Chronic exposure to low concentrations of chlorpyrifos affects normal cyclicity and histology of the uterus in female rats.

Chlorpyrifos (CPF), the most used insecticide in Argentina, can act as an endocrine disruptor at low doses. We previously demonstrated that chronic exposure to CPF induces hormonal imbalance in vivo. The aim of this work was to study the effects of low concentrations of CPF (0.01 and 1 mg/kg/day) on the reproductive system of virgin adult rats. In the ovary, we studied the effects of CPF on steroidogenesis by determining steroid hormone content by RIA and CYP11 and CYP19 enzyme expression by qRT-PCR. The estrous cycle was evaluated by microscopic observation of vaginal smear, as well as by changes in uterine histology. In endometrium, we determined the fractal dimension and expression of PCNA, ERα and PR by IHC. Our results showed that chronic exposure to CPF affects ovarian steroid synthesis, causing alterations in the normal cyclicity of animals. In addition, CPF induced proliferative changes in the uterus, suggesting that it could affect reproduction or act as a risk factor in the development of uterine proliferative pathologies.

The Rhinella arenarum transcriptome: de novo assembly, annotation and gene prediction.

The common toad Rhinella arenarum is widely distributed in Argentina, where it is utilised as an autochthonous model in ecotoxicological research and environmental toxicology. However, the lack of a reference genome makes molecular assays and gene expression studies difficult to carry out on this non-model species. To address this issue, we performed a genome-wide transcriptome analysis on R. arenarum larvae through massive RNA sequencing, followed by de novo assembly, annotation, and gene prediction. We obtained 57,407 well-annotated transcripts representing 99.4% of transcriptome completeness (available at http://rhinella.uncoma.edu.ar). We also defined a set of 52,800 high-confidence lncRNA transcripts and demonstrated the reliability of the transcriptome data to perform phylogenetic analysis. Our comprehensive transcriptome analysis of R. arenarum represents a valuable resource to perform functional genomic studies and to identify potential molecular biomarkers in ecotoxicological research.

Comprehensive analysis of renal arsenic accumulation using images based on X-ray fluorescence at the tissue, cellular, and subcellular levels.

Exposure to arsenic (As) through drinking water results in accumulation of As and its methylated metabolites in several organs, promoting adverse health effects, particularly potential development of cancer. Arsenic toxicity is a serious global health concern since over 200 million people are chronically exposed worldwide. Abundant biochemical and epidemiological evidence indicates that the kidney is an important site of uptake and accumulation of As, and mitochondrial damage plays a crucial role in arsenic toxicity. However, non-destructive analyses and in situ images revealing As fate in renal cells and tissue are scarce or almost non-existent. In this work, kidney tissue from exposed rats was analyzed by EDXRF (Energy dispersive X-ray fluorescence), micro-SRXRF (micro X-ray Fluorescence using Synchrotron Radiation), SRTXRF (SRXRF in total reflection condition), SEM-EDX (Scanning Electron Microscope in combination with EDXRF) and SRXRF-XANES (SRXRF in combination with X-ray Absorption Near Edge Spectroscopy). Our results provide evidence of renal cortex distribution of As with periglomerular localization, co-localization of S, Cu and As in subcellular compartment of proximal tubule cells, mono-methylarsonous acid accumulation in renal cortex mitochondria, and altered subcellular concentration and distribution of other elements.

Arsenic absorption and excretion in chronically exposed developing toad Rhinella arenarum.

We assessed the toxicodynamics of As in developing Rhinella arenarum toad embryos and larvae exposed from fertilization to 0.01-10mgAsL-1. We determined As content in toad embryos and larvae by X-ray fluorescence spectrometry. Toad embryos and larvae actively bioaccumulated As, reaching tissue concentrations more than one-thousand higher than control levels after 23d-exposure to 10mgAsL-1. The bioconcentration factors also increased up to fifty times higher levels in toad larvae respect to media levels. Once recovered in As-free media, the larvae rapidly excreted the bioaccumulated As with a half-life of 1.6d. By calcein transport competition assays, we infer that As is excreted through ABCC-like transporters, probably conjugated with GSH. These results are relevant for comprehending the risks posed by As exposure in this autochthonous aquatic species that develops in water courses from Argentina, that may contain As levels ranging between 10-15,000µgL-1.

Molecular effectors in the chronic exposure to arsenic as early and sensitive biomarkers in developing Rhinella arenarum toads.

Arsenic, a natural element of ecological relevance, is one of the most toxic elements present in various regions of the world. It can be found in natural water sources throughout Argentina in concentrations between 0.01 and 15mgL-1. The Argentinean autochthonous toad Rhinella arenarum was selected to study the molecular mechanisms involved in the effects and response to the chronic As exposure along its embryonic and larval development. We evaluated the effects on MAPK signal transduction pathway and transcription factors c-FOS and c-JUN, and the regulation of the expression at protein levels of different antioxidant enzymes. Our results indicated that As is modulating the MAPK pathway, increasing MEK and ERK levels both in the nuclear and post-nuclear fraction along the embryonic development and mainly at the beginning of the larval stage. Through this pathway, As can upregulate transcription factors like c-FOS and c-JUN, impacting the antioxidant response of the exposed embryos and larvae through antioxidant enzymes and recycling of GSH. Arsenic triggered specifically the synthesis of antioxidant enzymes in exposed R. arenarum embryo and larvae. In particular, the expression levels of SOD, CAT and GST enzymes analyzed by Western blot showed a similar behavior to their enzymatic activities in our previous work. This fact suggests that not only the synthesis of these antioxidant enzymes but also their rapid degradation after inactivation would be regulated in response to ROS levels. Antioxidant enzymes may show dual responses of induction and inactivation followed by degradation depending on the levels of oxidative stress and impact on ROS targets when the exposure is sustained in time and intensity. We also performed a probability of exceedence analysis including our previous results to visualize a progression of the response in time and also established the best early-responding biomarkers at the lowest As concentrations. As a conclusion, the molecular biomarkers such as the MAPKs MEK and ERK and transcription factors c-FOS and c-JUN are early induced in the response of developing toad embryos exposed to very low As concentrations in water. The advantage of counting with molecular biomarkers early responding to low concentrations of As in a chronic exposure is that they may anticipate the irreversible damage at later developmental stages due to the constant oxidative challenge.

Chronic toxicity of arsenic during Rhinella arenarum embryonic and larval development: Potential biomarkers of oxidative stress and antioxidant response.

The Argentinean autochthonous toad Rhinella arenarum was selected to study the chronic toxicity of arsenic (As) and the biochemical responses elicited by exposure to As in water during embryonic and larval development. Significant decreases in the total reactive antioxidant potential and in catalase activity were observed in individuals exposed chronically to sublethal concentrations of As, which is indicative of an oxidative stress situation. However, an antioxidant response was elicited during chronic exposure to As, as evidenced by the increase in endogenous reduced glutathione content and glutathione-related enzymatic activities such as glutathione S-transferase (GST) and glutathione reductase. This protective response might prevent a deeper decline in the antioxidant system and further oxidative damage. Alternatively, it might be linked to As conjugation with reduced glutathione for its excretion. Considering the sustained increase in GST activity and the decrease in the total antioxidant reactive potential observed, the authors propose them as good candidates to be used as biomarkers during As exposure. Interestingly, glutathione reductase activity was inhibited at a very low concentration of As considered safe for aquatic life. Environ Toxicol Chem 2017;36:1614-1621. © 2016 SETAC.

Toxicity of the insecticide chlorpyrifos to the South American toad Rhinella arenarum at larval developmental stage.

Chlorpyrifos (CPF) is an insecticide widely used for pest control in the fruit-productive region of North Patagonia, Argentina, where it is found in superficial waters. The aim of this study was to establish the toxic effects of CPF in Rhinella arenarum toad larvae as a potentially exposed species. We determined the 96 h-LC50 (1.46 ± 0.27 mg/L), the LOEC (0.81 mg/L, LC10) and NOEC (0.43 mg/L, LC1) for CPF lethality as endpoint. We also analyzed biochemical biomarkers in larvae exposed to sublethal CPF concentrations. The IC50 for cholinesterase was 0.113 ± 0.026 mg/L, one order of magnitude lower than the LC50. Carboxylesterase activity was inhibited, buffering OP toxicity on cholinesterase. Reduced glutathione increased after 24h as an antioxidant response, and decreased at 96 h together with catalase activity, due to oxidative stress. These biochemical effects suggest that environmentally relevant CPF concentrations pose a threat to R. arenarum larvae progression.

Acute toxicity of arsenic and oxidative stress responses in the embryonic development of the common South American toad Rhinella arenarum.

Arsenic (As), a natural element of ecological relevance, is found in natural water sources throughout Argentina in concentrations between 0.01 mg/L and 15 mg/L. The autochthonous toad Rhinella arenarum was selected to study the acute toxicity of As and the biochemical responses elicited by the exposure to As in water during its embryonic development. The median lethal concentration (LC50) value averaged 24.3 mg/L As and remained constant along the embryonic development. However, As toxicity drastically decreased when embryos were exposed from heartbeat-stage on day 4 of development, suggesting the onset of detoxification mechanisms. Given the environmental concentrations of As in Argentina, there is a probability of exceeding lethal levels at 1% of sites. Arsenic at sublethal concentrations caused a significant decrease in the total antioxidant potential but generated an increase in endogenous glutathione (GSH) content and glutathione S-transferase (GST) activity. This protective response might prevent a deeper decline in the antioxidant system and further oxidative damage. Alternatively, it might be linked to As conjugation with GSH for its excretion. The authors conclude that toad embryos are more sensitive to As during early developmental stages and that relatively high concentrations of this toxic element are required to elicit mortality, but oxidative stress may be an adverse effect at sublethal concentrations.