Sherif Z. Abdel-Rahman

A Pharmacogenetic versus a Clinical Algorithm for Warfarin Dosing

BACKGROUND The clinical utility of genotype-guided (pharmacogenetically based) dosing of warfarin has been tested only in small clinical trials or observational studies, with equivocal results. METHODS We randomly assigned 1015 patients to receive doses of warfarin during the first 5 days of therapy that were determined according to a dosing algorithm that included both clinical variables and genotype data or to one that included clinical variables only. All patients and clinicians were unaware of the dose of warfarin during the first 4 weeks of therapy. The primary outcome was the percentage of time that the international normalized ratio (INR) was in the therapeutic range from day 4 or 5 through day 28 of therapy. RESULTS At 4 weeks, the mean percentage of time in the therapeutic range was 45.2% in the genotype-guided group and 45.4% in the clinically guided group (adjusted mean difference, [genotype-guided group minus clinically guided group], -0.2; 95% confidence interval, -3.4 to 3.1; P=0.91). There also was no significant between-group difference among patients with a predicted dose difference between the two algorithms of 1 mg per day or more. There was, however, a significant interaction between dosing strategy and race (P=0.003). Among black patients, the mean percentage of time in the therapeutic range was less in the genotype-guided group than in the clinically guided group. The rates of the combined outcome of any INR of 4 or more, major bleeding, or thromboembolism did not differ significantly according to dosing strategy. CONCLUSIONS Genotype-guided dosing of warfarin did not improve anticoagulation control during the first 4 weeks of therapy. (Funded by the National Heart, Lung, and Blood Institute and others; COAG ClinicalTrials.gov number, NCT00839657.).

A multiplex PCR procedure for polymorphic analysis of GSTM1 and GSTT1 genes in population studies

A deletion polymorphism in glutathione S-transferase theta (GSTT1) gene was recently discovered in humans. Similar to the GSTM1 gene, GSTT1 is also recognized as a risk modifier in exposed populations. To evaluate the role of genetic polymorphism in health effects, the combined genetic polymorphism of different genes should be taken into consideration. In the present study, we have developed a multiplex PCR approach for simultaneous replication of both genes for molecular analysis. The multiplex PCR protocol was validated using donor DNA with different polymorphic combinations for both genes from two different ethnic populations (North Americans and Egyptians). The prevalence of the GSTM1 null genotype was 51% among North Americans and 44% among Egyptians. The prevalence of the GSTT1 null genotype was 15% among North Americans and 14.7% among Egyptians. Combined polymorphism analysis of both genes revealed that 6.3% of North Americans harbor the deleted genotype of both genes compared to 8.8% of the Egyptians. The data indicate that there is no major difference in allelic distribution of both genes between the ethnic populations. The multiplex PCR assay used in this study has the advantage of reducing the time, effort and cost required to carry out such analysis. It will also significantly enhance the ability to use genetic screening techniques as a potential tool for early detection of health outcomes in exposed populations.

The 399Gln polymorphism in the DNA repair gene XRCC1 modulates the genotoxic response induced in human lymphocytes by the tobacco-specific nitrosamine NNK

DNA repair plays a critical role in protecting the genome of the cell from the insults of cancer-causing agents such as those found in tobacco smoke. Reduced DNA repair capacity would, therefore, constitute a significant risk factor for smoking-related cancers. Recently, a number of polymorphisms in several DNA repair genes have been discovered, and it is possible that these polymorphisms may affect DNA repair capacity and thus modulate cancer susceptibility in exposed populations. In the current study, we explored the relationship between two polymorphisms in the DNA repair gene XRCC1 (polymorphisms in codons 194 and 399) and the genotoxic response induced by the tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). The sister chromatid exchange (SCE) assay was used as a marker of genetic damage. Our results, using whole blood cultures from 47 volunteers, indicated that treatment of cells with 0.24, 0.72 and 1.44 mM of NNK induced a concentration-dependent increase in the mean number of SCE (P<0.001). There was a significant difference (P<0.05) in response to NNK treatment between cells from individuals with the 399Gln allele (either homozygous or heterozygous) and cells from individuals with the homozygous 399 Arg/Arg genotype. Treatment of cells that have the 399Gln allele with 0.24, 0.72 and 1.44 mM NNK resulted in 22.8, 35.8 and 52.8% increases in NNK-induced SCE, respectively. Treatment of cells with the 399 Arg/Arg genotype using the same NNK concentrations resulted in 16.0, 15.5 and 32.6% increases in NNK-induced SCE, respectively. In contrast, no significant difference in NNK-induced SCE was observed between cells with the codon 194 Arg/Arg genotype and cells with the codon 194 Arg/Trp genotype at all concentrations of NNK tested. These data suggest that the Arg399Gln amino acid change may alter the phenotype of the XRCC1 protein, resulting in deficient DNA repair. Our study underscores the important role of polymorphisms in DNA repair genes in influencing the genotoxic responses to environmental mutagens, and justifies additional studies to investigate their potential role in susceptibility to cancer.

Inheritance of the 194Trp and the 399Gln variant alleles of the DNA repair gene XRCC1 are associated with increased risk of early-onset colorectal carcinoma in Egypt.

Patients under age 40 constitute 35.6% of all colorectal cancer cases in Egypt, an unusual disease pattern to which both environmental exposures and inefficient DNA repair may contribute. While a number of polymorphisms in DNA repair genes have been recently identified, their role as cancer risk modifiers is yet to be determined. In a pilot case-control study, we tested the hypothesis that polymorphisms in the gene for the DNA repair enzyme XRCC1 are associated with increased risk of colorectal cancer among Egyptians. Using a multiplex polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methodology, allelic variants of the XRCC1 gene at codons 194 (Arg-->Trp) (194Trp) and 399 (Arg-->Gln) (399Gln), were analyzed in DNA from lymphocytes of 48 newly-diagnosed colorectal cancer cases and 48 age- and sex-matched controls. Overall, the inheritance of 194Trp allele (Arg/Trp genotype) and 399Gln allele (combined Arg/Gln and Gln/Gln genotypes) was associated with increased colorectal cancer risk (odds ratio (OR)=2.56, 95% confidence limits (CL) 0.73-9.40, and P=0. 08 for 194Trp allele and OR=3.98, 95% CL 1.50-10.6, and P<0.001 for 399Gln allele). Interestingly, the frequencies of 194Trp and 399Gln genotypes were higher in colorectal cancer cases under age 40 than in corresponding controls, and an association between both polymorphisms and early age of disease onset was observed (OR=3.33, 95% CL 0.48-35.90, and P=0.16 for 194Trp and OR=11.90, 95% CL 2.30-51.50, and P=0.0003 for 399Gln). Analysis of the data after adjustment for place of residence indicated that the frequencies of the genotypes with the 194Trp and the 399Gln alleles were higher among urban residents (OR=3.33, 95% CL 0.48-35.90, and P=0.16 for 194Trp and OR=9.97, 95% CL 1.98-43.76, and P<0.001 for 399Gln) than among rural residents (OR=2.00, 95% CL 0.36-26.00, and P=0.30 for 194Trp and OR=1.90, 95% CL 0.50-7.53, and P=0.20 for 399Gln). These findings support our hypothesis and suggest that polymorphisms in the XRCC1 gene, in conjunction with place of residence, may modify disease risk. This first demonstration that polymorphisms in DNA repair genes may contribute to colorectal cancer susceptibility and may increase the risk of early onset of the disease opens the door for future studies in that direction.

β-Carotene: a cancer chemopreventive agent or a co-carcinogen?

Evidence from both epidemiological and experimental observations have fueled the belief that the high consumption of fruits and vegetables rich in carotenoids may help prevent cancer and heart disease in humans. Because of its well-documented antioxidant and antigenotoxic properties, the carotenoid beta-carotene (betaCT) gained most of the attention in the early 1980s and became one of the most extensively studied cancer chemopreventive agents in population-based trials supported by the National Cancer Institute. However, the results of three randomized lung cancer chemoprevention trials on betaCT supplementation unexpectedly contradicted the large body of epidemiological evidence relating to the potential benefits of dietary carotenoids. Not only did betaCT show no benefit, it was associated with significant increases in lung cancer incidence, cardiovascular diseases, and total mortality. These findings aroused widespread scientific debate that is still ongoing. It also raised the suspicion that betaCT may even possess co-carcinogenic properties. In this review, we summarize the current data on the co-carcinogenic properties of betaCT that is attributed to its role in the induction of carcinogen metabolizing enzymes and the over-generation of oxidative stress. The data presented provide convincing evidence of the harmful properties of this compound if given alone to smokers, or to individuals exposed to environmental carcinogens, as a micronutrient supplement. This has now been directly verified in a medium-term cancer transformation bioassay. In the context of public health policies, while the benefits of a diet rich in a variety of fruits and vegetables should continue to be emphasized, the data presented here point to the need for consideration of the possible detrimental effects of certain isolated dietary supplements, before mass cancer chemoprevention clinical trials are conducted on human subjects. This is especially important for genetically predisposed individuals who are environmentally or occupationally exposed to mutagens and carcinogens, such as those found in tobacco smoke and in industrial settings.

Polymorphism of metabolizing genes and lung cancer histology: Prevalence of CYP2E1 in adenocarcinoma

The relationship between genetic predisposition and development of specific cancers has not been adequately elucidated. In this study, the involvement of three polymorphic genes (CYP2E1, GSTM1, and GSTT1) in the development of different histological types of lung cancer was investigated. DNA was extracted from peripheral blood lymphocytes of lung cancer patients who have been long-term cigarette smokers (n = 52). Allelic variants of CYP2E1 were detected using PCR followed by PstI restriction enzyme digest and RFLP analysis, which detects a specific mutation causing over-expression of the gene. GSTM1 and GSTT1 genotypes were detected using two separate differential PCR methods. Our results indicate a 13.5% allele frequency for the CYP2E1 rare PstI site among the lung cancer patients which represents a 3.4-fold increase over the normal controls (OR = 3.5, 95% CL = 0.65-25.8). A novel observation is that all the patients with this polymorphism had adenocarcinomas only, resulting in a significant association between them (OR = 16.17, 95% CL = 0.95-73, P = 0.02). The frequency of the null GSTM1 gene was 42.3% among the lung cancer patients with no preferential tendency towards developing squamous cell carcinoma versus adenocarcinoma (OR = 1.10, 95% CL = 0.3-4.14, P = 0.5). The GSTT1 gene was absent in 21.1% of the patients with a non-significant tendency towards developing squamous cell carcinoma (OR = 1.23, 95% CL = 0.25-6.1, P = 0.5). Another important observation is the significant predominance of the three predisposing polymorphic alleles among the adenocarcinoma patients (OR = 3.4, 95% CL = 0.78-16.1, P = 0.05) compared with the squamous cell carcinoma patients. The results of this study indicate that the inheritance of several polymorphic metabolizing genes, particularly the CYP2E1 gene, contributes not only to the development of lung cancer but also to the development of specific types of cancer.

Single nucleotide polymorphisms of the DNA repair gene XPD/ERCC2 alter mRNA expression

Objectives Epidemiological studies documented associations between single nucleotide polymorphisms (SNPs) in the nucleotide excision repair gene XPD/ERCC2 and cancer risk. Little is known, however, about the underlying mechanisms for these associations. We explored a novel mechanism that could further explain the reported risk-modifying effect of these SNPs on disease susceptibility. Methods Using quantitative real-time polymerase chain reaction, we examined the relationship between three SNPs in the XPD gene (R156R in exon 6, D312N in exon 10 and K751Q in exon 23) and mRNA levels as a potential mechanism by which these SNPs could alter DNA repair capacity and affect disease risk. To further investigate the mechanism(s) by which these SNPs alter mRNA transcription levels, we performed a localized Mfold structure analysis on the mRNA sequence surrounding the studied SNPs. Results All three SNPs studied, alone and in combination, significantly decreased constitutive XPD mRNA levels (P<0.003) in lymphocytes of healthy subjects. The decrease in mRNA levels was significantly greater in smokers and was exacerbated by smoking duration and intensity. The decrease was more pronounced in older than in younger subjects. The R156R and the K751Q polymorphisms were predicted to alter mRNA secondary structure, indicating that these SNPs potentially affect local folding and mRNA stability. Conclusions Our results provide novel mechanistic explanations for epidemiological studies linking these SNPs to elevated cancer risk and emphasize the importance of comprehensively investigating the effect of both synonymous and nonsynonymous SNPs as risk modifiers by considering their potential effects on gene expression, protein translation and functions.

Caffeic acid phenethyl ester, a promising component of propolis with a plethora of biological activities: A review on its anti‐inflammatory, neuroprotective, hepatoprotective, and cardioprotective effects

Caffeic acid phenethyl ester (CAPE) is an important active component of honey bee propolis that possesses a plethora of biological activities. Propolis is used safely in traditional medicine as a dietary supplement for its therapeutic benefits. This review highlights the recently published data about CAPE bioavailability, anti‐inflammatory, neuroprotective; hepatoprotective and cardioprotective activities. CAPE showed promising efficacy both in vitro and in vivo studies in animal models with minimum adverse effects. Its effectiveness was demonstrated in multiple target organs. Despite this fact, it has not been yet investigated as a protective agent or a potential therapy in humans. Investigation of CAPE efficacy in clinical trials is strongly encouraged to elucidate its therapeutic benefit for different human diseases after performing full preclinical toxicological studies and gaining more insights into its pharmacokinetics.

Modulation of human placental P-glycoprotein expression and activity by MDR1 gene polymorphisms

The ABC transporter P-glycoprotein is a product of the MDR1 gene and its function in human placenta is to extrude xenobiotics from the tissue thus decreasing fetal exposure. The goal of this investigation was to examine the effect of three polymorphisms in the MDR1 gene on the expression and activity of placental P-gp. In 199 term placentas examined, the C1236T variant was associated with 11% lower P-gp protein expression than wild-type, while the C3435T and G2677T/A variants each were associated with a 16% reduction (p<0.05). Homozygotes for the C1236T and C3435T variant allele (TT) were associated with 42% and 47% increase in placental P-gp transport activity, respectively (p=0.04 and p=0.02) of the prototypic substrate, [(3)H]-paclitaxel. These findings indicate that the C3435T and G2677T/A SNPs in MDR1 are significantly associated with decreased placental P-gp protein expression, while the C1236T and C3245T homozygous variants are significantly associated with an increase in its efflux activity.

Polymorphisms in the DNA repair gene XRCC1 and susceptibility to alcoholic liver cirrhosis in older Southeastern Brazilians

The population of Southeastern Brazil has a very high mortality rate from liver cirrhosis, a disease that is considered an irreversible pre-malignant condition. This is largely due to the high prevalence of alcohol abuse in the region. Chronic alcohol consumption is associated with the production of free radical intermediates that can cause several DNA lesions. Reduced repair of these DNA lesions would, therefore, constitute a significant risk factor for liver cirrhosis and subsequent cancer. Recently, a number of polymorphisms in several DNA repair genes have been discovered, and it is possible that these polymorphisms may affect DNA repair capacity and thus modulate susceptibility to the disease. In this study, we tested the hypothesis that polymorphisms in the DNA repair gene XRCC1 are associated with increased risk of liver cirrhosis in Southeastern Brazilians. We conducted a pilot case-control study of 97 liver cirrhosis cases and 96 controls (matched for age, sex, and ethnicity) to investigate the role of two allelic variants coding for amino acid changes in the XRCC1 gene (the Arg194Trp and the Arg399Gln polymorphisms). Overall, we observed a 1.8-fold increase in the relative risk of liver cirrhosis associated with the 399Gln allele (either the heterozygous Arg/Gln or the homozygous Gln/Gln genotypes). The adjusted odds ratio (OR) was 1.82 (95% confidence limit (CL) 1.10-3.30). The relative risk appears to be highest among the Mestiso ethnic group (OR 2.60, 95% CL 0.92-7.34). There was a significant association between the 399Gln polymorphism and the risk of liver cirrhosis in older individuals over the age of 45 years (OR 2.70 (95% CL 1.14-6.48) compared to an OR of 1.24 (95% CL 0.55-2.78) for those under 45 years of age. No association was observed between the XRCC1 194Trp polymorphism and risk of liver cirrhosis. These preliminary results suggest that the XRCC1 399Gln polymorphism may be a significant risk modifier for alcoholic liver cirrhosis and justifies additional studies in that direction.