Distortion product otoacoustic emission sensitivity to different solvents in a population of industrial painters.

Objective: To evaluate the ototoxic effect of the exposure to different organic solvents and noise using distortion product otoacoustic emissions (DPOAEs).Design: The exposure to different solvents was evaluated by measuring, before and at the end of the work-shift, the urinary concentrations of solvent metabolites used as dose biomarkers. The urinary concentrations of DNA and RNA oxidation products were also measured as biomarkers of oxidative damage. The simultaneous exposure to noise was also evaluated. DPOAEs and pure tone audiometry (PTA) were used as outcome variables, and were correlated to the exposure variables using mixed effect linear regression models.Study sample: Seventeen industrial painters exposed to a solvent mixture in a naval industry. A sample size of 15 was estimated from previous studies as sufficient for discriminating small hearing level and DPOAE level differences (5 dB and 2 dB, respectively) at a 95% confidence level.Results: Statistically significant associations were found between the DPOAE level and the urinary dose biomarkers and the oxidative damage biomarkers. DPOAE level and the logarithm of the metabolite concentration showed a significant negative correlation.Conclusions: DPOAE are sensitive biomarkers of exposure to ototoxic substances and can be effectively used for the early detection of hearing dysfunction.

Asbestos exposure affects poly(ADP-ribose) polymerase-1 activity: role in asbestos-induced carcinogenesis.

Asbestos is known to induce malignant mesothelioma (MM) and other asbestos-related diseases. It is directly genotoxic by inducing DNA strand breaks and cytotoxic by promoting apoptosis in lung target cells. Poly(ADP-ribose) polymerase-1 (PARP1) is a nuclear zinc-finger protein with a function as a DNA damage sensor. To determine whether PARP1 is involved in asbestos-induced carcinogenesis, PARP1 expression and activity as well as DNA damage and repair were evaluated in circulating cells of asbestos-exposed subjects, MM patients and age-matched controls. PARP1 expression and activity were also evaluated in pleural biopsies of MM patients and compared with normal tissue. Accumulation of the pre-mutagenic 8-hydroxy-2'-deoxyguanosine and elevated PARP1 expression were found both in asbestos-exposed subjects and MM patients. Although PARP1 was highly expressed, its activity was relatively low. Low DNA repair efficiency was observed in lymphocytes from MM patients. High expression of PARP1 associated with low PARP activity was also found in MM biopsies. To mimic PARP1 dysfunction, PARP1 expression and activity were induced in immortalised mesothelial cells by their exposure to asbestos in the presence of a PARP1 inhibitor, which resulted in transformation of the cells. We propose that exposure to asbestos inhibits the PARP1 activity possibly resulting in higher DNA instability, thus causing malignant transformation.

Occupational Exposure in Industrial Painters: Sensitive and Noninvasive Biomarkers to Evaluate Early Cytotoxicity, Genotoxicity and Oxidative Stress.

This study aimed to identify sensitive and noninvasive biomarkers of early cyto-genotoxic, oxidative and inflammatory effects for exposure to volatile organic compounds (VOCs) in shipyard painters. On 17 (11 spray and 6 roller) painters (previously characterized for VOCs exposure to toluene, xylenes, ethylbenzene, ethyl acetate) and on 18 controls, we performed buccal micronucleus cytome (BMCyt) assay; Fpg-comet assay on lymphocytes; detection of urinary 8-oxoGua (8-oxo-7,8-dihydroguanine), 8-oxodGuo (8-oxo-7,8-dihydro-2'-deoxyguanosine) and 8-oxoGuo (8-oxo-7,8-dihydroguanosine), and cytokines release on serum. We found induction of cyto-genotoxicity by BMCyt assay and inflammatory effects (IL-6 and TNFα) in roller painters exposed to lower VOC concentrations than spray painters. In contrast, in both worker groups, we found direct and oxidative DNA damage by comet assay (with slightly higher oxidative DNA damage in roller) and significant increase of 8-oxoGuo and decrease of 8-oxodGuo and 8-oxoGua in respect to controls. The cyto-genotoxicity observed only on buccal cells of roller painters could be related to the task's specificity and the different used protective equipment. Although limited by the small number of subjects, the study shows the usefulness of all the used biomarkers in the risk assessment of painters workers exposed to complex mixtures.

Direct and Oxidative DNA Damage in a Group of Painters Exposed to VOCs: Dose - Response Relationship.

Volatile organic compounds (VOCs) are present in several working activities. This work is aimed at comparing oxidative stress and DNA damage biomarkers to specific VOCs in the occupational exposure of painters. Dose-response relationships between biomarkers of oxidative stress and of dose were studied. Unmetabolized VOCs and their urinary metabolites were analyzed. Urinary Methylhyppuric acids (MHIPPs, xylenes metabolite), Phenylglyoxylic and Mandelic acid (PGA, MA ethylbenzene metabolites), S-Benzylmercapturic acid (SBMA, toluene metabolite), and S-Phenylmercapturic acid (SPMA, benzene metabolite) were quantified at the end of work-shift. Oxidative stress was determined by: urinary excretion of 8-oxodGuo, 8-oxoGua and 8-oxoGuo and direct/oxidative DNA damage in blood by Fpg-Comet assay. Multivariate linear regression models were used to assess statistical significance of the association between dose and effect biomarkers. The regressions were studied with and without the effect of hOGG1 and XRCC1 gene polymorphisms. Statistically significant associations were found between MHIPPs and both 8-oxoGuo and oxidative DNA damage effect biomarkers measured with the Comet assay. Oxidative DNA damage results significantly associated with airborne xylenes and toluene, whilst 8-oxodGuo was significantly related to urinary xylenes and toluene. Direct DNA damage was significantly associated to SBMA. XRCC1 wild-type gene polymorphism was significantly associated with lower oxidative and total DNA damage with respect to heterozygous and mutant genotypes. The interpretation of the results requires some caution, as the different VOCs are all simultaneously present in the mixture and correlated among them.

A Predictive Model Assessing Genetic Susceptibility Risk at Workplace.

(1) Background: The study of susceptibility biomarkers in the immigrant workforce integrated into the social tissue of European host countries is always a challenge, due to high individual heterogeneity and the admixing of different ethnicities in the same workplace. These workers having distinct cultural backgrounds, beliefs, diets, and habits, as well as a poor knowledge of the foreign language, may feel reluctant to donate their biological specimens for the biomonitoring research studies. (2) Methods: A model predicting ethnicity-specific susceptibility based on principal component analysis has been conceived, using the genotype frequency of the investigated populations available in publicly accessible databases. (3) Results: Correlations among ethnicities and between ethnic and polymorphic genes have been found, and low/high-risk profiles have been identified as valuable susceptibility biomarkers. (4) Conclusions: In the absence of workers' consent or access to blood genotyping, ethnicity represents a good indicator of the subject's genotype. This model, associating ethnicity-specific genotype frequency with the susceptibility biomarkers involved in the metabolism of toxicants, may replace genotyping, ensuring the necessary safety and health conditions of workers assigned to hazardous jobs.

Susceptibility biomarker detection in urine exfoliate DNA.

AIM: The occupational biomonitoring of exposures to carcinogens is carried out by measuring dose (metabolites) and susceptibility biomarkers (gene polymorphisms) in two biological matrices: urine for metabolite detection and blood for genotyping. Blood is the most common substrate but has some disadvantages including: invasiveness of the harvesting technique; need of specialized staff and equipment; and high infection risk. METHODS & RESULTS: We propose our in-house approach using urine as single sample in 20 volunteers for simultaneous detection of dose and susceptibility biomarkers in order to verify efficacy and feasibility. CONCLUSION: Despite the low number of subjects, interindividual and gender variability in DNA yield, urine genomic DNA is a valuable source for gene polymorphism studies when blood samples are not available. [Formula: see text].

Is it possible to use biomonitoring for the quantitative assessment of formaldehyde occupational exposure?

The European classification, labeling and packaging classified formaldehyde as human carcinogen Group 1B and mutagen 2, fostering the re-evaluation of the exposure risk in occupational settings. Although formaldehyde exposure is traditionally measured in air, many efforts were made to identify specific exposure biomarkers: urinary formaldehyde, formic acid and DNA damage indicators. Though used in combination, none of these seems satisfactory. The influence of the metabolism on exogenous formaldehyde levels, the exposure to other xenobiotics, the difference in genetic background and metabolism efficiency, misled the relationship between genotoxicity and exposure data. Nevertheless, the limitation of adverse effects to the local contact sites hampers biomonitoring. Here we discuss the feasibility of formaldehyde biomonitoring and the use of DNA, DNA-protein cross-links and protein adducts as potential biomarkers.

Biomarkers of susceptibility following benzene exposure: influence of genetic polymorphisms on benzene metabolism and health effects.

Benzene is a ubiquitous occupational and environmental pollutant. Improved industrial hygiene allowed airborne concentrations close to the environmental context (1-1000 µg/m(3)). Conversely, new limits for benzene levels in urban air were set (5 µg/m(3)). The biomonitoring of exposure to such low benzene concentrations are performed measuring specific and sensitive biomarkers such as S-phenylmercapturic acid, trans, trans-muconic acid and urinary benzene: many studies referred high variability in the levels of these biomarkers, suggesting the involvement of polymorphic metabolic genes in the individual susceptibility to benzene toxicity. We reviewed the influence of metabolic polymorphisms on the biomarkers levels of benzene exposure and effect, in order to understand the real impact of benzene exposure on subjects with increased susceptibility.

Utility of checklist to describe experimental methods for investigating molecular biomarkers.

INTRODUCTION: In research articles, detailed description of experimental methods and reagents is fundamental for correct reproducibility of the published data. This becomes even more important when such data contribute to identify molecular targets and toxicity biomarkers whose role is crucial in the physiology and pathology of human health. Methods & Objectives: To achieve good reproducibility of data we took advantage of others' experiences and analyzed molecular biology and immunodetection techniques in 32 journal articles investigating the human NRF2 and Keap1 genes involved in the cell response to oxidative stress. RESULTS & CONCLUSIONS: In conclusion of the analysis, we assessed deficiency of information in the published methods, making it difficult to select appropriate protocols. Underlining the importance of assay reproducibility, this paper proposes the utility of a minimum information checklist of methods for biomarker detection.

Influence of genetic polymorphisms of styrene-metabolizing enzymes on the levels of urinary biomarkers of styrene exposure.

Styrene exposure is still present in different occupational settings including manufacture of synthetic rubber, resins, polyesters and plastic. The aim of this work was to investigate the effects of polymorphic genes CYP2E1, EPHX1, GSTT1, and GSTM1 on the urinary concentrations of the styrene metabolites mandelic acid (MA), phenylglyoxylic acid (PGA) and on the concentration ratios between (MA+PGA) and urinary styrene (U-Sty) and airborne styrene (A-Sty), in 30 workers from two fiberglass-reinforced plastic manufacturing plants and 26 unexposed controls. Personal air sampling and biological monitoring results revealed that sometimes exposure levels exceeded both the threshold limit value (TLV) and the biological exposure index (BEI) suggested by the American Conference of Governmental Industrial Hygienists. A significantly reduced excretion of styrene metabolites (MA+PGA) in individuals carrying the CYP2E1*5B and CYP2E1*6 heterozygote alleles, with respect to the homozygote wild type, was observed only in the exposed group. A reduction was also detected, in the same group, in subjects carrying the slow allele EPHX1 (codon 113), through the lowering of (MA+PGA)/urinary styrene concentration ratio. In addition, the ratio between MA+PGA and the personal airborne styrene concentration appeared to be modulated by the predicted mEH activity, in the exposed group, as evidenced by univariate linear regression analysis. Our results confirm some previous hypotheses about the role of the polymorphism of genes coding for enzymes involved in the styrene detoxification pathway: this may significantly reduce the levels of excreted metabolites and therefore it must be taken into account in the interpretation of the biological monitoring results for occupational exposure.

Influence of genetic polymorphism on t,t-MA/S-PMA ratio in 301 benzene exposed subjects.

This study investigated the effect of polymorphic genes GSTT1, GSTM1, GSTA1, EHPX1, NQO1, CYP2E1, CYP1A and MPO on the urinary concentrations and ratio (R) of the benzene metabolites trans,trans-muconic acid (t,t-MA) and S-phenyl mercapturic acid (S-PMA) in 301 oil refinery workers. The metabolites' concentrations are lower and R is higher (100.66) in non-smokers (n=184) than in smokers (n=117, R=36.54). Non-smokers have lower S-PMA and a higher R in GSTT1 null genotypes than in positive, and a higher S-PMA and a lower R in GSTA1 wild type genotypes. In smokers the GSTT1 null genotype effect on both S-PMA and R is confirmed, and is also shown in GSTM1 null, but not in GSTA1 wild type genotypes. GSTT1 null polymorphism reduces the conjugation rate of benzene epoxide with GSH, and to a lesser extent also GSTTA1 mutant, GSTM1 null and NQO1 mutant genotypes. The activity of one GST is compensated by another in GSTM1 and GSTA1 defective subjects, but not in GSTT1 null genotypes, whose average S-PMA excretion is about 50% with respect to the positive ones, for the same benzene exposure. R showed to be a more sensitive marker for these effects than the metabolite levels.

In vitro effect of aspartame in angiogenesis induction.

Aspartame (APM) is the most widely used artificial sweetener and is added to a wide variety of foods, beverages, drugs, and hygiene products. In vitro and in vivo tests have reported contradictory data about APM genotoxicity. We evaluated the angiogenic effect of APM in an in vitro model using blood vessel development assay (Angio-Kit), cultured endothelial cells and fibroblasts. The release of IL-6, VEGF-A, and their soluble receptors sIL-R6 and sVEGFR-2 were determined over time in the conditioned medium of the Angio-Kit system, endothelial cells and cell lines with fibroblast properties after APM treatment. Reactive oxygen species (ROS) formation, cell viability, and stimulation of the extracellular signal-regulated kinases (erk1/2) and protein p38 were also evaluated. Exposure to APM induced blood vessel formation. ROS production was observed in endothelial cells after APM treatment, which was associated with a slight cell cytotoxicity. Neither intracellular ROS formation nor cell death was observed in fibroblasts. APM increases the levels of inflammatory mediator IL-6, VEGF and their soluble receptors released from endothelial cells into the medium. APM treatment induces VEGF-pathway activation by erk1/2 and p38 phosphorylation. APM at low doses is an angiogenic agent that induces regenerative cytokine production leading to the activation of MAPKs and resulting in the formation of new blood vessels.

Angiogenic effect induced by mineral fibres.

Due to the toxic effect of asbestos, other materials with similar chemical-physical characteristics have been introduced to substitute it. We evaluate the angiogenic effect of certain asbestos substitute fibres such as glass fibres (GFs), ceramic fibres (CFs) and wollastonite fibres (WFs) and then compare angiogenic responses to those induced by crocidolite asbestos fibres (AFs). An in vitro model using human endothelial cells in small islands within a culture matrix of fibroblasts (Angio-Kit) was used to evaluate vessel formation. The release of IL-6, sIL-R6, IL-8, VEGF-A and their soluble receptors, sVEGFR-1, sVEGFR-2, was determined in the conditioning medium of Angio-Kit system after fibre treatment. ROS formation and cell viability were evaluated in cultured endothelial cells (HUVEC). To evaluate the involvement of intracellular mechanisms, EGFR signalling, ROS formation and nuclear factor-κB (NFκB) pathway were then inhibited by incubating HUVEC cells with AG1478, NAC and PDTC respectively, and the cytokine and growth factor release was analyzed in the culture medium after 7 days of fibre incubation. Among the mineral fibres tested, WFs markedly induced blood vessel formation which was associated with release of IL-6 and IL-8, VEGF-A and their soluble receptors. ROS production was observed in HUVEC after WFs treatment which was associated with cell cytotoxicity. The EGFR-induced ERK phosphorylation and ROS-mediated NFκB activation were involved in the cytokine and angiogenic factor release. However, only the EGFR activation was able to induce angiogenesis. The WFs are potential angiogenic agents that can induce regenerative cytokine and angiogenic factor production resulting in the formation of new blood vessels.

Association of MiR-126 with soluble mesothelin-related peptides, a marker for malignant mesothelioma.

BACKGROUND: Improved detection methods for diagnosis of malignant pleural mesothelioma (MPM) are essential for early and reliable detection as well as treatment. Since recent data point to abnormal levels of microRNAs (miRNAs) in tumors, we hypothesized that a profile of deregulated miRNAs may be a marker of MPM and that the levels of specific miRNAs may be used for monitoring its progress. METHODS AND RESULTS: miRNAs isolated from fresh-frozen biopsies of MPM patients were tested for the expression of 88 types of miRNA involved in cancerogenesis. Most of the tested miRNAs were downregulated in the malignant tissues compared with the normal tissues. Of eight significantly downregulated, three miRNAs were assayed in cancerous tissue and adjacent non-cancerous tissue sample pairs collected from 27 formalin-fixed, paraffin-embedded MPM tissues by quantitative RT-PCR. Among the miRNAs tested, only miR-126 significantly remained downregulated in the malignant tissues. Furthermore, the performance of the selected miR-126 as biomarker was evaluated in serum samples of asbestos-exposed subjects and MPM patients and compared with controls. MiR-126 was not affected by asbestos exposure, whereas it was found strongly associated with VEGF serum levels. Levels of miR-126 in serum, and its levels in patients' serum in association with a specific marker of MPM, SMRPs, correlate with subjects at high risk to develop MPM. CONCLUSIONS AND SIGNIFICANCE: We propose miR-126, in association with SMRPs, as a marker for early detection of MPM. The identification of tumor biomarkers used alone or, in particular, in combination could greatly facilitate the surveillance procedure for cohorts of subjects exposed to asbestos.

Activation of alpha-diacylglycerol kinase is critical for the mitogenic properties of anaplastic lymphoma kinase.

Oncogenic rearrangements of the tyrosine kinase receptor anaplastic lymphoma kinase (ALK), most commonly represented by the nucleophosmin/ALK fusion protein (NPM/ALK), are involved in the pathogenesis of anaplastic large-cell lymphomas (ALCLs). In an effort to identify new intracellular transducers operative in ALK-positive malignancies, we have investigated the potential involvement of diacylglycerol kinase (DGK). Here we show that alphaDGK is constitutively activated in the NPM/ALK-positive ALCL-derived cell line Karpas 299 and in NPM/ALK-infected 32D hematopoietic cells. These results were further validated in fibroblastic NIH-3T3 cells expressing a previously described chimeric epidermal growth factor receptor (EGFR)/ALK molecule that allows dissection of ALK enzymatic function under conditions of controlled ligand-induced activation. In this cell system, we also show that ALK-mediated alphaDGK activation is dependent on p60src tyrosine kinase, with which alphaDGK forms a complex. The specific inhibition of alphaDGK, obtained by cell treatment with R59949, significantly reduced cellular growth in all cell lines. This result was further confirmed in Karpas 299 cells following specific down-regulation of alphaDGK by RNA interference. Overall, our data indicate that alphaDGK activation is involved in the control of ALK-mediated mitogenic properties.

A ligand-inducible epidermal growth factor receptor/anaplastic lymphoma kinase chimera promotes mitogenesis and transforming properties in 3T3 cells.

Oncogenic rearrangements of the anaplastic lymphoma kinase (ALK) gene, encoding a receptor type tyrosine kinase, are frequently associated with anaplastic large cell lymphomas. Such rearrangements juxtapose the intracellular domain of ALK to 5'-end sequences belonging to different genes and create transforming fusion proteins. To understand how the oncogenic versions of ALK contribute to lymphomagenesis, it is important to analyze the biological effects and the biochemical properties of this receptor under controlled conditions of activation. To this aim, we constructed chimeric receptor molecules in which the extracellular domain of the ALK kinase is replaced by the extracellular, ligand-binding domain of the epidermal growth factor receptor (EGFR). Upon transfection in NIH 3T3 fibroblasts, the EGFR/ALK chimera was correctly synthesized and transported to the cell surface, where it was fully functional in forming high versus low affinity EGF-binding sites and transducing an EGF-dependent signal intracellularly. Overexpression of the EGFR/ALK chimera in NIH 3T3 was sufficient to induce the malignant phenotype; the appearance of the transformed phenotype was, however, conditionally dependent on the administration of EGF. Moreover, the EGFR/ALK chimera was significantly more active in inducing transformation and DNA synthesis than the wild type EGFR when either was expressed at similar levels in NIH 3T3 cells. Comparative analysis of the biochemical pathways implicated in the transduction of mitogenic signals did not show any increased ability of the EGFR/ALK to phosphorylate PLC-gamma and MAPK compared with the EGFR. On the contrary, EGFR/ALK showed to have a consistently greater effect on phosphatidylinositol 3-kinase activity compared with the EGFR, indicating that this enzyme plays a major role in mediating the mitogenic effects of ALK in NIH 3T3 cells.