Evaluation of Systemic Genotoxic/Oxidative and Proinflammatory Effects in Workers of a Titanium Dioxide Production Plant.

This study is aimed at evaluating whether the occupational exposure to TiO2 during the industrial production process is able to induce genotoxic, oxidative, and inflammatory effects on blood, biomonitoring the same workers that showed micronucleus induction in the exfoliated buccal cells, as previous published. The final aim was to find sensitive and suitable biomarkers to evaluate potential early toxicity of occupational exposure to TiO2. On the same 40 workers involved in the manufacture of TiO2 pigment, 5 office workers, and 18 controls previously studied, we used formamidopyrimidine glycosylase- (Fpg-) comet assay on lymphocytes to evaluate genotoxic/oxidative effects and detected cytokine (IL-6, IL-8, and TNFα) release by ELISA to evaluate proinflammation. Moreover, we studied the possible influence of single nucleotide polymorphisms of XRCC1 and hOGG1 DNA repair genes and of GST metabolism-related genes (GSTT1 and GSTM1) on the evaluated effects. We did not find statistically significant differences in the mean values of the analysed Fpg-comet assay parameters; only the percentage of DNA damaged cells appearing in the test as comets (% comets) resulted higher in the exposed workers compared to controls. Also, the data analysed taking into account the specific task (bagging, industrial cleaning, mobile operations, maintaining, and production) showed differences only for % comets which resulted higher in industrial cleaners compared to controls. We found variations of IL-6 and IL-8 levels in the exposed workers with concentrations that were lower for IL-6 and higher for IL-8 compared to the control group. XRCC1, hOGG1, and GSTT1 polymorphisms did not influence neither comet parameters nor cytokine release. These findings demonstrate that TiO2 production process is able to induce slight proinflammatory effects in terms of IL-8 increased release but not significant genotoxic/oxidative effects on lymphocytes, which do not seem to be a target of TiO2, prevalently inhalable particles, generated in the studied production site.

Contribution of Genetic Polymorphisms in Human Health.

Human health is influenced by various factors; these include genetic inheritance, behavioral lifestyle, socioeconomic and environmental conditions, and public access to care and therapies in case of illness, with the support of the national health system. All these factors represent the starting point for the prevention and promotion of a healthy lifestyle. However, it is not yet clear to what extent these factors may actually affect the health of an entire population. The exposures to environmental and occupational factors are several, most of which might be poorly known, contributing to influencing individual health. Personal habits, including diet, smoking, alcohol, and drug consumption, together with unhealthy behaviors, may inevitably lead people to the development of chronic diseases, contributing to increasing aging and decreasing life expectancy. In this article, we highlight the role of susceptibility biomarkers, i.e., the genetic polymorphisms of individuals of different ethnicities, with particular attention to the risk factors in the response to specific exposures of Europeans. Moreover, we discuss the role of precision medicine which is representing a new way of treating and preventing diseases, taking into account the genetic variability of the individual with each own clinical history and lifestyle.

A follow-up study on workers involved in the graphene production process after the introduction of exposure mitigation measures: evaluation of genotoxic and oxidative effects.

During nanomaterial (NM) production, workers could be exposed, particularly by inhalation, to NMs and other chemicals used in the synthesis process, so it is important to have suitable biomarkers to monitor potential toxic effects. Aim of this study was to evaluate the effectiveness of the introduction of exposure mitigation measures on workers unintentionally exposed to graphene co-pollutants during production process monitoring the presumable reduction of workplace NM contamination and of early genotoxic and oxidative effects previously found on these workers. We used Buccal Micronucleus Cytome (BMCyt) assay and Fpg-comet test, resulted the most sensitive biomarkers on our first biomonitoring work, to measure the genotoxic effects. We also detected urinary oxidized nucleic acid bases 8-oxoGua, 8-oxoGuo and 8-oxodGuo to evaluate oxidative damage. The genotoxic and oxidative effects were assessed on the same graphene workers (N = 6) previously studied, comparing the results with those found in the first biomonitoring and with the control group (N = 11). This was achieved 6 months after the installation of a special filter hood (where to perform the phases at higher risk of NM emission) and the improvement of environmental and personal protective equipment. Particle number concentration decreased after the mitigation measures. We observed reduction of Micronucleus (MN) frequency and oxidative DNA damage and increase of 8-oxodGuo excretion compared to the first biomonitoring. These results, although limited by the small subject number, showed the efficacy of adopted exposure mitigation measures and the suitability of used sensitive and noninvasive biomarkers to bio-monitor over time workers involved in graphene production process.

MicroRNA expression is associated with auditory dysfunction in workers exposed to ototoxic solvents and noise.

This study is part of a project on early hearing dysfunction induced by combined exposure to volatile organic compounds (VOCs) and noise in occupational settings. In a previous study, 56 microRNAs were found differentially expressed in exposed workers compared to controls. Here, we analyze the statistical association of microRNA expression with audiometric hearing level (HL) and distortion product otoacoustic emission (DPOAE) level in that subset of differentially expressed microRNAs. The highest negative correlations were found; for HL, with miR-195-5p and miR-122-5p, and, for DPOAEs, with miR-92b-5p and miR-206. The homozygous (mut) and heterozygous (het) variants of the gene hOGG1 were found disadvantaged with respect to the wild-type (wt), as regards the risk of hearing impairment due to exposure to VOCs. An unsupervised artificial neural network (auto contractive map) was also used to detect and show, using graph analysis, the hidden connections between the explored variables. These findings may contribute to the formulation of mechanistic hypotheses about hearing damage due to co-exposure to noise and ototoxic solvents.

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.

Occupational exposure to volatile organic compounds affects microRNA profiling: Towards the identification of novel biomarkers.

In the framework of a project aimed at finding novel predictive biomarkers of VOCs exposure-related diseases, the effect of exposure to ethylbenzene, toluene, and xylene has been analyzed in a group of painters (spray- and roller-painters) working in the shipyard industry. Airborne levels of solvents were higher in spray- than in roller-painters, and comparable to the Occupational Exposure Limits (OELs), particularly for toluene and xylene. The urinary concentration of each volatile organic compound (VOC) and of the corresponding metabolites were also concurrently measured. A set of oxidative stress biomarkers, i.e., the products of DNA and RNA oxidation, RNA methylation, and protein nitration, were measured, and found significantly higher at the end of the work shift. MicroRNA (MiRNA) expression was analyzed in the VOC-exposed workers and in a control group, finding 56 differentially expressed (DE) miRNAs at a statistically significant level (adjusted p ≤ 0.01). The Receiver-Operating Characteristic curves, computed for each identified miRNA, showed high sensitivity and specificity. A pathway analysis in the Kyoto Encyclopedia of Genes and Genomes (KEGG) showed that miRNA-1, which was found downregulated in exposed workers, is involved in the lung cancer oncogenesis. A subset of 10 miRNAs (out of the 56 DE) was selected, including those with the highest correlation to the urinary dose biomarkers measured at the end of work-shift. Multivariate ANOVA analysis showed a statistically significant correlation between the urinary dose biomarkers (both the VOCs urinary concentration and the VOCs' metabolite concentration), and the identified miRNA subset, indicating that the exposure to low VOC doses may be sufficient to activate the miRNA response. Four miRNAs belonging to the subset strongly related to the VOCs and VOCs' metabolites concentration were individuated, miR-589-5p, miR-941, miR-146b-3p and miR-27a-3p, with well-known implications in oxidative stress and inflammation processes.

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.

Confronting two biomolecular techniques to detect NRF2 gene polymorphism biomarkers.

AIM: Gene polymorphism biomarkers identify individual susceptibility to environmental and occupational hazards. The conventional approach considers polymerase chain reaction (PCR) followed by restriction fragment length polymorphism analysis (RFLP), a reliable but expensive and time-consuming two-step procedure. Therefore we evaluated the simpler method confronting two-pair primers (CTPP)-PCR for its robustness and applicability to epidemiologic studies. MATERIALS & METHODS: We compared CTPP-PCR and PCR-RFLP techniques to detect two NRF2 polymorphisms in a set of biological samples. RESULTS: CTPP-PCR produced contradictory results and required the orthogonal technique for confirming the data. CONCLUSION: In contrast to PCR-RFLP, CTPP-PCR of NRF2 polymorphisms resulted in ambiguous genotyping which strongly jeopardized heterozygosis classification. The necessity of long optimization and control procedures nullified the potential advantages of CTPP-PCR in terms of costs and time.

Circulating microRNAs as potential biomarkers of occupational exposure to low dose organic solvents.

Circulating microRNAs (miRNAs) have been recently acknowledged as novel and non-invasive biomarkers of exposure to environmental and occupational hazardous substances. This preliminary study investigates the potential role of blood miRNAs as molecular biomarkers of exposure to the most common organic solvents (ethylbenzene, toluene, xylene) used in the shipyard painting activity. Despite the low number of recruited workers, a two-tail standard Students' test with Holm-Bonferroni adjusted p-value shows a significant up-regulation of two miRNAs (miR_6819_5p and miR_6778_5p) in exposed workers with respect to controls. A correlation analysis between miRNA, differentially expressed in exposed workers and in controls and urinary dose biomarkers i.e. methylhyppuric acid (xylenes metabolite), phenylglyoxylic and mandelic acid (ethylbenzene metabolites) S-benzyl mercapturic acid (toluene metabolite) and S-phenylmercapturic acid (benzene metabolite) measured at the end of the work-shift, allowed the identification of high correlation (0.80-0.99) of specific miRNAs with their respective urinary metabolites. MiRNA_671_5p correlated with methylhippuric, S-phenylmercapturic and S-benzyl mercapturic acid while the miRNA best correlating with the phenylglioxylic acid was miRNA_937_5p. These findings suggest miRNA as sensitive biomarkers of low dose exposure to organic chemicals used at workplace. Urinary DNA and RNA repair biomarkers coming from the oxidation product of guanine have been also associated to the different miRNAs. A significant negative association was found between 8-oxo-7,8-dihydroguanine (8-oxoGua) urinary concentration and miR_6778_5p. The findings of the present pilot study deserve to be tested on a larger population with the perspective of designing a miRNA based test of low dose exposure to organic solvents.

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.

Intramuscular DNA vaccination protocols mediated by electric fields.

Vaccination is historically one of the most important methods for preventing infectious diseases in humans and animals. New insights in the biology of the immune system allow a more rational design of vaccines, and new vaccination strategies are emerging. DNA vaccines have been proposed as a promising approach for introducing foreign antigens into the host for inducing protective immunity against infectious and cancer diseases. Nevertheless, because of their poor immunogenicity, plasmid DNA vaccination strategies need further implementations. Recent data suggest electrotransfer as a useful tool to improve DNA-based vaccination protocols, being able to stimulate both the humoral and cellular immune responses. In preclinical trials, gene electrotransfer is successfully used in prime-boost combination protocols and its tolerability and safety has been demonstrated also in Phase I clinical trials. In this chapter, we report a short comment supporting electrotransfer as an effective strategy to improve DNA-based vaccination protocols and describe the vaccination procedures by plasmid DNA in combination with electrotransfer and hyaluronidase pretreatment in use in our laboratory.

Hyaluronidase contributes to early inflammatory events induced by electrotransfer in mouse skeletal muscle.

Electrotransfer of genes is one of the preferred strategies used to deliver plasmid DNA into skeletal muscle. In our experience, the combination of hyaluronidase (HYA) with electrotransfer (ET) of DNA vaccine enhances transfection of muscular fibers and increases expression of the encoded antigen. However, the contribution of HYA to the inflammatory reaction induced by ET, and its role in supporting ET adjuvancy, has never been investigated. We analyzed the events occurring in the first 2 weeks after electrotransfer to mouse muscle in the presence of HYA, to verify whether HYA contributes to the local inflammatory response induced by ET. Our results demonstrate that HYA amplifies the ET effect in terms of inflammatory cell recruitment enhancing the early release of interleukin (IL)-1ß, tumor necrosis factor-α, and IL-6 cytokines. In contrast, HYA does not induce helper T cell type 1 and 2 cytokine production, confirming that the DNA vaccine is indispensable to induce mediators of antigen-specific immune responses. We observed inflammatory cell migration in the muscle treated with HYA plus ET in a time window between days 4 and 7 after cytokine induction. These observations are important in the choice of prime-boost intervals for optimizing ET-based DNA vaccination protocols. Because HYA contributes to vaccine spread and enhances the proinflammatory effect of ET in muscle we strongly support the use of HYA to potentiate DNA vaccine efficacy.

Electroporation in DNA vaccination protocols against cancer.

Since conventional therapeutic approaches in cancer are highly invasive they hardly prolong patient survival for more than few months. Having the ability to stimulate both cellular and humoural immune responses, immunisation with naked plasmid DNA encoding tumour-associated antigens or tumour-specific antigens has recently reported a plethora of advantages, and the improvement of vaccine efficacy has emerged as a goal in the development of DNA vaccination as anti-tumour therapy. Nevertheless, because of their poor immunogenicity when administered as unformulated intramuscular injections, plasmid DNA vaccines need to be improved. Recent data suggest that the DNA vaccine efficacy may significantly be increased by electroporation. This review highlights the recent literature that supports electroporation as an effective strategy to improve DNA-based vaccination protocols, investigating the most relevant studies, recently developed for the applications of DNA vaccine electrotransfer against tumours in pre-clinical and clinical studies.

Comparison and critical analysis of robotized technology for monoclonal antibody high-throughput production.

We have previously demonstrated how to transform the conventional method of hybridoma production and screening into a fast, high-throughput technology. Nevertheless, there were still open questions related to automated procedures and immunization protocols that we address now by comparing the hybridoma production work-flow in automated and manually executed processes. In addition, since the animals' antibody responses to single or multiple antigen challenge affect monoclonal antibody throughput, different immunization and fusion strategies were tested. Specifically, the results obtained with multiplexing (multiple target antigens injected into a single animal) and single antigen immunization followed by splenocyte pooling immediately before fusion were compared with conventional methods. The results presented here demonstrate that the optimal protocol consists of automated somatic-cell fusion and hybridoma dilution followed by manual plating of hybridoma cells. Additionally, more specific and productive hybridoma clones were obtained with multiplexed immunization in a single animal with respect to the splenocyte pooling from single antigen immunized animals. However, in terms of overall antibody yield, the conventional method consisting of single immunization for each single animal assured ten times more specific hybridoma cell lines than the strategy based on the multiple antigen immunization followed by separate fusion step. In conclusion, the most productive approach for recovering a large number of suitable antibodies relies on single antigen immunization followed by automated fusion and dilution steps and manual plating.

Production, novel assay development and clinical applications of monoclonal antibodies.

Since the advent of hybridoma technology 35 years ago, research on monoclonal antibodies has developed enormously. Monoclonal antibodies of mouse origin were the first to be produced and continue to be the most popular affinity reagents for investigating the proteome of all organisms. For their adaptability to a variety of biological assays monoclonal antibodies are key tools for basic research as well as for diagnosis and therapy of human diseases. Recently, the expanding demand of high-quality antibodies with better specificities has resulted in a significant improvement in traditional hybridoma production methods. Owing to the ability of these affinity reagents to selectively target tumour cells, cancer has been a major focus of programmes for monoclonal antibody development. This review focuses on patents related to the advances made in the monoclonal antibody manufacture, showing how the traditional production techniques were turned into alternative, faster and more effective methods. Other patents are focussed on new technologies in which monoclonal antibodies are employed for the development of high-performance screening assays. A conclusive series of patents is related to monoclonal antibodies which find application to the diagnosis and the treatment of specific cancer diseases such as haematological malignancies and solid tumours.

Application of electroporation in DNA vaccination protocols.

Vaccination is historically one of the most important methods for preventing infectious diseases in humans and animals. Due to recent advances in understanding the biology of the immune system, a more rational design of vaccines and vaccination strategies such as those based on gene transfer have been proposed. In particular, naked DNA vaccination is emerging as a promising approach for introducing foreign antigens into the host, inducing protective immunity against infectious diseases and malignant tumours. Plasmid DNA vaccines offer several advantages in comparison to traditional vaccines such as safety, tolerability and feasibility in manufacture. Nevertheless, because of their poor immunogenicity, plasmid DNA vaccines need further implementation. Recent data suggest electroporation as useful strategy to improve DNA-based vaccination protocols, being able to stimulate both the humoural and cellular immune responses. In pre-clinical trials, electroporation is successfully used in prime-boost combination protocols and its efficacy and tolerability has been demonstrated in Phase I clinical trials. Since these initial results appear promising, in the next future we will assist to further developments of naked DNA vaccination associated to the electroporation technology. This approach not only provides the basis for human studies but also a practical application to veterinary medicine.

Antigenic features of protein carriers commonly used in immunisation trials.

An aluminium hydroxide adjuvant induced a more elevated and rapid immune responses against short peptides conjugated to the Keyhole Lympet Hemocyanin carrier than immuneasy adjuvant. Furthermore, since carrier proteins may compete with the fused or chemically linked polypeptides in eliciting antigen-specific immune response, we classified the immunogenicity of the most common carrier proteins used in molecular biology for antigen expression and mouse immunisation. The disulfide isomerase protein A gave a carrier with the lowest immunogenicity whilst disulfide isomerase protein C gave the highest immunogenicity and therefore should be avoided as a fusion partner. Using this protein as a model, we identified and located the immunodominant epitopes along its sequence. These results now enable the combination of carrier and immunisation conditions to be optimized.