RESUMO
Complex mixtures of chemicals present in groundwater at legacy-contaminated industrial sites can pose significant risks to adjacent surface waters. The combination of short-term molecular and chronic apical effect assessments is a promising approach to characterize the potential hazard of such complex mixtures. The objectives of this study were to: (1) assess the apical effects (survival, growth, development, and liver histopathology) after chronic exposure of early life stages (ELSs) of fathead minnows (FHM; Pimephales promelas) to contaminated groundwater from a legacy-contaminated pesticide manufacturing and packaging plant, and (2) identify possible molecular mechanisms responsible for these effects by comparing results to mechanistic outcomes previously determined by a short-term reduced transcriptome assay (EcoToxChips). This study revealed a significant increase in mortality and prevalence of spinal curvatures, as well as a significant reduction in the length of FHMs exposed to the groundwater mixtures in a concentration-dependent manner. There was an increasing trend in the prevalence of edema in FHMs, though not significantly different from controls. Additionally, no histopathological effects were observed in the liver of FHMs exposed to the groundwater mixtures. Short-term molecular outcomes determined in a parallel study were found to be informative of chronic apical outcomes, including cardiotoxicity, spinal deformities, and liver toxicity. Overall, the results observed in this study demonstrated that short-term transcriptomics analyses could support the hazard assessment of complex contaminated sites.
Assuntos
Cyprinidae , Água Subterrânea , Poluentes Químicos da Água , Poluentes Químicos da Água/toxicidade , Água Subterrânea/química , Animais , Monitoramento Ambiental , Fígado/efeitos dos fármacos , Praguicidas/toxicidadeRESUMO
Traditional risk assessment methods face challenges in the determination of drivers of toxicity for complex mixtures such as those present at legacy-contaminated sites. Bioassay-driven analysis across several levels of biological organization represents an approach to address these obstacles. This study aimed to apply a novel transcriptomics tool, the EcoToxChip, to characterize the effects of complex mixtures of contaminants in adult fathead minnows (FHMs) and to compare molecular response patterns to higher-level biological responses. Adult FHMs were exposed for 4 and 21 days to groundwater mixtures collected from a legacy-contaminated site. Adult FHM showed significant induction of micronuclei in erythrocytes, decrease in reproductive capacities, and some abnormal appearance of liver histology. Parallel EcoToxChip analyses showed a high proportion of upregulated genes and a few downregulated genes characteristic of compensatory responses. The three most enriched pathways included thyroid endocrine processes, transcription and translation cellular processes, and xenobiotics and reactive oxygen species metabolism. Several of the most differentially regulated genes involved in these biological pathways could be linked to the apical outcomes observed in FHMs. We concluded that molecular responses as determined by EcoToxChip analysis show promise for informing of apical outcomes and could support risk assessments of complex contaminated sites.
Assuntos
Cyprinidae , Poluentes Químicos da Água , Animais , Poluentes Químicos da Água/análise , Reprodução , Fígado/metabolismo , Cyprinidae/metabolismo , Misturas ComplexasRESUMO
Mechanistic toxicology approaches represent a promising alternative to traditional live animal testing; however, the often-noted uncertainties concerning the linkages between effects observed at molecular and apical levels curtails the adoption of such approaches. The objective of this study was to apply a novel transcriptomics tool, EcoToxChips, to characterize the effects of complex mixtures of contaminants in fish and to compare molecular response patterns to higher-level biological responses including swimming behavior, deformities, and mortality. Fathead minnow (FHM) embryos were exposed for seven days to increasing concentrations of groundwater collected from moderate (MIAZ) and high (HIAZ) industrial activity zones of a legacy contaminated site. There was a concentration-dependent disruption of photo-dependent swimming responses associated with avoidance behavior patterns and spinal deformities (HIAZ and MIAZ), and an induction of pericardial edema and mortality (HIAZ-10%). Parallel EcoToxChip analyses showed a shift from a majority of upregulated genes at lower concentrations to a majority of downregulated genes at higher concentrations for both treatment conditions. Many of the significantly differentially regulated genes were involved in biological pathways including induction of oxidative stress, activating of several metabolic processes and growth, cell death, and inhibition of signal transduction signaling processes. Several contaminants present in the groundwater mixtures could have contributed to an exceedance of antioxidant system capacities that possibly led to the deformities, altered swimming behaviours, and mortality observed in FHMs. Therefore, molecular response patterns could be linked to apical outcomes observed in this study. Overall, the results observed in this study demonstrate that transcriptomics approaches such as the EcoToxChip system could be supportive of risk assessment of complex contaminated sites.
Assuntos
Cyprinidae , Poluentes Químicos da Água , Animais , Larva , Poluentes Químicos da Água/toxicidade , Cyprinidae/metabolismo , Natação , Perfilação da Expressão GênicaRESUMO
UV-induced formation of photoproducts in DNA is a major initiating event of skin cancer. Consequently, many analytical tools have been developed for their quantification in DNA. In the present work, we extended our previous liquid chromatography-mass spectrometry method to the quantification of the short DNA fragments containing photoproducts that are released from cells by the repair machinery. We designed a robust protocol including a solid-phase extraction step (SPE), an enzymatic treatment aimed at releasing individual photoproducts, and a liquid chromatography method combining on-line SPE and ultra-high-performance liquid chromatography for optimal specificity and sensitivity. We also added relevant internal standards for a better accuracy. The method was validated for linearity, repeatability, and reproducibility. The limits of detection and quantification were found to be in the fmol range. The proof of concept of the use of excreted DNA repair products as biomarkers of the genotoxicity of UV was obtained first in in vitro studies using cultured HaCat cells and ex vivo on human skin explants. Further evidence was obtained from the detection of pyrimidine dimers in the urine of human volunteers collected after recreational exposure in summer. An assay was designed to quantify the DNA photoproducts released from cells within short fragments by the DNA repair machinery. These oligonucleotides were isolated by solid-phase extraction and enzymatically hydrolyzed. The photoproducts were then quantified by on-line SPE combined with UHPLC-MS/MS with isotopic dilution.