Systemic administration of the iron chelator deferiprone protects against light-induced photoreceptor degeneration in the mouse retina

Oxidative stress plays a key role in a light-damage (LD) model of retinal degeneration as well as in age-related macular degeneration (AMD). Since iron can promote oxidative stress, the iron chelator deferiprone (DFP) was tested for protection against light-induced retinal degeneration. To accomplish this, A/J mice were treated with or without oral DFP and then were placed in constant bright white fluorescent light (10,000 lx) for 20 h. Retinas were evaluated at several time points after light exposure. Photoreceptor apoptosis was assessed using the TUNEL assay. Retinal degeneration was assessed by histology 10 days after exposure to damaging white light. Two genes upregulated by oxidative stress, heme oxygenase 1 (Hmox1) and ceruloplasmin (Cp), as well as complement component 3 (C3) were quantified by RT-qPCR. Cryosections were immunolabeled for an oxidative stress marker (nitrotyrosine), a microglial marker (Iba1), as well as both heavy (H) and light (L) ferritin. Light exposure resulted in substantial photoreceptor-specific cell death. Dosing with DFP protected photoreceptors, decreasing the numbers of TUNEL-positive photoreceptors and increasing the number of surviving photoreceptors. The retinal mRNA levels of oxidative stress-related genes and C3 were upregulated following light exposure and diminished by DFP treatment. Immunostaining for nitrotyrosine indicated that DFP reduced the nitrative stress caused by light exposure. Robust H/L-ferritin-containing microglial activation and migration to the outer retina occurred after light exposure and DFP treatment reduced microglial invasion. DFP is protective against light-induced retinal degeneration and has the potential to diminish oxidative stress in the retina.     

Eicosapentaenoic acid metabolism by cytochrome P450 enzymes of the CYP2C subfamily

CYP2C enzymes epoxidize arachidonic acid (AA) to metabolites involved in the regulation of vascular and renal function. We tested the hypothesis that eicosapentaenoic acid (EPA), a n-3 polyunsaturated fatty acid, may serve as an alternative substrate. Human CYP2C8 and CYP2C9, as well as rat CYP2C11 and CYP2C23, were co-expressed with NADPH-CYP reductase in a baculovirus/insect cell system. The recombinant enzymes showed high EPA and AA epoxygenase activities and the catalytic efficiencies were almost equal comparing the two substrates. The 17,18-double bond was the preferred site of EPA epoxidation by CYPs 2C8, 2C11, and 2C23. 17(R),18(S)-Epoxyeicosatetraenoic acid was produced with an optical purity of about 70% by CYPs 2C9, 2C11, and 2C23 whereas CYP2C8 showed the opposite enantioselectivity. These results demonstrate that EPA is an efficient substrate of CYP2C enzymes and suggest that n-3 PUFA-rich diets may shift the CYP2C-dependent generation of physiologically active eicosanoids from AA- to EPA-derived metabolites.     

Lack of causal relationship between clusterin expression and photoreceptor apoptosis in light-induced retinal degeneration

Induction of apoptosis in the retina leads to cellular death by molecular mechanisms that are not well understood. Clusterin expression is increased in tissues undergoing apoptosis, including retinal neurodegenerative states, but the causal relationships remain to be clarified. To gain insight into clusterin's role in photoreceptor apoptosis, the cellular distribution of clusterin mRNA was compared with the pattern of apoptotic nuclear labelling in a rat model of light-induced retinal degeneration. In control retinal sections, clusterin mRNA was localized to the retinal pigment epithelium cells, photoreceptor inner segments, inner nuclear layer, and ganglion cell layer. Clusterin expression decreased in photoreceptors and retinal pigment epithelium cells, which progressively degenerated, and increased in preserved inner nuclear layer, in proportion to the duration of light exposure in both cyclic light- and dark-reared animals. These results suggest that clusterin is not causally involved in apoptotic mechanisms of photoreceptor death, but may relate to cytoprotective functions.     

Tauroursodeoxycholic acid (TUDCA) protects photoreceptors from cell death after experimental retinal detachment

Background

Detachment of photoreceptors from the underlying retinal pigment epithelium is seen in various retinal disorders such as retinal detachment and age-related macular degeneration and leads to loss of photoreceptors and vision. Pharmacologic inhibition of photoreceptor cell death may prevent this outcome. This study tests whether systemic administration of tauroursodeoxycholic acid (TUDCA) can protect photoreceptors from cell death after experimental retinal detachment in rodents.

Methodology/Principal Findings

Retinal detachment was created in rats by subretinal injection of hyaluronic acid. The animals were treated daily with vehicle or TUDCA (500 mg/kg). TUNEL staining was used to evaluate cell death. Photoreceptor loss was evaluated by measuring the relative thickness of the outer nuclear layer (ONL). Macrophage recruitment, oxidative stress, cytokine levels, and caspase levels were also quantified. Three days after detachment, TUDCA decreased the number of TUNEL-positive cells compared to vehicle (651±68/mm² vs. 1314±68/mm², P = 0.001) and prevented the reduction of ONL thickness ratio (0.84±0.03 vs. 0.65±0.03, P = 0.002). Similar results were obtained after 5 days of retinal detachment. Macrophage recruitment and expression levels of TNF-a and MCP-1 after retinal detachment were not affected by TUDCA treatment, whereas increases in activity of caspases 3 and 9 as well as carbonyl-protein adducts were almost completely inhibited by TUDCA treatment.

Conclusions/Significance

Systemic administration of TUDCA preserved photoreceptors after retinal detachment, and was associated with decreased oxidative stress and caspase activity. TUDCA may be used as a novel therapeutic agent for preventing vision loss in diseases that are characterized by photoreceptor detachment.     

Rabbit aorta converts 15-HPETE to trihydroxyeicosatrienoic acids: potential role of cytochrome P450

Previous work showed that rabbit aorta metabolizes arachidonic acid via 15-lipoxygenase to 15-hydroperoxyeicosatetraenoic acid (15-HPETE), which undergoes an enzymatic rearrangement to 11-hydroxy-14,15-epoxyeicosatrienoic acid (11-H-14,15-EETA) and 15-hydroxy-11,12-epoxyeicosatrienoic acid (15-H-11,12-EETA). Hydrolysis of the epoxy group results in the formation of 11,14,15- and 11,12,15-trihydroxyeicosatrienoic acids (THETAs). Endothelial cells have several heme-containing enzymes including cytochromes P450 (CYP), nitric oxide synthase (eNOS), and prostacyclin (PGI(2)) synthase that catalyze the rearrangement of 15-HPETE to HEETAs. Incubation of arachidonic acid and 15-lipoxygenase, or 15-HPETE with rabbit aortic microsomes or rat liver microsomes, a rich source of CYP, resulted in the formation of a product that comigrated with THETAs and HEETAs on HPLC. Immunoblot analysis showed the presence of CYP2C8 and CYP2J2 in aortic tissue and when CYP2J2 or CYP2C8 was incubated with arachidonic acid and 15-lipoxygenase, the major products were 11,12,15- and 11,14,15-THETAs. Incubation of purified hematin, CYP2C11, eNOS or PGI(2) synthase enzymes with arachidonic acid and 15-lipoxygenase produced a different pattern of metabolites from rabbit aortic microsomes. Clotrimazole, a non-specific CYP inhibitor, and ebastine and terfenadone, specific CYP2J2 inhibitors, blocked the ability of aortic microsomes to produce THETAs while specific inhibitors of PGI(2) synthase, eNOS or CYP2C8/2C9 had no effect on THETA production. We suggest that a CYP, possibly CYP2J2, may function as the hydroperoxide isomerase converting 15-HPETE to HEETAs in rabbit vascular tissue. Further hydrolysis of the epoxy group of the HEETAs results in the formation of 11,12,15- and 11,14,15-THETAs. The HEETAs and THETAs are both vasodilators and may function as important regulators of vascular tone.     

Linkage between the CYP2C8 and CYP2C9 genetic polymorphisms

Cytochrome P450 (CYP) 2C8 and 2C9 are polymorphic enzymes. The CYP2C8*3 and CYP2C9*2 are the major variant alleles in Caucasian populations. The enzymes encoded by these variant alleles have impaired function for the metabolism of several drug substrates. In the present study 1468 subjects that were used as population-based controls in the Stockholm Heart Epidemiology Program (SHEEP) were genotyped by allelic discrimination using a 5'-nuclease assay for CYP2C8*1, 2C8*3, 2C9*1, 2C9*2, and 2C9*3 variant alleles in which the frequencies appeared to be 0.91, 0.095, 0.83, 0.11, and 0.066, respectively. Approximately, 96% of the subjects with CYP2C8*3 allele also carried a CYP2C9*2 and 85% of the subjects that had CYP2C9*2 variant also carried a CYP2C8*3. The number of subjects carrying both of the CYP2C8*1*3 and CYP2C9*1*2 was 4.5-fold higher than expected. This strong association may be of importance especially for the metabolism of common substrates of CYP2C8 and CYP2C9 like arachidonic acid that produces physiologically active metabolites.     

Biosynthesis of epoxyeicosatrienoic acids varies between polymorphic CYP2C enzymes

Arachidonic acid is oxidized by cytochromes P450 2C (CYP2C) to epoxyeicosatrienoic acids (EETs), possessing vasoactive properties, with 11,12-EET as the endothelium derived hyperpolarization factor. Genetic variants of CYP2C enzymes have altered drug metabolizing capacity. Our primary aim was to determine whether EET biosynthesis differed in human liver microsomes with known CYP2C genotypes. Human liver microsomes (n = 25) of different CYP2C genotypes or yeast-expressed CYP2C enzymes were used. Analysis of metabolites was performed by liquid chromatography/mass spectrometry. Samples genotyped as CYP2C8*3/*3/CYP2C9*2/*2 exhibited a 34% (p < 0.05) decreased EET biosynthesis, compared to other CYP2C8/CYP2C9 haplotypes. Inhibition experiments suggested CYP2C8 and CYP2C9 to be the predominant catalysts of EETs. We found no differences between the three recombinantly expressed CYP2C9 variants, but CYP2C8.1 had lower K_m than these isoforms. In conclusion, there are genetic differences in the CYP2C-dependent oxidation of arachidonic acid to vasoactive metabolites, of which the relevance to cardiovascular pathophysiology is still unclear.     

Cytochrome p-450 epoxygenase products contribute to attenuated vasoconstriction after chronic hypoxia

The systemic vasculature exhibits attenuated vasoconstriction following chronic hypoxia (CH) that is associated with endothelium-dependent vascular smooth muscle (VSM) cell hyperpolarization. We hypothesized that increased production of arachidonic acid metabolites such as the cyclooxygenase product prostacyclin or cytochrome p-450 (CYP) epoxygenase-derived epoxyeicosatrienoic acids (EETs) contributes to VSM cell hyperpolarization following CH. VSM cell resting membrane potential (Em) was measured in superior mesenteric artery strips isolated from rats with control barometric pressure (Pb, congruent with 630 Torr) and CH (Pb, 380 Torr for 48 h). VSM cell Em was normalized between groups following administration of the CYP inhibitors 17-octadecynoic acid and SKF-525A. VSM cell hyperpolarization after CH was not altered by cyclooxygenase inhibition, whereas the selective CYP2C9 inhibitor sulfaphenazole normalized VSM cell Em between groups. Iberiotoxin also normalized VSM cell Em, which suggests that large-conductance, Ca2+-activated K+ (BKCa) channel activity is increased after CH. Sulfaphenazole administration restored phenylephrine-induced and myogenic vasoconstriction and Ca2+ responses of mesenteric resistance arteries isolated from CH rats to control levels. Western blot experiments demonstrated that CYP2C9 protein levels were greater in mesenteric arteries from CH rats. In addition, 11,12-EET levels were elevated in endothelial cells from CH rats compared with controls. We conclude that enhanced CYP2C9 expression and 11,12-EET production following CH contributes to BKCa channel-dependent VSM cell hyperpolarization and attenuated vasoreactivity.     

DNA repair in photoreceptor survival

Light triggers a sequence of events that damage photoreceptor cells within the superior central portion of the retina, resulting in apoptotic cell death. This damage is mediated by energy absorbed by rhodopsin and the intermediates of the rhodopsin-bleaching process. Furthermore, inhibition of the visual cycle and the re-isomerization of all-trans retinol preserve photoreceptors. We have recently shown light-induced DNA fragmentation to occur only within photoreceptors, and, in time-courses following light treatment, these cells exhibit two peaks of damage, approx 24 h apart. This was also observed by quantification of nucleosome-length DNA fragments and their multimers (DNA ladders) as well as by highly repetitive short interspersed nuclear element (SINE) analysis. This bimodal pattern of photoreceptor DNA fragmentation suggests two populations of cells, and each of these were affected by light at a different rate or time. However, the rat retina is composed of 500 nm-sensitive rods, and approx 2% cones, suggesting that a two-cell-type hypothesis is incorrect. Thus, there is a possibility that light-induced DNA fragmentation is triggered and that some photoreceptors are able to initiate a repair mechanism, resulting in a temporary decrease in DNA damage followed by another wave of fragmentation that ultimately leads to cell death. Subsequently, we observed that the repair enzyme DNA polymerase beta was upregulated following light treatment, again suggesting the presence of a repair mechanism. Our results suggest that a DNA-repair mechanism exists within photoreceptors, and indicate that manipulation of this process may provide additional protection and/or recovery from events that trigger DNA fragmentation and apoptotic cell death in photoreceptors.     

Overproduction of bioactive retinoic acid in cells expressing disease-associated mutants of retinol dehydrogenase 12

Retinol dehydrogenase 12 (RDH12) is an NADP(+)-dependent oxidoreductase that in vitro catalyzes the reduction of all-trans-retinaldehyde to all-trans-retinol or the oxidation of retinol to retinaldehyde depending on substrate and cofactor availability. Recent studies have linked the mutations in RDH12 to severe early-onset autosomal recessive retinal dystrophy. The biochemical basis of photoreceptor cell death caused by mutations in RDH12 is not clear because the physiological role of RDH12 is not yet fully understood. Here we demonstrate that, although bi-directional in vitro, in living cells, RDH12 acts exclusively as a retinaldehyde reductase, shifting the retinoid homeostasis toward the increased levels of retinol and decreased levels of bioactive retinoic acid. The retinaldehyde reductase activity of RDH12 protects the cells from retinaldehyde-induced cell death, especially at high retinaldehyde concentrations, and this protective effect correlates with the lower levels of retinoic acid in RDH12-expressing cells. Disease-associated mutants of RDH12, T49M and I51N, exhibit significant residual activity in vitro, but are unable to control retinoic acid levels in the cells because of their dramatically reduced affinity for NADPH and much lower protein expression levels. These results suggest that RDH12 acts as a regulator of retinoic acid biosynthesis and protects photoreceptors against overproduction of retinoic acid from all-trans-retinaldehyde, which diffuses into the inner segments of photoreceptors from illuminated rhodopsin. These results provide a novel insight into the mechanism of retinal degeneration associated with mutations in RDH12 and are consistent with the observation that RDH12-null mice are highly susceptible to light-induced retinal apoptosis in cone and rod photoreceptors.     

Metabolism of eicosapentaenoic and docosahexaenoic acids by recombinant human cytochromes P450

Epoxidation and hydroxylation of arachidonic acid (AA) are both catalyzed by cytochromes P450s (CYPs). The oxidized metabolites are known to be involved in the regulation of vascular tone and renal function. By using a panel of 15 human recombinant CYPs, this study demonstrates that other polyunsaturated long-chain fatty acids (PUFA-LC), especially the ω3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are also epoxidised. The regioselectivity of epoxidation of four PUFA-LC by CYPs was investigated. Among the several CYPs tested, CYP2C9/2C19 and 1A2 were the most efficient in EPA and DHA epoxidations. It ensued that 10 μM of these two ω3 fatty acids decreased by more than 80% and 60%, respectively, the formation by CYP2C9 of AA-epoxidised derivatives. These findings suggest that some physiological effects of ω3 fatty acids may be due to a shift in the generation of active epoxidised metabolites of AA through CYP-mediated catalysis.     

Arachidonic acid release in renal proximal tubule cell injuries and death

Arachidonic acid release and the effect of phospholipase inhibitors on various types of cell injuries and death to rabbit renal proximal tubule suspensions were determined. Proximal tubules were exposed to the mitochondrial inhibitor antimycin A (0.1 μM), the protonophore carbonyl cyanide ρ-trifluoromethoxypheitylhydrazone (1 μM FCCP), the oxidant tertbutyl hydroperoxide (0.5 mM TBHP), or the calcium ionophore ionomycin (5 μM) in the absence or presence of the putative phospholipase inhibitors dibucaine, mepacrine, chlorpromazine, or U-26384. The phospholipase inhibitors had no effect on the proximal tubule lactate dehydrogenase (LDH) release (a marker of cell death) produced by FCCP, antimycin A, or ionomycin after 1,2, or 2 hours of exposure, respectively. Only dibucaine and mepacrine decreased LDH release in TBHP-treated proximal tubules without decreasing TBHP-induced lipid peroxidation. Antimycin A and ionomycin did not release arachidonic acid from proximal tubules prelabeled with [1-¹⁴C] arachidonic acid. In contrast, TBHP released arachidonic acid from proximal tubules prior to the onset of cell death, and dibucaine and mepacrine decreased the TBHP-induced release. Thus, phospholipase inhibitors were cytoprotective in those injuries that produced arachidonic acid release. These results suggest that arachidonic acid release and phospholipase A₂ activation play a contributing role in oxidant-induced renal proximal tubule cell injury and death but not in mitochondrial inhibitor- or calcium ionophore-induced proximal tubule cell injury and death.     

Reactive oxygen species as mediators of photoreceptor apoptosis in vitro

Retinitis pigmentosa is a heterogeneous group of retinal degenerations characterized by a progressive loss of photoreceptors through the process of apoptosis. The apoptotic cell death of photoreceptors appears to represent a final common pathway in the pathology of retinitis pigmentosa. Previous studies have reported the ability of antioxidants to ameliorate light-induced retinal degeneration, suggesting a role for oxidative stress in photoreceptor cell death. This study demonstrates an early and sustained increase in intracellular reactive oxygen species accompanied by a rapid depletion of intracellular glutathione in an in vitro model of photoreceptor apoptosis. These early changes in the cellular redox state precede disruption of mitochondrial transmembrane potential, nuclear condensation, DNA nicking, and cell shrinkage, all of which are well-characterized events of apoptotic cell death. The ability of zinc chloride and pyrrolidine dithiocarbamate, two established antioxidants, to inhibit photoreceptor apoptosis through the scavenging of intracellular reactive oxygen species establishes a role for reactive oxygen species as possible mediators of in vitro photoreceptor apoptosis. This study provides a molecular basis for the inhibition of photoreceptor apoptosis by antioxidants.     

亚致死剂量乙草胺对蚯蚓的毒性效应及敏感生物标记物

本研究以赤子爱胜蚓为受试生物,采用外源添加污染物的方法,将受试生物暴露于含亚致死剂量乙草胺(添加浓度分别为1、2、4、8 mg·kg^-1)的土壤中7 d,研究蚯蚓生长抑制率、细胞色素P450同工酶(CYP1A2、2C9和3A4)活力及代谢组学对乙草胺的响应,从个体、酶、小分子标记物3个层次探讨亚致死剂量乙草胺对蚯蚓的毒性效应,初步推断其毒性作用阈值,筛选敏感生物标记物,探讨其致毒机理。结果表明: 乙草胺暴露下,与对照相比,蚯蚓体重抑制率无明显差异,但CYP1A2、2C9和3A4活力受到明显抑制,10组小分子代谢物(1, 6-二磷酸果糖、胞苷酸、尿苷酸、腺苷酸、腺苷、黄嘌呤、延胡索酸、二羟基戊二酸、鸟氨酸与16-羟二十烷四烯酸)水平显著降低;另有6组小分子代谢物(腺苷琥珀酸、琥珀酸、精氨酸、色氨酸、天冬酰胺与苯丙氨酸)水平在2～8 mg·kg^-1乙草胺暴露下显著升高。乙草胺暴露导致蚯蚓受到氧化损伤,糖酵解功能减弱,三羧酸循环失衡,嘌呤及嘧啶代谢紊乱,氨基酸代谢受损。与个体水平的受试终点相比,CYP同工酶活力与上述16个小分子代谢物对乙草胺暴露的响应更为敏感。建议将CYP同工酶(1A2、2C9及3A4)活力与上述小分子代谢物为一组生物标记物,可以多指标、多层次联合诊断土壤乙草胺污染的生态毒性效应。其诊断结果将更为精准。     

Hyperactivation of retina by light in mice leads to photoreceptor cell death mediated by VEGF and retinal pigment epithelium permeability

Light toxicity is suspected to enhance certain retinal degenerative processes such as age-related macular degeneration. Death of photoreceptors can be induced by their exposure to the visible light, and although cellular processes within photoreceptors have been characterized extensively, the role of the retinal pigment epithelium (RPE) in this model is less well understood. We demonstrate that exposition to intense light causes the immediate breakdown of the outer blood–retinal barrier (BRB). In a molecular level, we observed the slackening of adherens junctions tying up the RPE and massive leakage of albumin into the neural retina. Retinal pigment epithelial cells normally secrete vascular endothelial growth factor (VEGF) at their basolateral side; light damage in contrast leads to VEGF increase on the apical side – that is, in the neuroretina. Blocking VEGF, by means of lentiviral gene transfer to express an anti-VEGF antibody in RPE cells, inhibits outer BRB breakdown and retinal degeneration, as illustrated by functional, behavioral and morphometric analysis. Our data show that exposure to high levels of visible light induces hyperpermeability of the RPE, likely involving VEGF signaling. The resulting retinal edema contributes to irreversible damage to photoreceptors. These data suggest that anti-VEGF compounds are of therapeutic interest when the outer BRB is altered by retinal stresses.     

Identification of cytochrome P450 2D6 and 2C9 substrates and inhibitors by QSAR analysis

This paper presents four new QSAR models for CYP2C9 and CYP2D6 substrate recognition and inhibitor identification based on human clinical data. The models were used to screen a large data set of environmental chemicals for CYP activity, and to analyze the frequency of CYP activity among these compounds. A large fraction of these chemicals were found to be CYP active, and thus potentially capable of affecting human physiology. 20% of the compounds within applicability domain of the models were predicted to be CYP2C9 substrates, and 17% to be inhibitors. The corresponding numbers for CYP2D6 were 9% and 21%. Where the majority of CYP2C9 active compounds were predicted to be both a substrate and an inhibitor at the same time, the CYP2D6 active compounds were primarily predicted to be only inhibitors. It was demonstrated that the models could identify compound classes with a high occurrence of specific CYP activity. An overrepresentation was seen for poly-aromatic hydrocarbons (group of procarcinogens) among CYP2C9 active and mutagenic compounds compared to CYP2C9 inactive and mutagenic compounds. The mutagenicity was predicted with a QSAR model based on Ames in vitro test data.     

Analysis of diclofenac and its metabolites by high-performance liquid chromatography: relevance of CYP2C9 genotypes in diclofenac urinary metabolic ratios

In humans, diclofenac is metabolised to 4'-hydroxy (OH), 3'-OH and 5-OH metabolites. The polymorphic CYP2C9 is involved in the metabolism of diclofenac to 4'-OH diclofenac and 3'-OH diclofenac. The aim of the present study was to develop a high-performance liquid chromatographic method to simultaneously measure diclofenac and its metabolites in urine, suitable for metabolic studies. After liquid-liquid extraction the compounds were separated in a reversed-phase column and measured by ultraviolet absorption at 282 nm. For all compounds intra-day and inter-day variations were less than 7%, and the limits of quantitation were 0.25 mg/l. No analytical interference with endogenous compounds was found. The relationship between diclofenac metabolic ratios among different CYP2C9 genotypes is reported. The CYP2C9*3/*3 subject had the highest diclofenac/4'-OH ratios. However no difference was found between CYP2C9*2/*2 and *1/*1 genotypes. The chromatographic method developed was sensitive and reliable for the measurement of diclofenac and its metabolites simultaneously in human urine, and is suitable for use in diclofenac metabolism studies.     

Role of CYP2C9 and CYP2C19 polymorphisms in patients with atherosclerosis

The arachidonic acid metabolizing CYP enzymes with prominent roles in vascular regulation are epoxygenases of the two gene family which generate epoxyeicosatrienoic acids. Carriers of CYP2C9 mutant alleles exhibit a diminished CYP2C9 metabolic capacity leading to decreased endothelium-derived hyperpolarizing factors (EDHF) synthesis and an increased risk for atherosclerosis. We investigated whether the polymorphisms of CYP2C9/19 are related with atherosclerosis. We examined 108 patients having angioraphically > or =70 coronary artery narrowing and 90 healthy controls. CYPC2C9/19*2 and CYP2C9/19*3 alleles were investigated in both patients and controls by a real time PCR instrument. There was no significant difference in the distribution of the CYP2C9*2/*3 alleles between cases and the controls. We found that smoker patients having CYP2C9*2 heterozygote genotype have 3.7-fold risk of developing atherosclerosis. CYP2C19*3 heterozygote alleles are more frequent in patients than in controls (10.2%, 5.6% respectively) and it is related with a three-fold risk of atherosclerosis (odds ratio (OR) = 3.75, confidence interval (CI) = 0.75-18.65). It becomes clear that cigarette smoking can cause almost all major diseases prevalent today, such as cancer or heart disease. This inter-subject variability in cigarette-induced pathologies is partly mediated by genetic variants of genes that may participate in detoxification processes, e.g., cytochrome P450 (CYP), cellular susceptibility to toxins, such as p53, or disease development such as atherosclerosis.