/- 2.6 compared with 4.9 +/- 3.1; P = 0.03). CONCLUSIONS: These data indicate an overall elevation of oxidative DNA damage in CAD patients correlated with the severity of the disease and some atherogenic risk factors, suggesting a possible role of oxidative genetic damage in the pathogenesis of atherosclerosis.