Development of a unified method for the determination of legacy and metabolites of current pesticides in serum for exposure assessment.

The use of pesticides is often regarded as a fundamental aspect of conventional agriculture. However, these compounds have gained recognition as some of the oldest and most widely employed xenobiotic contaminants, necessitating effective strategies for human biomonitoring. In this context, a method was developed for the determination of 16 legacy organochlorine pesticides, 6 metabolites of current pesticides (2,4-D, malathion, parathion, fipronil, pyraclostrobin, cypermethrin, permethrin, cyfluthrin), and 1 triazine herbicide (atrazine) in serum. Samples were prepared with water, formic acid, acetonitrile, and ultrasound irradiation, followed by solid-phase extraction with Oasis Prime HLB. Subsequently, metabolites from current pesticides underwent derivatization using MTBSTFA with 1% TBDMSCl for analysis via gas chromatography-tandem mass spectrometry (GC-MS/MS), employing an SLB-5MS fused silica capillary column. Analytical curves were generated with limits of quantification from 0.3 to 4.0 ng.mL-1. Accuracy ranged from 69 to 124%, and the coefficient of variation from 2 to 28%. Moreover, determining 1-(4-chlorophenyl)-1H-pyrazol-3-ol was suggested as a biomarker for pyraclostrobin biomonitoring. This analytical approach facilitated the determination of both legacy and metabolites of current pesticides in the same serum sample, presenting an interesting and cost-effective option for large cohorts, and multi-omics studies that evaluate time-dependent biomarkers in blood samples, thereby enabling biomonitoring within the same matrix. Furthermore, a proof-of-concept involving 10 volunteers demonstrated exposure to 9 pesticides at mean concentrations measured in ng mL-1, consistent with findings from various biomonitoring initiatives.

Hazard assessment of antineoplastic drugs and metabolites using cytotoxicity and genotoxicity assays.

Antineoplastic drugs are among the most toxic pharmaceuticals. Their release into the aquatic ecosystems has been reported, giving rise to concerns about the adverse effects, including cytotoxicity and genotoxicity, that they may have on exposed organisms. In this study, we analyzed the cytotoxicity and genotoxicity of 5-fluorouracil (5-FU) and its metabolite alpha-fluoro-beta-alanine (3-NH2-F); gemcitabine (GEM) and its metabolite 2'-deoxy-2',2'-difluorouridine (2-DOH-DiF); as well as cyclophosphamide (CP) on the HepG2 cell line. Drug concentrations were based on those previously observed in the effluent of a major cancer hospital in Brazil. The study found that GEM, 2-DOH-DiF and 5-FU resulted in reduced cell viability. No reduction in cell viability was observed for CP and 3-NH2-F. Genotoxic assessment revealed damage in the form of nucleoplasmic bridges for CP and 3-NH2-F. The tested concentrations of all compounds resulted in significantly increased MNi and NBUDs. The results showed that these compounds induced cytotoxic and genotoxic effects in HepG2 cells at concentrations found in the environment. To the best of our knowledge, this study is the first to report on the cytogenotoxic impacts of the metabolites 3-NH2-F and 2-DOH-DiF in HepG2 cells. These findings may help in the development of public policies that could minimize potential environmental contamination.

Analysis of mitochondrial DNA copy number variation in Brazilian farmers occupationally exposed to pesticides.

The use of pesticide use has been linked to the higher production of reactive oxygen species, resulting in oxidative stress, which in turn can cause genomic instability. A marker for instability is the copy number variation of the mitochondrial genome (mtDNAcn), which has been found to be altered in diverse human diseases, including tumors. This research aimed to examine the variation of mtDNAcn in individuals occupationally exposed to pesticides. Real-time PCR assays were conducted on 154 individuals (78 exposed and 76 non-exposed). Pesticide-exposed ndividuals exhibited a significant reduction in mtDNAcn (1.11 ± 0.37mtDNAcn/genome) compared to non-exposed individuals (1.30 ± 0.33mtDNAcn/genome; p = 0.001). The multivariate analysis indicated that individuals who reported using haloxyfop and copper sulfate demonstrated an increase (ß = 0.200, p = 0.053) and a decrease (ß=-0.2, p = 0.021), respectively, in mtDNAcn. In conclusion, our findings suggest that chronic exposure to pesticides results in changes in mtDNAcn.

The Usefulness of an Online Simplified Screening Questionnaire (SSQ) in Identifying Work-Related Cancers.

To obtain a history of occupational exposure in the workplace, the questionnaire is one of the main sources of information. The aim of this study was to develop an online questionnaire using the REDCap data management platform based on the Work-Related Cancer Surveillance Guidelines, reported by the Brazilian National Cancer Institute. Several issues were taken into consideration for its routine application. It should be simple, easy, capable of being applied in a short time and used in the clinical setting of collecting information on the occupational history of the cancer patient. Consequently, this could enable the compulsory notification of work-related cancer. The questionnaire was developed based on questions about the use of and exposure to carcinogenic factors at work and due to smoking. An entirely electronic version of the cancer patient interview was performed using tablets. The online questionnaire was applied at the Barretos Cancer Hospital, Barretos, to newly diagnosed patients from July 2016 to 2018. A total of 1063 patients were included, and 550 indicated positively when asked "Do you work, or have you worked with this substance and/or in this function?/job?" Of these potentially notified patients, 38 subsequently had compulsorily reported work-related cancer. Another important result of this study was the creation and development of a website. In conclusion, we developed an online tool that could facilitate hospital routines, contributing to generating data for the compulsory notification of work-related cancer and triggering investigations and surveillance actions in Brazil.

Occupational Exposures and Risks of Non-Hodgkin Lymphoma: A Meta-Analysis.

Non-Hodgkin lymphoma (NHL) is a heterogeneous group with different types of diseases. It remains unclear as to what has led to an increase in incidences of NHL, however, chemical substance exposure is known to be one of the risk factors for the disease. Therefore, we performed a systematic review and meta-analysis including case-control, cohort, and cross-sectional observational epidemiological studies to verify the association between occupational exposure to carcinogens and NHL risk. Articles between the years 2000 and 2020 were collected. Two different reviewers performed a blind selection of the studies using the Rayyan QCRI web app. Post-completion, the selected articles were extracted and analyzed via the RedCap platform. Our review resulted in 2719 articles, of which 51 were included in the meta-analysis, resulting in an overall OR of 1.27 (95% CI 1.04-1.55). Furthermore, it was observed that the main occupation associated with the increased risk of NHL was that in which workers are exposed to pesticides. We therefore conclude that the evidence synthesis of the epidemiological literature supports an increased risk for NHL, regardless of subtype, considering occupational exposure to certain chemical compounds, mainly pesticides, benzene, and trichlorethylene, and certain classes of work, primarily in the field of agriculture.

Detection of anti-cancer drugs and metabolites in the effluents from a large Brazilian cancer hospital and an evaluation of ecotoxicology.

The use of chemotherapy agents has been growing worldwide, due to the increase number of cancer cases. In several countries, mainly in Europe countries, these drugs have been detected in hospitals and municipal wastewaters. In Brazil this issue is poorly explored. The main goal of this study was to assess the presence of three anti-cancer drugs, 5-fluorouracil (5-FU), gemcitabine (GEM) and cyclophosphamide (CP), and two metabolites, alpha-fluoro-beta-alanine (3-NH2-F) and 2'-deoxy-2',2'-difluorouridine (2-DOH-DiF), in effluents from a large cancer hospital, in the municipal wastewater treatment plant (WWTP) influent and effluent, and also to evaluate toxicity of the mixtures of these compounds by ecotoxicological testing in zebrafish. The sample collections were performed in Barretos Cancer Hospital of the large cancer center in Brazil. After each collection, the samples were filtered for subsequent Liquid Chromatography Mass Spectrometry analysis. The presence of CP, GEM, and both metabolites (3-NH2-F and 2-DOH-DiF) were detected in the hospital wastewater and the WWTP influent. Three drugs, GEM, 2-DOH-DiF and CP, were detected in the WWTP effluent. Two drugs were detected below the limit of quantification, 2-DOH-DiF:

Evaluation of DNA Methylation Changes and Micronuclei in Workers Exposed to a Construction Environment.

Methylation levels in tumor-suppressor genes and repetitive sequences have previously been used to study the relationship between environmental air pollution and epigenetic changes related to cancer. In this study, we measured the methylation profiles of the promoter regions CDKN2A, MLH1 and APC and the repetitive sequence LINE-1 in 59 workers exposed to the construction environment and in 49 unexposed workers. We also evaluated the micronuclei frequency and levels of trace elements in the blood of all workers. We evaluated of levels of particulate matter and polycyclic aromatic hydrocarbons (PAHs) at the construction site to characterize the environmental exposure. Our findings demonstrated that exposed workers exhibited significantly higher average levels of promoter methylation of CDKN2A, APC, and MLH1 genes and increased hypomethylation of the LINE-1 in comparison to unexposed workers (all p < 0.05). A higher frequency of micronuclei was observed in the exposed group (2 ± 2) compared to the unexposed group (1 ± 1) with p < 0.001. High levels of particulate matter (51⁻841 µg/m³) and some PAHs were found in samples from the construction environment. In summary, we provide evidence of increased DNA damage and altered DNA methylation of exposed workers, suggesting that genomic approaches to biomonitoring may be an effective way of estimating future cancer risk for construction workers.

MicroRNA expression as risk biomarker of breast cancer metastasis: a pilot retrospective case-cohort study.

BACKGROUND: MicroRNAs (miRNAs) are small, non-coding RNA molecules involved in post-transcriptional gene regulation and have recently been shown to play a role in cancer metastasis. In solid tumors, especially breast cancer, alterations in miRNA expression contribute to cancer pathogenesis, including metastasis. Considering the emerging role of miRNAs in metastasis, the identification of predictive markers is necessary to further the understanding of stage-specific breast cancer development. This is a retrospective analysis that aimed to identify molecular biomarkers related to distant breast cancer metastasis development. METHODS: A retrospective case cohort study was performed in 64 breast cancer patients treated during the period from 1998-2001. The case group (n = 29) consisted of patients with a poor prognosis who presented with breast cancer recurrence or metastasis during follow up. The control group (n = 35) consisted of patients with a good prognosis who did not develop breast cancer recurrence or metastasis. These patient groups were stratified according to TNM clinical stage (CS) I, II and III, and the main clinical features of the patients were homogeneous. MicroRNA profiling was performed and biomarkers related to metastatic were identified independent of clinical stage. Finally, a hazard risk analysis of these biomarkers was performed to evaluate their relation to metastatic potential. RESULTS: MiRNA expression profiling identified several miRNAs that were both specific and shared across all clinical stages (p ≤ 0.05). Among these, we identified miRNAs previously associated with cell motility (let-7 family) and distant metastasis (hsa-miR-21). In addition, hsa-miR-494 and hsa-miR-21 were deregulated in metastatic cases of CSI and CSII. Furthermore, metastatic miRNAs shared across all clinical stages did not present high sensitivity and specificity when compared to specific-CS miRNAs. Between them, hsa-miR-183 was the most significative of CSII, which miRNAs combination for CSII (hsa-miR-494, hsa-miR-183 and hsa-miR-21) was significant and were a more effective risk marker compared to the single miRNAs. CONCLUSIONS: Women with metastatic breast cancer, especially CSII, presented up-regulated levels of miR-183, miR-494 and miR-21, which were associated with a poor prognosis. These miRNAs therefore represent new risk biomarkers of breast cancer metastasis and may be useful for future targeted therapies.

Analysis and functional annotation of an expressed sequence tag collection for tropical crop sugarcane.

To contribute to our understanding of the genome complexity of sugarcane, we undertook a large-scale expressed sequence tag (EST) program. More than 260,000 cDNA clones were partially sequenced from 26 standard cDNA libraries generated from different sugarcane tissues. After the processing of the sequences, 237,954 high-quality ESTs were identified. These ESTs were assembled into 43,141 putative transcripts. Of the assembled sequences, 35.6% presented no matches with existing sequences in public databases. A global analysis of the whole SUCEST data set indicated that 14,409 assembled sequences (33% of the total) contained at least one cDNA clone with a full-length insert. Annotation of the 43,141 assembled sequences associated almost 50% of the putative identified sugarcane genes with protein metabolism, cellular communication/signal transduction, bioenergetics, and stress responses. Inspection of the translated assembled sequences for conserved protein domains revealed 40,821 amino acid sequences with 1415 Pfam domains. Reassembling the consensus sequences of the 43,141 transcripts revealed a 22% redundancy in the first assembling. This indicated that possibly 33,620 unique genes had been identified and indicated that >90% of the sugarcane expressed genes were tagged.