Base excision repair pathway: PARP1 genotypes as modulators of therapy response in cervical cancer patients

Context: Genetic polymorphisms in genes of the base excision repair (BER) pathway appear to modulate the therapy response of cancer patients. PARP1 protein recognizes the DNA strand damage and facilitates the subsequent recruitment of BER proteins. Few studies have reported an association between PARP1 Val762Ala polymorphism (rs1136410) and cancer therapy response.

Objective: The purpose of our study was to determine whether PARP1 Val762Ala polymorphism have prognostic value in patients with cervical cancer.

Materials and methods: Two hundred and sixty adult patients, with histologically confirmed cervical cancer, at FIGO-stages IB2-IVA, primarily treated with concurrent chemotherapy (cisplatin) and radiotherapy. Overall survival (OS) and disease-free survival (DFS) were the primary end points of the analysis. The PARP1 Val762Ala genetic variants were analyzed by allelic discrimination by real-time PCR.

Results: We observed that peri- and postmenopausal women carrying the C-allele present a statistically significant lower OS and DFS (log-rank test, p?=?0.008 and p?=?0.006, respectively) among those with early stage cervical cancer. Cox regression analysis confirmed these results, after adjustment for other prognostic factors (for OS: HR, 3.70; 95%CI, 1.32–10.38; p?=?0.013 and for DFS: HR, 3.97; 95%CI, 1.59–9.93; p?=?0.003).

Conclusions: This is the first study evaluating the effect of PARP1 Val762Ala polymorphism in treatment response in cervical cancer patients. PARP1 genotypes may contribute as an independent prognostic factor in cervical cancer, being useful in predicting the clinical outcome.     

Detection and identification of 4‐hydroxy‐2‐nonenal Schiff‐base adducts along with products of Michael addition using data‐dependent neutral loss‐driven MS3 acquisition: Method evaluation through an in vitro study on cytochrome c oxidase modifications

We report a data‐dependent neutral‐loss‐driven MS³ acquisition to enhance, in addition to abundant Michael adducts, the detection of Schiff‐base adducts of proteins and 4‐hydroxy‐2‐nonenal, a reactive end product of lipid peroxidation. In vitro modification of cytochrome c oxidase, a mitochondrial protein complex, was used as a model to evaluate the method. The technique allowed for a confident validation of modification sites and also identified a Schiff‐base adduct in subunit Vb of the protein complex.     

Heritable variation in the foliar secondary metabolite sideroxylonal in <Emphasis Type="Italic">Eucalyptus</Emphasis> confers cross-resistance to herbivores

Plants encounter a broad range of natural enemies and defend themselves in diverse ways. The cost of defense can be reduced if a plant secondary metabolite confers resistance to multiple herbivores. However, there are few examples of positively correlated defenses in plants against herbivores of different types. We present evidence that a genetically variable chemical trait that acts as a strong antifeedant to mammalian herbivores of Eucalyptus also deters insect herbivores, suggesting a possible mechanism for cross-resistance. We provide field confirmation that sideroxylonal, an important antifeedant for mammalian herbivores, also determines patterns of damage by Christmas beetles, a specialist insect herbivore of Eucalyptus. In a genetic progeny trial of Eucalyptus tricarpa, we found significant heritabilities of sideroxylonal concentration (0.60), overall insect damage (0.34), and growth traits (0.30–0.53). Population of origin also had a strong effect on each trait. Negative phenotypic correlations were observed between sideroxylonal and damage, and between damage and growth. No relationship was observed between sideroxylonal concentration and any growth trait. Our results suggest that potential for evolution by natural selection of sideroxylonal concentrations is not strongly constrained by growth costs and that both growth and defense traits can be successfully incorporated into breeding programs for plantation trees.     

Antioxidant inhibits tamoxifen-DNA adducts in endometrial explant culture

Fresh human endometrial explants were incubated for 24h at 37 degrees C with either tamoxifen (10-100 micro M) or the vehicle (0.1% ethanol). Three metabolites namely, alpha-hydroxytamoxifen, 4-hydroxytamoxifen, and N-desmethyltamoxifen were identified in the culture media. Tissue size was limited but DNA adducts formed by the alpha-hydroxytamoxifen pathway were detected using authentic alpha-(deoxyguanosyl-N(2)) tamoxifen standards. Relative DNA-adduct levels of 2.45, 1.12, and 0.44 per 10(6) nucleotides were detected following incubations with 100, 25, and 10 micro M tamoxifen, respectively. The concurrent exposure of the explants to 100 micro M tamoxifen with 1mM ascorbic acid reduced the level of alpha-hydroxytamoxifen substantially (68.9%). The formation of tamoxifen-DNA adducts detectable in the explants from the same specimens exposed to 100 micro M tamoxifen with 1mM ascorbic acid were also inhibited. These results support the role of oxidative biotransformation of tamoxifen in the subsequent formation of DNA adducts in this tissue.     

Resveratrol protects SR-B1 levels in keratinocytes exposed to cigarette smoke

Cigarette smoking (CS) has been strongly linked to several health conditions including heart disease, lung cancer, and other respiratory and circulatory ailments. Deleterious effects of cigarette smoking on skin have also been well documented, but unlike effects on other organs, damage does not depend upon inhalation. The upper layer of the skin, the stratum corneum (rich in cholesterol fatty acids and ceramide), is very susceptible to damage induced by exposure to environmental stressors that can modify its lipid composition and thereby affect its function of protecting skin from dehydration. Scavenger receptor B1 (SR-B1) is involved in the uptake of cholesterol in several tissues including skin. We previously demonstrated that CS exposure induces formation of aldehyde (HNE) adducts that decrease SR-B1 expression. As topical resveratrol, a well-known polyphenolic stilbene, has been demonstrated to show benefits against skin disorders, we investigated its possible role as a protective agent against CS-induced reduction of SR-B1 expression in cutaneous tissue. In this study, we demonstrate that resveratrol at doses ranging from 0.5 to 10 μM is not toxic and is able to increase SR-B1 protein levels in a dose-dependent manner in human keratinocytes. Moreover, when the cells that were pretreated with various doses of resveratrol were exposed to CS, the loss of SR-B1 was prevented in a dose-dependent manner. In addition, in keratinocytes, resveratrol was also able to prevent an increase in HNE–protein adducts induced by CS. In particular resveratrol was able to prevent HNE–SR-B1 adduct formation. Thus, resveratrol seems to be a natural compound that could provide skin with a defense against exogenous stressors by protecting the essential cholesterol receptor, SR-B1.     

Glutathione-S-transferase pi as a model protein for the characterisation of chemically reactive metabolites

Chemically reactive metabolites (CRMs) are thought to be responsible for a number of adverse drug reactions through modification of critical proteins. Methods that defined the chemistry of protein modification at an early stage would provide invaluable tools for drug safety assessment. Here, human GST pi (GSTP) was exploited as a model target protein to determine the chemical, biochemical and functional consequences of exposure to the hepatotoxic CRM of paracetamol (APAP), N-acetyl-p-benzoquinoneimine (NAPQI). Site-specific, dose-dependent modification of Cys47 in native and His-tagged GSTP was revealed by MS, and correlated with inhibition of glutathione (GSH) conjugating activity. In addition, the adaptation of iTRAQ labelling technology to define precisely the quantitative relationship between covalent modification and protein function is described. Multiple reaction monitoring (MRM)-MS of GSTP allowed high sensitivity detection of modified peptides at physiological levels of exposure. Finally, a bioengineered mutant cytochrome P450 with a broad spectrum of substrate specificities was used in an in vitro reaction system to bioactivate APAP: in this model, GSTP trapped the CRM and exhibited both reduced enzyme activity and site-specific modification of the protein. These studies provide the foundation for the development of novel test systems to predict the toxicological potential of CRMs produced by new therapeutic agents.     

Risk assessment and the importance of ochratoxins

The multi-faceted and potent toxicity of ochratoxin A to experimental animals, its accepted role as disease determinant in mycotoxic porcine nephropathy, its putative role in the idiopathic human disease Balkan endemic nephropathy and associated urinary tract tumours, and its widespread occurrence usually in trace amounts in foodstuffs is focussing attention on minimising human intake. Conservative risk assessment based on porcine renal disease data has set tolerable intake values rather similar to actual average human intake. However, translating experimental rat tumour data to possible human risk is proving difficult partly because, in the author's view, the finding of a low incidence of DNA adducts could arise from an artificial surge in serum concentration following rapid uptake from aqueous intragastric administration, and this probably exaggerates what would occur during more natural slow uptake from food. The need for experimental data that can assist meaningful evaluation of risk of natural intake of ochratoxin A is emphasised, but prospective legislation should be balanced against socio-economic consequences of setting over-strict limits on contamination of relevant agricultural commodities, particularly in vulnerable specialist sectors such as coffee.     

Ecogenotoxicology in earthworms： A review

Pollutant dynamics and bioavailability greatly differ in soil and aquatic systems. Therefore, specific approaches and models are needed to assess the impact of soil contamination to terrestrial ecosystems. Earthworms among other soil invertebrates have received more attention because of their ecological importance. They represent a dominant part of the soil biomass and are soil engineers regulating important soil processes, notably fertilization. The release in soils of pollutants known for their persistence and/or their toxicity is a concern. Exposure of terrestrial species to pollutants that may alter genomic function has become an increasing topic of research in the last decade. Indeed, genome disturbances due to genetic and epigenetic mechanisms may impair growth, as well as reproduction and population dynamics in the long term. Despite their importance in gene expres- sion, epigenetic mechanisms are not yet understood in soil invertebrates. Until now, pollutant-induced changes in genome expression in natural biota are still being studied through structural alteration of DNA. The first biomarker relating to genotoxicant exposure in earthworms from multi-contaminated soils reported is DNA adducts measurements. It has been replaced by DNA breakage measured by the Comet assay, now more commonly used. Functional genomic changes are now being explored owing to molecular ＂omic＂ technologies. Approaches, objectives and results are overviewed herein. The focus is on studies dealing with genotoxicity and populational effects established from environmentally-relevant experiments and in situ studies [Current Zoology 60 （2）： 255-272, 2014].     

Reversed-phase HPLC-ESI/MS analysis of birch leaf proanthocyanidins after their acidic degradation in the presence of nucleophiles

Mountain birch leaves contain large amounts of structurally variable polymeric proanthocyanidins. Their isolation procedure was enhanced by the addition of liquid-liquid extractions prior to column chromatography over Sephadex LH-20. Isolated polymeric proanthocyanidins were depolymerised by acid-catalysis in the presence of benzyl mercaptan or phloroglucinol in order to study their composition. The resulting degradation products, flavan-3-ols and flavan-3-ol adducts, were analysed with reversed-phase high-performance liquid chromatography using UV photodiode array detection for quantification and electrospray ionisation mass spectrometry for identification. The results showed that polymeric proanthocyanidins contained (epi)gallocatechins and (epi)catechins as the extension units and, mainly, (+)-catechin as the terminal unit. The mean degree of polymerisation was found to be 26 based on thiolysis and 31 based on phloroglucinol degradation.     

Determination of isocyanate biomarkers in construction site workers

4,4′-Methylenediphenyl diisocyanate (MDI) is the most important isocyanate in the manufacture of polyurethanes, dyes, pigments and adhesives. High concentrations of isocyanates are a potent respiratory irritant. Therefore, it is important to develop methods to monitor exposure to such compounds. We monitored biological samples from 40 non-exposed and 45 exposed construction site workers. 4,4′-Methylenedianiline (MDA) and N-acetyl-4,4′-MDA (AcMDA) were determined from untreated urine (U-MDA, U-AcMDA) and MDA was analysed from acid-treated urine (U-MDA-tot). Haemoglobin (Hb) adducts of MDA (Hb-MDA) were determined in all workers. The levels of biomarkers decreased in the following order: U-MDA-tot>U-AcMDA>U-MDA>Hb-MDA. The same order was found for the percentage of samples, which were found positive in exposed workers: 100%, 91%, 91%, 27%. The urine levels U-MDA-tot correlate with U-MDA, U-AcMDA and Hb-MDA with r=0.79, 0.86 and 0.39, respectively (Spearman rank order, p<0.01). U-AcMDA correlates with U-MDA and Hb-MDA with r=0.77 and 0.47, respectively (p<0.01). U-MDA correlates with Hb-MDA (r=0.38, p<0.05). The levels in the controls were significantly lower than in the exposed workers for all compounds (Mann–Whitney test, p<0.01). The median isocyanate-specific IgE-level was higher in the exposed workers, but the difference was statistically not significant. The change of the biomarker levels was compared in a group of workers (n=20), which were analysed prior to isocyanate exposure and after the exposure for ～4–7 months. All urine MDA metabolites and the Hb-adduct levels increased significantly (Wilcoxon sign test, p<0.01). Total IgE increased significantly after the exposure with isocyanate activity (p<0.01). With the present work it could be shown that outdoor workers are exposed to a similar extent as workers from a MDI factory.