A Quantitative Adverse Outcome Pathway for Embryonic Activation of the Aryl Hydrocarbon Receptor of Fishes by Polycyclic Aromatic Hydrocarbons Leading to Decreased Fecundity at Adulthood.

Quantitative adverse outcome pathways (qAOPs) describe the response-response relationships that link the magnitude and/or duration of chemical interaction with a specific molecular target to the probability and/or severity of the resulting apical-level toxicity of regulatory relevance. The present study developed the first qAOP for latent toxicities showing that early life exposure adversely affects health at adulthood. Specifically, a qAOP for embryonic activation of the aryl hydrocarbon receptor 2 (AHR2) of fishes by polycyclic aromatic hydrocarbons (PAHs) leading to decreased fecundity of females at adulthood was developed by building on existing qAOPs for (1) activation of the AHR leading to early life mortality in birds and fishes, and (2) inhibition of cytochrome P450 aromatase activity leading to decreased fecundity in fishes. Using zebrafish (Danio rerio) as a model species and benzo[a]pyrene as a model PAH, three linked quantitative relationships were developed: (1) plasma estrogen in adult females as a function of embryonic exposure, (2) plasma vitellogenin in adult females as a function of plasma estrogen, and (3) fecundity of adult females as a function of plasma vitellogenin. A fourth quantitative relationship was developed for early life mortality as a function of sensitivity to activation of the AHR2 in a standardized in vitro AHR transactivation assay to integrate toxic equivalence calculations that would allow prediction of effects of exposure to untested PAHs. The accuracy of the predictions from the resulting qAOP were evaluated using experimental data from zebrafish exposed as embryos to another PAH, benzo[k]fluoranthene. The qAOP developed in the present study demonstrates the potential of the AOP framework in enabling consideration of latent toxicities in quantitative ecological risk assessments and regulatory decision-making. Environ Toxicol Chem 2024;43:2145-2156. © 2024 SETAC.

The Combined Effect of Copper Nanoparticles and Microplastics on Transcripts Involved in Oxidative Stress Pathway in Rainbow Trout (Oncorhynchus Mykiss) Hepatocytes.

Copper nanoparticles (CuNPs) and microplastics (MPs) are two emerging contaminants of freshwater systems. Despite their co-occurrence in many water bodies, the combined effects of CuNPs and MPs on aquatic organisms are not well-investigated. In this study, primary cultures of rainbow trout hepatocytes were exposed to dissolved Cu, CuNPs, MPs, or a combination of MPs and CuNPs for 48 h, and the transcript abundances of oxidative stress-related genes were investigated. Exposure to CuNPs or dissolved Cu resulted in a significant increase in the transcript abundances of two antioxidant enzymes, catalase (CAT) and superoxide dismutase (SOD). Exposure to CuNPs also led to an upregulation in the expression of Na+/K+ ATPase alpha 1 subunit (ATP1A1). Microplastics alone or in combination with CuNPs did not have a significant effect on abundances of the target gene transcripts. Overall, our findings suggested acute exposure to CuNPs or dissolved ions may induce oxidative stress in hepatocytes, and the Cu-induced effect on target gene transcripts was not associated with MPs.

Inhibition of Oocyte Maturation by Malathion and Structurally Related Chemicals in Zebrafish (Danio rerio) After In Vitro and In Vivo Exposure.

Oogenesis is the process by which a primary oocyte develops into a fertilizable oocyte, making it critical to successful reproduction in fish. In zebrafish (Danio rerio), there are five stages of oogenesis. During the final step (oocyte maturation), the maturation-inducing hormone 17α,20ß-dihydroxy-4-pregnen-3-one (MIH) activates the membrane progestin receptor, inducing germinal vesicle breakdown. Using in vitro assays, it has been shown that anthropogenic stressors can dysregulate MIH-induced oocyte maturation. However, it is unknown whether the in vitro assay is predictive of reproductive performance after in vivo exposure. We demonstrate that a known inhibitor of oocyte maturation, malathion, and a structurally related chemical, dimethoate, inhibit oocyte maturation. However, malaoxon and omethoate, which are metabolites of malathion and dimethoate, did not inhibit oocyte maturation. Malathion and dimethoate inhibited maturation to a similar magnitude when oocytes were exposed for 4 h in vitro or 10 days in vivo, suggesting that the in vitro zebrafish oocyte maturation assay might be predictive of alterations to reproductive performance. However, when adult zebrafish were exposed to malathion for 21 days, there was no alteration in fecundity or fertility in comparison with control fish. Our study supports the oocyte maturation assay as being predictive of the success of in vitro oocyte maturation after in vivo exposure, but it remains unclear whether inhibition of MIH-induced oocyte maturation in vitro correlates to decreases in reproductive performance. Environ Toxicol Chem 2022;41:1381-1389. © 2022 SETAC.

The brominated flame retardant, TBCO, impairs oocyte maturation in zebrafish (Danio rerio).

The brominated flame retardant, 1,2,5,6-tetrabromocyclooctane (TBCO), has been shown to decrease fecundity in Japanese medaka (Oryzias latipes) and there is indirect evidence from analysis of the transcriptome and proteome that this effect might be due to impaired oogenesis. An assay for disruption of oocyte maturation by chemical stressors has not been developed in Japanese medaka. Thus, using zebrafish (Danio rerio) as a model, objectives of the present study were to determine whether exposure to TBCO has effects on maturation of oocytes and to investigate potential mechanisms. Sexually mature female zebrafish were given a diet of 35.3 or 628.8 µg TBCO / g food for 14 days after which, stage IV oocytes were isolated to assess maturation in response to maturation inducing hormone. To explore potential molecular mechanisms, abundances of mRNAs of a suite of genes that regulate oocyte maturation were quantified by use of quantitative real-time PCR, and abundances of microRNAs were determined by use of miRNAseq. Ex vivo maturation of oocytes from fish exposed to TBCO was significantly less than maturation of oocytes from control fish. The percentage of oocytes which matured from control fish and those exposed to low and high TBCO were 89, 71, and 67%, respectively. Among the suite of genes known to regulate oocyte maturation, mRNA abundance of insulin like growth factor-3 was decreased by 1.64- and 3.44-fold in stage IV oocytes from females given the low and high concentrations of TBCO, respectively, compared to the control group. Abundances of microRNAs regulating the expression of proteins that regulate oocyte maturation, including processes related to insulin-like growth factor, were significantly different in stage IV oocytes from fish exposed to TBCO. Overall, results of this study indicated that impaired oocyte maturation might be a mechanism of reduced reproductive performance in TBCO-exposed fish. Results also suggested that effects of TBCO on oocyte maturation might be due to molecular perturbations on insulin-like growth factor signaling and expression of microRNAs.

Dicamba elevates concentrations of S-adenosyl methionine but does not induce oxidative stress or alter DNA methylation in rainbow trout (Oncorhynchus mykiss) hepatocytes.

Dicamba is a benzoic acid herbicide used to target woody and broadleaf weeds in industrial, domestic, and municipal spheres. Because of its widespread use, dicamba is frequently detected in surface waters near sites of application. However, little is known regarding the effects of dicamba on freshwater fishes. In the present study, primary cultures of hepatocytes from rainbow trout (Oncorhynchus mykiss) were exposed to either an environmentally relevant (0.22 or 2.2 µg L-1) or supra-environmental (22 µg L-1) concentration of dicamba for 48 h to investigate if oxidative stress is a mechanism of toxicity. mRNA abundances of genes involved in the response to oxidative stress, levels of lipid peroxidation, and concentrations of glutathione and s-adenosyl methionine (SAM) were quantified. Results indicate that dicamba does not induce oxidative stress. However, exposure to 2.2 µg L-1 of dicamba did cause a 5.24-fold increase in concentrations of SAM. To investigate the mechanisms of increased SAM, effects of dicamba on global and genome-wide DNA methylation were quantified. Dicamba did not cause changes to DNA methylation. Overall, dicamba was not acutely toxic to hepatocytes and did not cause oxidative stress or changes in DNA methylation at environmentally relevant concentrations.