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THE ASSOCIATION OF OGG1 Ser326Cys POLYMORPHISM AND URINARY 8-OHdG LEVELS WITH LUNG CANCER SUSCEPTIBILITY: A HOSPITAL- BASED CASE-CONTROL STUDY IN TURKEY

The Association of OGG1 Ser326Cys Polymorphism and Urinary 8-OHdG Levels With Lung Cancer Susceptibility: A Hospital-Based Case-Control Study in Turkey High incidence and poor prognosis of lung cancer make it a major health problem worldwide. Although smoking is a major cause of lung cancer, only some smokers develop lung cancer, which suggests that there is a genetic predisposition in some individuals. 8-OHG is an important oxidative base lesion and may elevate due to cancer and smoking. It is repaired by 8-hydroxyguanine DNA glycosylase 1 (OGG1), which has several polymorphisms. Although the Ser326Cys polymorphism is consistently associated with a range of cancers, findings about this polymorphism and lung cancer risk are contradictory. To date, no study has examined this association in the Turkish population. We conducted a case-control study to investigate the association between OGG1 Ser326Cys polymorphism and the risk of lung cancer using PCR-RFLP. We also evaluated gene-smoking interaction and excretion of urinary 8-OHdG. Our results suggest that the OGG1 Ser326Cys polymorphism is not a genetic risk factor for lung cancer, and that the heterozygous genotype is associated with a significantly reduced risk for lung cancer. The levels of 8-OHdG did not correlate with the polymorphism and smoking. Larger association studies are needed to validate our findings, and mechanistic studies are needed to elucidate the underlying molecular mechanisms of this association. Povezanost OGG1 Ser326Cys polimorfizma i razina 8-OHdG u mokraći sa sklonosti obolijevanju od karcinoma pluća: rezultati ispitivanja na bolesnicima i kontrolnoj populaciji u Turskoj Karcinom pluća velik je javnozdravstveni problem u čitavom svijetu zbog svoje visoke učestalosti i loše prognoze. Premda je navika pušenja jedan od glavnih uzročnika karcinoma pluća, od ove bolesti oboli samo dio populacije pušača, što govori u prilog postojanju genetske predispozicije za njezin nastanak. 8-OHG je oksidativno oštećenje baze u molekuli DNA čija se učestalost može povećati zbog zloćudnih tumora i pušenja. U popravku tog oštećenja sudjeluje enzim 8-hidroksigvanin DNA-glikozilaza (OGG1) za koji je dokazano postojanje polimorfizma. Iako se polimorfizam Ser326Cys često dovodi u vezu s različitim vrstama zloćudnih bolesti, dosadašnji su rezultati o vezi između polimorfizma tog enzima i rizika od pojave karcinoma pluća kontradiktorni. Do danas na turskoj populaciji nisu provedena istraživanja koja bi dala jasne odgovore o toj povezanosti. Ovo je istraživanje usporedo provedeno u bolesnika i u zdravoj populaciji primjenom metode PCR-RFLP s ciljem utvrđivanja moguće povezanosti polimorfizma OGG1 Ser326Cys i rizika od karcinoma pluća. Nadalje, istražena je interakcija gena i navike pušenja te ekskrecija 8-OHdG u mokraći. Dobiveni rezultati pokazuju da polimorfizam OGG1 Ser326Cys nije genetski čimbenik rizika od pojave karcinoma pluća, a pokazalo se da je heterozigotni genotip povezan sa značajno nižim rizikom od karcinoma pluća. Razine 8-OHdG izmjerene u mokraći nisu bile u korelaciji ni s polimorfizmom ni s navikom pušenja. Zaključujemo da su za vrednovanje dobivenih rezultata potrebna istraživanja na još većem broju ispitanika te mehanistička istraživanja koja bi mogla razjasniti molekularne mehanizme koji su u pozadini ove povezanosti.

Effects of the anti-malarial compound cryptolepine and its analogues in human lymphocytes and sperm in the Comet assay.

Malaria is a mosquito-borne infectious disease caused by the genus Plasmodium. It causes one million deaths per year in African children under the age of 5 years. There is an increasing development of resistance of malarial parasites to chloroquine and other currently used anti-malarial drugs. Some plant products such as the indoloquinoline alkaloid cryptolepine have been shown to have potent activity against P. falciparum in vitro. On account of its toxicity, cryptolepine is not suitable for use as an antimalarial drug but a number of analogues of cryptolepine have been synthesised in an attempt to find compounds that have reduced cytotoxicity and these have been investigated in the present study in human sperm and lymphocytes using the Comet assay. The results suggest that cryptolepine and the analogues cause DNA damage in lymphocytes, but appear to have no effect on human sperm at the assessed doses. In the context of antimalarial drug development, the data suggest that all cryptolepine compounds and in particular 2,7-dibromocryptolepine cause DNA damage and therefore may not be suitable for pre clinical development as antimalarial agents.

The impact of OGG1, MTH1 and MnSOD gene polymorphisms on 8-hydroxy-2′-deoxyguanosine and cellular superoxide dismutase activity in myocardial ischemia–reperfusion

Ischemia–reperfusion (I/R) injury, by inducing oxidative DNA damage, is one of the leading causes of increased patient morbidity and mortality in coronary artery by-pass grafting (CABG) surgery. 8-Hydroxyguanine (8-OHG) is an important oxidative base lesion. The 8-oxoguanine glycosylase (hOGG1) and hMTH1, which have several polymorphisms, remove 8-OHdG from the nucleotide pool. We investigated whether there are any correlations the biomarkers of oxidative stress (superoxide dismutase; SOD and 8-OHdG in serum) with genotype for two DNA repair genes (OGG1 and MTH1) and an antioxidant enzyme gene (manganese superoxide dismutase; MnSOD). Therefore, we measured DNA damage (8-hydroxy-2-deoxyguanosine; 8-OHdG) and endogenous antioxidant activity (SOD) at five different time points (T1, before anesthesia; T2, after anesthesia; T3, after ischemia; T4, after reperfusion and T5, after surgery). and also, MnSOD and MutT homolog 1 (MTH1) genes polymorphisms were genotyped by polymerase chain reaction–restricted fragment length polymorphism (PCR–RFLP) in patients undergoing coronary artery by-pass grafting (CABG) surgery. No statistically significant differences were detected in the levels of 8-OHdG and SOD in serum in terms of OGG1 Ser326Cys, MTH1 Val83Met and MnSOD Ala16Val genetic polymorphisms. Our results suggest that OGG1, MTH1 and MnSOD gene polymorphisms are not genetic risk factors for I/R injury.

ADRA2A polymorphism and smoking in a Turkish population.

Adrenoceptors (ARs) consist of nine subtypes, which are involved in a wide spectrum of physiological functions and are the site of action for a considerable percentage of currently prescribed therapeutics. All AR subtypes (except α1D) can be polymorphic because of the genetic variations in the coding and non-coding regions. Sixteen sequence variations were identified in α-adrenergic 2A (ADRA2A) gene. Among them, ADRA2A C1291G polymorphism is one of the most important polymorphisms, which plays a major role in regulating neurotransmitter release, blood pressure, lipolysis, insulin secretion, and platelet aggregation. A C–G transversion results in an MspI restriction fragment length polymorphism located at 1291 bp upstream of the origin of transcription. Because Medline search showed no study showing the allelic frequencies, and no information is available on inter-individual variability of ADRA2A C1291G polymorphism in Turkish population, we genotyped 203 healthy Turkish subjects. Because of large genetic variation of the polymorphism, we aimed to find out the distribution of C1291G polymorphism in Turkish population. Furthermore, we evaluated the possible association between the C1291G polymorphism in the ADRA2A receptor gene and smoking. The frequencies for the 1291C and 1291G alleles were 64% and 36%, respectively. The genotype frequencies for C1291C, C1291G, and G1291G were 35.5%, 57.6%, and 6.9%, respectively, in Turkish population. The allelic frequencies (1291C and 1291G) and G1291G homozygous variant genotype were similar to those reported in different Caucasian populations; however, C1291C and C1291G genotypes were different. We also observed that the frequency of the G allele was slightly higher in smoker subjects and lower among controls. The ADRA2A G allele may play a role in the predisposition to smoking. There is a need for expanding genotype and haplotype studies because of its importance in various physiological disorders and to confirm the association of this polymorphism with smoking.

Determination of the impurities in drug products containing montelukast and in silico/in vitro genotoxicological assessments of sulfoxide impurity

Impurities affecting safety, efficacy, and quality of pharmaceuticals are of increasing concern for regulatory agencies and pharmaceutical industries, since genotoxic impurities are understood to play important role in carcinogenesis. The study aimed to analyse impurities of montelukast chronically used in asthma theraphy and perform genotoxicological assessment considering regulatory approaches. Impurities (sulfoxide, cis-isomer, Michael adducts-I&II, methylketone, methylstyrene) were quantified using RP-HPLC analysis on commercial products available in Turkish market. For sulfoxide impurity, having no toxicity data and found to be above the qualification limit, in silico mutagenicity prediction analysis, miniaturized bacterial gene mutation test, mitotic index determination and in vitro chromosomal aberration test w/wo metabolic activation system were conducted. In the analysis of different batches of 20 commercial drug products from 11 companies, only sulfoxide impurity exceeded qualification limit in pediatric tablets from 2 companies and in adult tablets from 7 companies. Leadscope and ToxTree programs predicted sulfoxide impurity as nonmutagenic. It was also found to be nonmutagenic in Ames MPF Penta I assay. Sulfoxide impurity was dose-dependent cytotoxic in human peripheral lymphocytes, however, it was found to be nongenotoxic. It was concluded that sulfoxide impurity should be considered as nonmutagenic and can be classified as ordinary impurity according to guidelines.

Micronucleus assay assessment of possible genotoxic effects in patients treated with titanium alloy endosseous implants or miniplates

The use of titanium and its alloys (Ti-6Al-4V) for oral surgery has increased dramatically in recent years. Ti is a stable biocompatible metal suitable for oral applications and it has been used for endosseous subperiosteal implants and miniplate fixation for more than 25 years. Dental implants are typically made of Ti or Ti alloys. The alloys are potentially toxic due to release of vanadium and aluminum. We tested the possible genotoxicity of Ti alloy endosseous implants and miniplates on the oral mucosal tissues of two groups of patients: 17 patients receiving Ti miniplate and screw fixation, and 37 endosseous dental implant placement patients. Preoperative and postoperative mucogingival cell samples were collected. Genotoxicity was assessed by the micronucleus assay (MN). There were slight but not statistically significant increases in the frequencies of MN (p=0.087 and p=0.047) post-operation in both groups. In summary, neither of the applications showed genotoxicity in the oral epithelial cells of patients.

Genotoxicity assessment of perfluoroalkyl substances on human sperm

Perfluoroalkyl and polyfluoroalkyl substances (PFAS) are synthetic chemicals that have been used in industry and consumer products. Because the presence of PFAS has been identified in humans and the environment in the last decade, human exposure to PFAS is a current public health concern. It has been shown that some PFAS lead to adverse health effects in the male reproductive system. However, there is no information about probable genotoxic effects of these chemicals on sperm cells. This study aimed to investigate the possible genotoxic damage on human sperm cells exposed to certain major PFAS compounds that were selected considering their extensive usage, high persistence in the environment, and high bioaccumulation in humans. These PFAS are perfluorooctanesulfonic acid (PFOS), perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), and perfluorohexanoic acid (PFHxA). The alkaline comet assay was used to detect the DNA damage to sperm. Sperm cells were treated with 0.1–1 mM of each PFAS at 32°C for 1 h to obtain optimal survival. As a result of the experiments, it was discovered that the exposure to PFOS, PFOA, PFNA, and PFHxA did not cause significant levels of cytotoxicity and did not cause damage to sperm DNA under these conditions. The results suggest that the exposure to these PFAS did not interfere with sperm DNA. Indirect toxicity mechanisms should be taken into account to assess the association between the PFAS exposure and male reproductive toxicity.

An investigation of the mutagenic activity of salamide – a major impurity of hydrochlorothiazide

Abstract Hydrochlorothiazide is a widely used antihypertensive agent and one of its major impurities, salamide (4-amino-6-chlorobenzene-1,3-disulphonamide), has a chemical structure containing a primary amino group, a functional group that has previously been reported to be associated with carcinogenic activity. It is known that hydrochlorothiazide purity is a challenging problem for the pharmaceutical industry. As there were no prior mutagenicity data for the impurity salamide, the aim was to investigate its mutagenicity in this study. Salamide was tested for mutagenic potential in Salmonella typhimurium TA98, TA100, TA 1535, TA 1537, and E. coli WP2 uvrA + E. coli WP2 [pKM101] strains at six different concentrations, the highest concentration being the 5000 μg/plate. In both the presence and absence of the metabolic activation system, no mutagenic activity was observed. Results indicated that salamide should be classified as an ordinary impurity and controlled according to Q3A(R2) and Q3B(R2) guidelines.

Comet Assay in Evaluating DNA Damage Associated With Ischaemia-Reperfusion Injury in Patients Undergoing Coronary Surgery

Comet Assay in Evaluating DNA Damage Associated With Ischaemia-Reperfusion Injury in Patients Undergoing Coronary Surgery Ischaemia-reperfusion (I/R) injury is responsible for a number of conditions such as coronary bypass and myocardial infarction, and deaths. Oxygen-free radicals formed during I/R have been proposed as the leading causes of tissue injury, and they play an important role in I/R injury. I/R induces oxidative DNA damage (such as purinic and pyrimidinic base lesions). Comet assay is a suitable and sensitive method for early detection of low-level DNA damage. We used modified alkaline comet assay in peripheral blood lymphocytes and evaluated I/R-induced DNA damage in patients undergoing coronary artery bypass graft (CABG) operation (in vivo model for I/R). No statistically significant difference in DNA damage levels was found before surgery, after anaesthesia, ischemia, reperfusion, and surgery. However, blood lactate levels (assessed in parallel with the comet assay) increased after I/R and did not return to the baseline level. Our findings showed that I/R injury did not induce DNA damage, but increased the lactate levels. This finding suggests that there might be reversible and uncommon necrosis that did not reflect on overall DNA base damage. Further studies are needed using this approach. Primjena kometnog testa u procjeni oštećenja DNA nastalih zbog ishemijsko-reperfuzijskih ozljeda u bolesnika podvrgnutih kirurškim zahvatima na koronarnim žilama Ishemijsko-reperfuzijska (I/R) ozljeda čest je uzročnik pobola i smrtnosti u slučajevima kao što su ugradnja koronarnih premosnica, infarkt miokarda i sl. Slobodni kisikovi radikali koji nastaju tijekom procesa ishemije i reperfuzije smatraju se jednim od glavnih uzročnika oštećenja tkiva i imaju važnu ulogu u I/R ozljedama. I/R ozljede izazivaju oksidativna oštećenja u DNA (primjerice oštećenja purinskih i pirimidinskih baza). Kometni test osjetljiva je metoda koja omogućuje utvrđivanje niskih razina primarnih oštećenja u molekuli DNA. U ovom smo istraživanju primjenom kometnog testa na bijelim krvnim stanicama procjenjivali razine oštećenja u DNA nastale zbog I/R ozljeda u bolesnika podvrgnutih ugradnji premosnice koronarne arterije (in vivo model za I/R). Rezultati istraživanja upućuju na to da nema značajnih razlika u razinama oštećenja DNA izmjerenim u uzorcima krvi uzimanima prije kirurškog zahvata, nakon anestezije te tijekom ishemije, reperfuzije i zahvata. Međutim, uočeno je da su nakon I/R ozljede porasle razine laktata u serumu koje se više nisu vratile na početne vrijednosti. Takve vrijednosti laktata u serumu upućuju na to da tijekom I/R nastupa neobična i reverzibilna nekroza koja se, međutim, ne odražava na stupanj oštećenja DNA. Za objašnjenje ovih zapažanja potrebna su daljnja istraživanja.