Deoxyguanosine In DNA Research Articles

Oxidative DNA damage estimated by urinary 8-Hydroxy-2’ –Deoxyguanosine (8-OHdG) and 8-Oxoguanine DNA Glycosylase (OGG1) in cigarette and non-cigarette smokers in South West Nigeria

BACKGROUND: The thousands of chemicals and compounds found in cigarette smoke, including many free radicals and oxidants, can cause oxidative damage to DNA either directly or by producing reactive oxygen species in cultured cells. This study was carried out to analyze the urinary 8-Oxoguanine DNA Glycosylase (OGG1) and 8-Hydroxy-2’ –Deoxyguanosine (8-OHdG) among cigarette and non-cigarette smokers in South-West, Nigeria. METHODS: Urine samples were collected from 250 cigarette smokers and 200 non-cigarette smokers who live in south-west, Nigeria. Questionnaire was administered first to both cigarette and non-cigarette smokers prior to specimen collection. About 10mls of urine samples were collected from each of the subjects and was used to carry out Oxoguanine Glycosylase and 8-Hydroxyl-2-deoxyguanosine using spectrophotometer at 450 nm wavelength. RESULTS: There was a significant (P < 0.05) increase in Urinary 8-OHdG (345.84±90.98 vs 223±68.48) and OGG1 (220.71±59.55 vs 97.20±56.14) of the cigarette smokers when compared with that of non-cigarette smokers. CONCLUSION: On the basis of this study, it has been observed and established that indulging in cigarette smoking can result in oxidative stress and DNA damage.

Recognition of 8-Oxo-2'-deoxyguanosine in DNA Using the Triphosphate of 2'-Deoxycytidine Connecting the 1,3-Diazaphenoxazine Unit, dCdapTP.

DNA is constantly damaged by various external and internal factors. In particular, oxidative damage occurs in a steady state, and 8-oxo-2'-deoxyguanosine (oxodG) is known as the main oxidative damage. OxodG is a strong genotoxic nucleoside and is thought to be involved in the pathogenesis of cancer and neurological diseases. However, a breakthrough method to detect the position of oxodG in DNA has not yet been developed. Therefore, we attempted to develop a novel method to detect oxodG in DNA using artificial nucleosides. Recently, we have succeeded in the recognition of oxodG in DNA by a single nucleotide elongation reaction using nucleoside derivatives based on a purine skeleton with a 1,3-diazaphenoxazine unit. In this study, we developed a new nucleoside derivative with a pyrimidine skeleton in order to further improve the recognition ability and enzymatic reaction efficiency. We, therefore, designed and synthesized 2'-deoxycytidine-1,3-diazaphenoxazine (Cdap) and its triphosphate derivatives. The results showed that it was incorporated into the primer strand relative to the dG template because of its cytidine skeleton, but it was more effective at the complementary position of the oxodG template. These results indicate that the new nucleoside derivative can be considered as one of the new candidates for the detection of oxodG in DNA.

Unprecedentedly High Level of Intracellular Vitamin C and DNA Epigenetic Marks in Prostate: Relevant for Male Fertility?

Seminal plasma composition is affected by the physiological state of the prostate, the major male reproductive gland. Semen components, like vitamin C, can modulate sperm function. Vitamin C is an effective scavenger of free radicals and is an essential component of enzymes such as TET proteins involved in the DNA demethylation process. In the present study, a broad range of parameters which may influence the metabolic state of the prostate gland were analysed including blood and prostate tissue vitamin C, epigenetic DNA modifications and 8-oxo-7,8-dihydro-2'-deoxyguanosine in DNA of leukocytes and prostate tissues. The experimental material were tissue samples from patients with benign prostatic hyperplasia (BPH), normal/marginal prostate tissues from prostate cancer patients, leukocytes from healthy donors, and blood plasma from BPH patients and healthy donors. We applied ultra-performance liquid chromatography methods with mass spectrometry and/or UV detection. We found an unprecedentedly high level of intracellular vitamin C in all analysed prostatic tissues (benign prostatic hyperplasia and normal, marginal ones), a value much higher than in leukocytes and most human tissues. DNA epigenetic patterns in prostate cells are similar to other soft tissues like the colon, however, its uniqueness is the unprecedentedly high level of 5-(hydroxymethyl)-2'-deoxyuridine and a significant increase in 5-formyl-2'-deoxycytidine value compared to aforementioned tissues. Moreover, the level of 8-oxo-7,8-dihydro-2'-deoxyguanosine, an established marker of oxidative stress, is significantly higher in prostate tissues than in leukocytes and many previously studied soft tissues. Our results pointed out that prostatic vitamin C (regarded as the main supplier of the vitamin C to seminal plasma) and the DNA modifications (which may be linked to the regeneration of prostate epithelium) may play important role to maintain the prostate health.

Cryptocaryone Promotes ROS-Dependent Antiproliferation and Apoptosis in Ovarian Cancer Cells.

Cryptocaryone (CPC) is a bioactive dihydrochalcone derived from Cryptocarya plants, and its antiproliferation was rarely reported, especially for ovarian cancer (OVCA). This study aimed to examine the regulation ability and mechanism of CPC on three histotypes of OVCA cells (SKOV3, TOV-21G, and TOV-112D). In a 24 h MTS assay, CPC showed antiproliferation effects to OVCA cells, i.e., IC50 values 1.5, 3, and 9.5 μM for TOV-21G, SKOV3, and TOV-112D cells. TOV-21G and SKOV3 cells showed hypersensitivity to CPC when applied for exposure time and concentration experiments. For biological processes, CPC stimulated the generation of reactive oxygen species and mitochondrial superoxide and promoted mitochondrial membrane potential dysfunction in TOV-21G and SKOV3 cells. Apoptosis was detected in OVCA cells through subG1 accumulation and annexin V staining. Apoptosis signaling such as caspase 3/7 activities, cleaved poly (ADP-ribose) polymerase, and caspase 3 expressions were upregulated by CPC. Specifically, the intrinsic and extrinsic apoptotic caspase 9 and caspase 8 were overexpressed in OVCA cells following CPC treatment. Moreover, CPC also stimulated DNA damages in terms of γH2AX expression and increased γH2AX foci. CPC also induced 8-hydroxy-2′-deoxyguanosine DNA damages. These CPC-associated principal biological processes were validated to be oxidative stress-dependent by N-acetylcysteine. In conclusion, CPC is a potential anti-OVCA natural product showing oxidative stress-dependent antiproliferation, apoptosis, and DNA damaging functions.

Multiple-turnover single nucleotide primer extension reactions to detect 8-oxo-2'-deoxyguanosine in DNA.

The identification of the position of 8-oxo-2'-deoxyguanosine (8-oxo-dG) in DNA is important to clarify the pathogenesis of many diseases. We herein developed a purine-1,3-diazaphenoxazine triphosphate (dPdapTP) and described the first example of detecting the presence of 8-oxo-dG by amplifying it several hundred times after the multiple-turnover single nucleotide primer extension reactions.

Blood chromium exposure, immune inflammation and genetic damage: Exploring associations and mediation effects in chromate exposed population

Both genetic damage and inappropriate immune function are relevant to cancer of hexavalent chromium [Cr(VI)]. However, its associations with immune response and genetic damage development are poorly understood. To explore their associations and mediating effects, 1249 participants were included from the Occupational Chromate Exposure Dynamic Cohort, and their blood Cr concentrations were measured as internal exposure. A set of biomarkers including urinary 8-hydroxy-2’ - deoxyguanosine (8-OHdG), micronucleus frequency (MNF) and mitochondrial DNA copy number (mtCN) was developed to evaluate the landscape of genetic damage of Cr(VI). Serum C-reactive protein (CRP) and first component of complement q (C1q) were measured to reflect immune inflammation. Multivariate linear regression and mediation analyses were applied to assess the potential associations and mediation effects. It was found that blood Cr level showed significant dose-dependent relationships with increasing of MNF and urinary 8-OHdG, while negative association with CRP and C1q. Furthermore, a 1-unit increase in CRP was associated with decreases of − 0.765 to − 0.254 in MNF, − 0.400 to − 0.051 in urinary 8-OHdG. 4.97% of the association between blood Cr level and the increased MNF was mediated by CRP. 11.58% of the relationship between concentration of blood Cr and urinary 8-OHdG was mediated by C1q. These findings suggested that Cr(VI) exposures might prompt genetic damage, possibly partial via worsening immune inflammation.

Dehydroepiandrosterone modulates oxidative DNA damage in pancreatic cancer: A case-control study.

Background and AimDehydroepiandrosterone (DHEA) has a protective role against several types of cancer, although its mechanisms of action are still unknown, it may be related to the antioxidant effect of DHEA. We hypothesized that DHEA has a preventive effect on the formation of the 8‐hydroxy‐2′‐deoxyguanosine (8‐OHdG) DNA adduct in pancreatic cancer patients.MethodsSerum DHEAs were quantified by the ELISA method in 50 pancreatic cancer patients with histopathological diagnosis of adenocarcinoma and 50 matched controls. The amount of 8‐OHdG was assessed in peripheral blood leukocyte extracted DNA using a 32P‐DNA postlabeling technique.ResultsPancreatic cancer patients had lower serum DHEA levels than healthy controls, although it did not differ significantly. Instead, the 8‐OHdG DNA adduct was significantly higher in the case than in the control (P = <0.001). Remarkably, the negative correlation between 8‐OHdG and DHEA was distinguished between cases (P = 0.025, r = −0.315) but not in controls (P = 0.078, r = −0.250). In the crude and corrected estimate for pancreatic cancer risk, a significant protective effect of DHEA against pancreatic cancer was found with increasing DHEA when 8‐OHdG is greater than its median (adjusted OR = 0, 79, 95% confidence intervals [CI]: 0.66–0.94). Similarly, a lower risk of pancreatic cancer was observed in the third tertile of DHEA (adjusted OR = 0.05, 95% CI: 0.004–0.69).ConclusionsThese results indicate that serum DHEA reduces the risk of pancreatic cancer with an anti‐DNA damage effect. Hence, the influence of DHEA to prohibit the accumulation of 8‐OHdG may be one of its physiological functions.

Estragole DNA adduct accumulation in human liver HepaRG cells upon repeated in vitro exposure

Accumulation of N2-(trans-isoestragol-3′-yl)-2′-deoxyguanosine (E-3′-N2-dG) DNA adducts derived from the alkenylbenzene estragole upon repeated dose exposure was investigated since the repair of this adduct was previously shown to be inefficient. To this end human HepaRG cells were exposed to repeating cycles of 2 h exposure to 50 μM estragole followed by 22 h repair to mimic daily exposure. The E-3′-N2-dG DNA adduct levels were quantified by LC–MS/MS after each cycle. The results show accumulation of E-3′-N2-dG DNA adducts at a rate of 17.53 adducts/108 nts/cycle. This rate at the dose level calculated by physiologically based kinetic (PBK) modeling to result in 50 μM was converted to a rate expected at average human daily intake of estragole. The predicted time estimated to reach adduct levels reported at the BMD10 of the related alkenylbenzene methyleugenol of 10−100 adducts /108 nts upon average human daily intake of estragole amounted to 8–80 (in rat) or 6–57 years (in human). It is concluded that the persistent nature of the E-3′-N2-dG DNA adducts may contribute to accumulation of substantial levels of DNA adducts upon prolonged dietary exposure.

Molecular Dynamics and In Vitro Quantification of Safrole DNA Adducts Reveal DNA Adduct Persistence Due to Limited DNA Distortion Resulting in Inefficient Repair.

The formation and repair of N2-(trans-isosafrol-3'-yl)-2'-deoxyguanosine (S-3'-N2-dG) DNA adduct derived from the spice and herbal alkenylbenzene constituent safrole were investigated. DNA adduct formation and repair were studied in vitro and using molecular dynamics (MD) simulations. DNA adduct formation was quantified using liquid chromatography-mass spectrometry (LCMS) in wild type and NER (nucleotide excision repair) deficient CHO cells and also in HepaRG cells and primary rat hepatocytes after different periods of repair following exposure to safrole or 1'-hydroxysafrole (1'-OH safrole). The slower repair of the DNA adducts found in NER deficient cells compared to that in CHO wild type cells indicates a role for NER in repair of S-3'-N2-dG DNA adducts. However, DNA repair in liver cell models appeared to be limited, with over 90% of the adducts remaining even after 24 or 48 h recovery. In our further studies, MD simulations indicated that S-3'-N2-dG adduct formation causes only subtle changes in the DNA structure, potentially explaining inefficient activation of NER. Inefficiency of NER mediated repair of S-3'-N2-dG adducts points at persistence and potential bioaccumulation of safrole DNA adducts upon daily dietary exposure.

Effect of Eight Weeks of Aerobic Exercise and Vitamin D Supplementation on 8-hydroxy-2'-deoxyguanosine and O6-methylguanine DNA methyltransferase in Lung of Rats Poisoned with Hydrogen Peroxide

Effect of Eight Weeks of Aerobic Exercise and Vitamin D Supplementation on 8-hydroxy-2'-deoxyguanosine and O6-methylguanine DNA methyltransferase in Lung of Rats Poisoned with Hydrogen Peroxide

Cellular levels and molecular dynamics simulations of estragole DNA adducts point at inefficient repair resulting from limited distortion of the double-stranded DNA helix

Estragole, naturally occurring in a variety of herbs and spices, can form DNA adducts after bioactivation. Estragole DNA adduct formation and repair was studied in in vitro liver cell models, and a molecular dynamics simulation was used to investigate the conformation dependent (in)efficiency of N2-(trans-isoestragol-3′-yl)-2′-deoxyguanosine (E-3′-N2-dG) DNA adduct repair. HepG2, HepaRG cells, primary rat hepatocytes and CHO cells (including CHO wild-type and three NER-deficient mutants) were exposed to 50 μM estragole or 1′-hydroxyestragole and DNA adduct formation was quantified by LC–MS immediately following exposure and after a period of repair. Results obtained from CHO cell lines indicated that NER plays a role in repair of E-3′-N2-dG adducts, however, with limited efficiency since in the CHO wt cells 80% DNA adducts remained upon 24 h repair. Inefficiency of DNA repair was also found in HepaRG cells and primary rat hepatocytes. Changes in DNA structure resulting from E-3′-N2-dG adduct formation were investigated by molecular dynamics simulations. Results from molecular dynamics simulations revealed that conformational changes in double-stranded DNA by E-3′-N2-dG adduct formation are small, providing a possible explanation for the restrained repair, which may require larger distortions in the DNA structure. NER-mediated enzymatic repair of E-3′-N2-dG DNA adducts upon exposure to estragole will be limited, providing opportunities for accumulation of damage upon repeated daily exposure. The inability of this enzymatic repair is likely due to a limited distortion of the DNA double-stranded helix resulting in inefficient activation of nucleotide excision repair.

Alterations in levels of 8-Oxo-2'-deoxyguanosine and 8-Oxoguanine DNA glycosylase 1 during a current episode and after remission in unipolar and bipolar depression

IntroductionPrevious studies showed significant increases in DNA base damage markers and significant alterations in base excision repair enzymes in patients with unipolar and bipolar depression. We aimed to investigate changes in urine 8-Oxo-2′-deoxyguanosine (8-oxo-dG) and gene expression levels of 8-Oxoguanine DNA glycosylase 1 (OGG1) during a current depressive episode and after remission in bipolar and unipolar disorders. MethodsTwenty-four acutely depressed bipolar (BD), 33 unipolar depression (UD) patients and 61 healthy controls were included in the study. Clinical evaluations, blood and urine sampling were completed at baseline and at remission after eight weeks. The urine 8-oxo-dG levels were assessed by liquid chromatography tandem mass spectrometry and adjusted for urine creatinine levels. The gene expression levels of OGG1 were determined from cDNA extracted from blood samples, using real time-polymerase chain reaction. ResultsAt baseline, patients presented significantly higher levels of 8-oxo-dG (p = 0.008), and lower gene expression of OGG1 (p = 0.024) compared to controls. Levels of either 8-oxo-dG or OGG1 expression did not differ between BD and UD. In patients who remitted by the 8th week (n = 30), 8-oxo-dG decreased significantly (p = 0.001), and gene expression levels of OGG1 increased by 2.95 times compared to baseline levels (p = 0.001). All comparisons were adjusted for age, sex, smoking status and body mass index. ConclusionOur results suggest that patients with bipolar and unipolar mood disorders present increased 8-oxo-dG and decreased gene expression levels of OGG1 in current depressive episodes, and that these changes might be reversed by the resolution of depressive symptoms. The causal relationship between DNA damage and repair requires further exploration.

Generation of reactive oxygen species by hydroxypyridone compound/iron complexes

ABSTRACT Objectives: Prooxidant properties of iron-binding hydroxypyridone compounds including deferiprone and mimosine were analyzed. Methods: Hydroxypyridone/iron-dependent production of reactive oxygen species was evidenced by the inactivation of aconitase, the most sensitive enzyme to oxidative stress in permeabilized yeast cells. Results and Discussion: Deferiprone and mimosine produced reactive oxygen species in the presence of ferrous sulfate. The inactivation required sodium azide the inhibitor of catalase, and addition of TEMPOL, a scavenger of superoxide radical, protected aconitase from the inactivation, suggesting that the superoxide radical produced from the hydroxypyridone/iron complex is responsible for the inactivation of aconitase. A principal role of superoxide radical was further supported by the finding that the hydroxypyridone/iron complex can inactivate aconitase in the presence of cyanide the inhibitor of superoxide dismutase. Deferiprone and mimosine stimulated the Fe2+ oxidation, resulting in the one-electron reduction of oxygen to form superoxide anion, which can inactivate aconitase by oxidizing the prosthetic iron-sulfur cluster. Mimosine further stimulated the ascorbate/iron-dependent formation of 8-hydroxy-2′-deoxyguanosine in DNA. Conclusion: Biological toxicity of mimosine and deferiprone reported previously can be accounted for by the prooxidant properties of hydroxypyridone compounds: coordination complex with iron generates reactive oxygen species resulting in the disturbance of mitochondrial energy metabolism and DNA damage.

Comparison of effects of 2.4 GHz Wi-Fi and mobile phone exposure on human placenta and cord blood

The aim of this study was to investigate the effects of radiofrequency radiation emitted from Wi-Fi systems and mobile phones on cord blood and placenta. The study included 149 pregnant women who were divided in subgroups: unexposed (control), mobile phone exposed, Wi-Fi exposed and mobile phone plus Wi-Fi exposed groups. Immediately after birth, placenta and cord blood samples were collected and protein carbonyl (PCO), malondialdehyde (MDA), total oxidant status (TOS), total antioxidant status, 8-hydroxy-2′-deoxyguanosine (8-OHdG) levels and DNA single strand breaks were analysed. The results of the study showed an increase in 8-OHdG, MDA, PCO and TOS in cord blood and placenta in the group exposed to mobile phones during gestation. However, the group exposed to Wi-Fi did not show alterations in the studied oxidative stress parameters. On the other hand, tail intensity and tail moment of DNA in the mobile phone exposure groups were higher than those in the control and Wi-Fi exposure groups. In conclusion, the results of this study indicated that mobile phone exposure during pregnancy could have an important potential to cause oxidative stress and DNA damage in cord blood and placenta. The results of this study also indicated that combined effects of Wi-Fi plus mobile phone exposure have a higher potential to cause synergistic harmful effects.

Triazolyl RuII, RhIII, OsII, and IrIII Complexes as Potential Anticancer Agents: Synthesis, Structure Elucidation, Cytotoxicity, and DNA Model Interaction Studies

Novel conjugated ruthenium(II), rhodium(III), and iridium(III) organometallic complexes of triazoles 1 and 2 synthesized and evaluated for anticancer activity against cervical (HeLa), kidney (HEK293), nonsmall lung cancer (A549), and leukemia (MT4) cancer cell lines are reported herein. The complexes are κ2-N,C coordinated and have the formula [ML(Ar)Cl] (where L is 1-benzyl-4-phenyl-1H-1,2,3-triazole for 1 and 1-benzyl-4-hydroxymethyl-1H-1,2,3-triazole for 2, Ar is p-cymene for RuII and OsII and Cp* for RhIII and IrIII, and M is metal). NMR studies, including HMBC and NOESY, were employed to unambiguously elucidate their structures and provide their conformational information in solution. Single-crystal X-ray diffraction data have been used to establish the solid-state structures of selected complexes, which further confirm the structural elucidation by NMR. Dynamic NMR studies, such as differential transferred NOE, have been employed to distinguish between isomers 1a_I and 1a_II of ruthenium(II) complexes of triazole 1. The rhodium(III) (1b) and iridium(III) (1c) complexes exhibited good cytotoxic activities (CC50 = 4–6 μM) comparable to that of the drug auranofin against lung cancer A549 cell lines (CC50 = 4.69 μM). While triazole 1 based ruthenium(II) (1a) and osmium(II) (1d) complexes displayed modest anticancer activities against HeLa and HEK293 cell lines, the analogous rhodium(III) and iridium(III) complexes exhibited good potential (CC50 = 9–54 μM versus auranofin (3–9 μM)) against these cancer cell lines. Insightful NMR studies on the interaction between the DNA model guanosine 5′-GMP and the complexes 1b,c reveal a possible mode of action of the aquated complexes involving carbenylation with DNA bases or purines through the triazolyl proton H-5. From the findings, these complexes could possibly confer their cytotoxic activities through intercalation with the DNA of pathological cells. Therefore, carbenylation of the triazolylrhodium(III) and iridium(III) complexes by DNA guanosine 5′-GMP is proposed as a novel mode of DNA intercalation of these complexes in cancer cells.

Substituting Inosine for Guanosine in DNA: Structural and Dynamic Consequences

Inosine differs from the guanosine nucleoside only by the absence of the N2 amino group. Both nucleosides also have similar electrostatic potentials. Therefore, substituting I for G has been used to probe various properties of nucleic acids and to facilitate the interpretation of binding studies. In particular, the absence of the amino group permits the assessment of its importance in the binding of ligands to the minor groove of duplex DNA. It has been known for some time that an I-C base pair is of lower stability than a regular G-C base pair, which needs to be considered when making DNA constructs containing inosine. However, it is generally assumed that both base pairs are structurally highly similar. To test this assumption in an identical sequence environment, we have determined the fine structure of two hairpin DNA substrates that differ only in the substitution of an I-C base pair for a G-C base pair. The structures have been solved using nuclear magnetic resonance (NMR) restraints in conjunction with Mardigras and molecular dynamics. The structural data are complemented with thermodynamic and dynamic data to get a comprehensive evaluation of the consequences of G-C vs I-C base pair substitutions. Our data show a strong similarity in the structures of the hairpins, but a significant difference in the melting temperatures, T m. This difference is also reflected in the drastically decreased base pair lifetime of 7.4 milliseconds compared to the G-C base pair lifetime of 155 milliseconds. The substitution of I-C for G-C is to probe for specific effect due to the amino group is satisfactory, as long as the lowered thermal stability and the drastically increased local dynamics are considered.

Phorate triggers oxidative stress and mitochondrial dysfunction to enhance micronuclei generation and DNA damage in human lymphocytes.

Herein, we studied phorate for its toxicological effects in human lymphocytes. Phorate treatment for 3 h has induced significant increase in the lymphocytic DNA damage. Compared to control, comet data from highest concentration of phorate (1000 µM) showed 8.03-fold increase in the Olive tail moment (OTM). Cytokinesis blocked micronucleus (CBMN) assay revealed 6.4-fold increase in binucleated micronucleated (BNMN) cells following the exposure with phorate (200 µM) for 24 h. The nuclear division index (NDI) in phorate (200 µM) treated cells reduced to 1.8 vis-à-vis control cells showed NDI of 1.94. Comparative to untreated control, 60.43% greater DCF fluorescence was quantitated in lymphocytes treated with phorate (500 µM), affirming reactive oxygen species (ROS) generation and oxidative stress. Flow cytometric data of phorate (200 µM) treated lymphocytes showed 81.77% decline in the fluorescence of rhodamine 123 (Rh123) dye, confirming the perturbation of mitochondrial membrane potential (ΔΨm). Calf thymus DNA (ct-DNA) treated with phorate (1000 µM) exhibited 2.3-fold higher 8-Hydroxy-2′-deoxyguanosine (8-oxodG) DNA adduct formation, signified the oxidative DNA damage. The alkaline unwinding assay revealed 4.0 and 6.5 ct-DNA strand breaks when treated to phorate and phorate-Cu (II) complex. Overall, the data unequivocally suggests the cyto- and genotoxic potential of phorate in human lymphocytes, which may induce comparable toxicological consequences in persons occupationally or non-occupationally exposed to insecticide phorate.

Effect of Cr(VI) and bisphenol A on the formation of 8-hydroxy-2’ deoxyguanosine DNA adduct from 2’ -deoxyguanosine through a Fenton-like reaction

In this research, we study the effect of the addition of bisphenol A (BPA) and Cr(VI) on the formation of the DNA adduct, 8-hydroxy-2’-deoxyguanosine (8-OHdG). The formation of 8-OHdG, which is monitored using reversed phase high performance liquid chromatography (HPLC) with a Ultraviolet-Visible (UV-Vis) detector at 254 nm, can be achieved through the reaction of 2’ -deoxyguanosine (dG) with BPA in the presence of Fenton-like reagents and vitamin C. The reaction is performed under different conditions, including several pH (7.4 and 8.4), temperatures (37 °C and 60 °C), and incubation times (7 and 12 h). To detect 8-OHdG, the minimum concentration was set as the limit of detection (LOD) value of 5.19 ppb, and the minimum concentration for its quantification was set as the limit of quantification (LOQ) value of 17.29 ppb. The results of this study indicate that the concentration of 8-OHdG increases upon addition of BPA and Cr(VI), and most of the concentration values obtained from the analysis are greater than the LOD value. The addition of Fenton-like reagent and vitamin C causes an increase in the concentration of 8-OHdG. For most samples, the conditions that cause the highest 8-OHdG concentration are a pH of 7.4, a temperature of 60 °C, and an incubation time of 12 h.

Role of Seroalbumin in the Cytotoxicity of cis-Dichloro Pt(II) Complexes with (N^N)-Donor Ligands Bearing Functionalized Tails.

Given the potent anticancer properties of cis-diamminedichloroplatinum(II) and knowing its mode of action, we synthesized four new cis-[PtCl2(N^N)] organoplatinum complexes, two with N-substituted pbi ligands (pbiR = 1-R-2-(2-pyridyl)benzimidazole) (namely, 1 and 2) and two more with 4,4'-disubstituted bpy ligands (bpy = 2,2'-bipyridine) (namely, 3 and 4). We explored their cytotoxicity and ability to bind to deoxyguanosine monophosphate (dGMP), DNA, and albumin models. By 1H NMR and UV-vis spectroscopies, circular dichroism, agarose gel electrophoresis, differential scanning calorimetry measurements, and density functional theory calculations, we verified that only 3 can form aquacomplex species after dimethyl sulfoxide solvation; surprisingly, 1, 2, and 3 can bind covalently to DNA, whereas 4 can form a noncovalent complex. Interestingly, only complexes 1 and 4 exhibit good cytotoxicity against human ovarian carcinoma (HeLa) cell line, whereas 2 and 3 are inactive. Although lung carcinoma (A549) cells are more resistant to the four platinum complexes than HeLa cells, when the protein concentration in the extracellular media is lower, the cytotoxicity becomes substantially enhanced. By native electrophoresis of bovine seroalbumin (BSA) and inductively coupled plasma mass spectrometry uptake studies we bear out, on one hand, that 2 and 3 can interact strongly with BSA and its cellular uptake is negligible and, on the other hand, that 1 and 4 can interact with BSA only weakly, its cellular uptake being higher by several orders. These results point up the important role of the protein binding features on their biological activity and cellular uptake of cis-"PtCl2" derivatives. Our results are valuable in the future rational design of new platinum complexes with improved biological properties, as they expose the importance not only of their DNA binding abilities but also of additional factors such as protein binding.

Cytochrome b5 plays a dual role in the reaction cycle of cytochrome P450 3A4 during oxidation of the anticancer drug ellipticine

Ellipticine is an anticancer agent that forms covalent DNA adducts after enzymatic activation by cytochrome P450 (CYP) enzymes, mainly by CYP3A4. This process is one of the most important ellipticine DNA-damaging mechanisms for its antitumor action. Here, we investigated the efficiencies of human hepatic microsomes and human recombinant CYP3A4 expressed with its reductase, NADPH:CYP oxidoreductase (POR), NADH:cytochrome b5 reductase and/or cytochrome b5 in Supersomes™ to oxidize this drug. We also evaluated the effectiveness of coenzymes of two of the microsomal reductases, NADPH as a coenzyme of POR, and NADH as a coenzyme of NADH:cytochrome b5 reductase, to mediate ellipticine oxidation in these enzyme systems. Using HPLC analysis we detected up to five ellipticine metabolites, which were formed by human hepatic microsomes and human CYP3A4 in the presence of NADPH or NADH. Among ellipticine metabolites, 9-hydroxy-, 12-hydroxy-, and 13-hydroxyellipticine were formed by hepatic microsomes as the major metabolites, while 7-hydroxyellipticine and the ellipticine N2-oxide were the minor ones. Human CYP3A4 in Supersomes™ generated only three metabolic products, 9-hydroxy-, 12-hydroxy-, and 13-hydroxyellipticine. Using the 32P-postlabeling method two ellipticine-derived DNA adducts were generated by microsomes and the CYP3A4-Supersome system, both in the presence of NADPH and NADH. These adducts were derived from the reaction of 13-hydroxy- and 12-hydroxyellipticine with deoxyguanosine in DNA. In the presence of NADPH or NADH, cytochrome b5 stimulated the CYP3A4-mediated oxidation of ellipticine, but the stimulation effect differed for individual ellipticine metabolites. This heme protein also stimulated the formation of both ellipticine-DNA adducts. The results demonstrate that cytochrome b5 plays a dual role in the CYP3A4-catalyzed oxidation of ellipticine: (1) cytochrome b5 mediates CYP3A4 catalytic activities by donating the first and second electron to this enzyme in its catalytic cycle, indicating that NADH:cytochrome b5 reductase can substitute NADPH-dependent POR in this enzymatic reaction and (2) cytochrome b5 can act as an allosteric modifier of the CYP3A4 oxygenase.Graphical abstract