Male-transmitted transgenerational effects of the herbicide linuron on DNA methylation profiles in Xenopus tropicalis brain and testis.

The herbicide linuron can cause endocrine disrupting effects in Xenopus tropicalis frogs, including offspring that were never exposed to the contaminant. The mechanisms by which these effects are transmitted across generations need to be further investigated. Here, we examined transgenerational alterations of brain and testis DNA methylation profiles paternally inherited from grandfathers developmentally exposed to an environmentally relevant concentration of linuron. Reduced representation bisulfite sequencing (RRBS) revealed numerous differentially methylated regions (DMRs) in brain (3060 DMRs) and testis (2551 DMRs) of the adult male F2 generation. Key genes in the brain involved in somatotropic (igfbp4) and thyrotropic signaling (dio1 and tg) were differentially methylated and correlated with phenotypical alterations in body size, weight, hind limb length and plasma glucose levels, indicating that these methylation changes could be potential mediators of the transgenerational effects of linuron. Testis DMRs were found in genes essential for spermatogenesis, meiosis and germ cell development (piwil1, spo11 and tdrd9) and their methylation levels were correlated with the number of germ cells nests per seminiferous tubule, an endpoint of disrupted spermatogenesis. DMRs were also identified in several genes central for the machinery that regulates the epigenetic landscape including DNA methylation (dnmt3a and mbd2) and histone acetylation (hdac8, ep300, elp3, kat5 and kat14), which may at least partly drive the linuron-induced transgenerational effects. The results from this genome-wide DNA methylation profiling contribute to better understanding of potential transgenerational epigenetic inheritance mechanisms in amphibians.

Embryonic Exposure to Benzotriazole Ultraviolet Stabilizer 327 Alters Behavior of Rainbow Trout Alevin.

Benzotriazole ultraviolet (UV) stabilizers (BUVSs) are used in great quantities during industrial production of a variety of consumer and industrial goods. As a result of leaching and spill, BUVSs are detectable ubiquitously in the environment. As of May 2023, citing concerns related to bioaccumulation, biomagnification, and environmental persistence, (B)UV(S)-328 was recommended to be listed under Annex A of the Stockholm Convention on Persistent Organic Pollutants. However, a phaseout of UV-328 could result in a regrettable substitution because the replacement chemical(s) could cause similar or unpredicted toxicity in vivo, relative to UV-328. Therefore, the influence of UV-327, a potential replacement of UV-328, was investigated with respect to early life development of newly fertilized rainbow trout embryos (Oncorhynchus mykiss), microinjected with environmentally relevant concentrations of UV-327. Developmental parameters (standard length), energy consumption (yolk area), heart function, blue sac disease, mortality, and behavior were investigated. Alevins at 14 days posthatching, exposed to 107 ng UV-327 g-1 egg, presented significant signs of hyperactivity; they moved on average 1.8-fold the distance and at 1.5-fold the velocity of controls. Although a substantial reduction in body burden of UV-327 was observed at hatching, it is postulated that UV-327, due to its lipophilic properties, interfered with neurological development and signaling from the onset of neurogenesis. If these results hold true across multiple taxa and species, a potential contributor to neurodevelopmental disorders might have been identified. These findings suggest that UV-327 poses an unknown hazard to rainbow trout embryos and alevins, rendering UV-327 a potential regrettable substitution to UV-328. However, a qualified statement on a regrettable substitution requires a comparative investigation on the teratogenic effects between the two BUVSs. Environ Toxicol Chem 2024;43:762-771. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Assessing the Toxicity of Benzotriazole Ultraviolet Stabilizers to Fishes: Insights into Aryl Hydrocarbon Receptor-Mediated Effects.

Benzotriazole ultraviolet stabilizers (BUVSs) are chemicals used to mitigate UV-induced damage to manufactured goods. Their presence in aquatic environments and biota raises concerns, as certain BUVSs activate the aryl hydrocarbon receptor (AhR), which is linked to adverse effects in fish. However, potencies of BUVSs as AhR agonists and species sensitivities to AhR activation are poorly understood. This study evaluated the toxicity of three BUVSs using embryotoxicity assays. Zebrafish (Danio rerio) embryos exposed to BUVSs by microinjection suffered dose-dependent increases in mortality, with LD50 values of 4772, 11 608, and 56 292 ng/g-egg for UV-P, UV-9, and UV-090, respectively. The potencies and species sensitivities to AhR2 activation by BUVSs were assessed using a luciferase reporter gene assay with COS-7 cells transfected with the AhR2 of zebrafish and eight other fishes. The rank order of potency for activation of the AhR2 from all nine species was UV-P > UV-9 > UV-090. However, AhR2s among species differed in sensitivities to activation by up to 100-fold. An approximate reversed rank order of species sensitivity was observed compared to the rank order of sensitivity to 2,3,7,8-tetrachlorodibenzo[p]dioxin, the prototypical AhR agonist. Despite this, a pre-existing quantitative adverse outcome pathway linking AhR activation to embryo lethality could predict embryotoxicities of BUVSs in zebrafish.

Effects of Alkylation on Potency of Benz[a]anthracene for AhR2 Transactivation in Nine Species of Freshwater Fish.

Polycyclic aromatic hydrocarbons (PAHs) are naturally occurring or anthropogenic organic chemicals that can activate the aryl hydrocarbon receptor 2 (AhR2) and induce toxicity in fishes. Alkyl PAHs are more abundant than nonalkylated PAHs in certain environmental matrices and there is growing evidence that alkylation can increase potency, dependent on the position of alkylation. However, it is unknown if the effect of alkylation on potency is conserved across species. In addition, relatively little is known regarding the extent of interspecies variation in sensitivity to PAHs and alkyl PAHs. Therefore, objectives of the present study were to characterize potency of benz[a]anthracene (BAA) and three alkylated homologues representing different alkylation positions in nine phylogenetically diverse species of fish using a standardized in vitro AhR2 transactivation assay. BAA and each alkylated homologue activated the AhR2 in a concentration-dependent manner in each species. Position-dependent effects on potency were observed in every species, but these effects were not consistent across species. Interspecies variation in sensitivity to AhR2 activation by each PAH was observed and ranged by up to 561-fold. Alkylation both increased and decreased the range of interspecies variation and sensitivity, but the potency of each alkylated homologue relative to BAA ranged by less than an order of magnitude among species. These results represent an early step toward the consideration of alkylated homologues for more objective ecological risk assessments of PAHs to native fishes. Environ Toxicol Chem 2023;42:1575-1585. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Endogenous AhR agonist FICZ accumulates in rainbow trout (Oncorhynchus mykiss) alevins exposed to a mixture of two PAHs, retene and fluoranthene.

Multiple studies have reported synergized toxicity of PAH mixtures in developing fish larvae relative to the additive effect of the components. From a toxicological perspective, multiple mechanisms are known to contribute to synergism, such as altered toxicodynamics and kinetics, as well as increased oxidative stress. An understudied contributor to synergism is the accumulation of endogenous metabolites, for example: the aryl hydrocarbon receptor 2 (AhR2) agonist and tryptophan metabolite 6-Formylindolo(3,2-b)carbazole (FICZ). Fish larvae exposed to FICZ, alongside knock-down of cytochrome p450 (cyp1a), has been reported to induced symptoms of toxicity similar to those observed following exposure to PAHs or the dioxin 2,3,7,8-tetrachlorodibenzo-p-dioxin. Here, we explored if FICZ accumulates in newly hatched rainbow trout alevins (Oncorhynchus mykiss) exposed to two PAHs with different properties: retene (potent AhR2 agonist) and fluoranthene (weak AhR2 agonist and Cyp1a inhibitor), either alone or as a binary mixture for 3 and 7 days. We found that exposure to the mixture resulted in accumulation of endogenous FICZ, synergized the blue sac disease index (BSD), and altered the body burden profiles of the PAHs, when compared to the alevins exposed to the individual components. It is thus very plausible that accumulation of endogenously derived FICZ contributed to the synergized BSD index and toxicity in exposed alevins. Accumulation of endogenously derived FICZ is a novel finding that extends our general understanding on PAHs toxicity in developing fish larvae, while at the same time highlighting why environmental risk assessment of PAHs should not be based solely results from the assessment of individual compounds.

Changes in cardiac proteome and metabolome following exposure to the PAHs retene and fluoranthene and their mixture in developing rainbow trout alevins.

Exposure to polycyclic aromatic hydrocarbons (PAHs) is known to affect developing organisms. Utilization of different omics-based technologies and approaches could therefore provide a base for the discovery of novel mechanisms of PAH induced development of toxicity. To this aim, we investigated how exposure towards two PAHs with different toxicity mechanisms: retene (an aryl hydrocarbon receptor 2 (Ahr2) agonist), and fluoranthene (a weak Ahr2 agonist and cytochrome P450 inhibitor (Cyp1a)), either alone or as a mixture, affected the cardiac proteome and metabolome in newly hatched rainbow trout alevins (Oncorhynchus mykiss). In total, we identified 65 and 82 differently expressed proteins (DEPs) across all treatments compared to control (DMSO) after 7 and 14 days of exposure. Exposure to fluoranthene altered the expression of 11 and 19 proteins, retene 29 and 23, while the mixture affected 44 and 82 DEPs by Days 7 and 14, respectively. In contrast, only 5 significantly affected metabolites were identified. Pathway over-representation analysis identified exposure-specific activation of phase II metabolic processes, which were accompanied with exposure-specific body burden profiles. The proteomic data highlights that exposure to the mixture increased oxidative stress, altered iron metabolism and impaired coagulation capacity. Additionally, depletion of several mini-chromosome maintenance components, in combination with depletion of several intermediate filaments and microtubules, among alevins exposed to the mixture, suggests compromised cellular integrity and reduced rate of mitosis, whereby affecting heart growth and development. Furthermore, the combination of proteomic and metabolomic data indicates altered energy metabolism, as per amino acid catabolism among mixture exposed alevins; plausibly compensatory mechanisms as to counteract reduced absorption and consumption of yolk. When considered as a whole, proteomic and metabolomic data, in relation to apical effects on the whole organism, provides additional insight into PAH toxicity and the effects of exposure on heart structure and molecular processes.

Exposure to retene, fluoranthene, and their binary mixture causes distinct transcriptomic and apical outcomes in rainbow trout (Oncorhynchus mykiss) yolk sac alevins.

Polycyclic aromatic hydrocarbons (PAHs) are widely spread environmental contaminants which affect developing organisms. It is known that improper activation of the aryl hydrocarbon receptor (AhR) by some PAHs contributes to toxicity, while other PAHs can disrupt cellular membrane function. The exact downstream mechanisms of AhR activation remain unresolved, especially with regard to cardiotoxicity. By exposing newly hatched rainbow trout alevins (Oncorhynchus mykiss) semi-statically to retene (32 µg l-1; AhR agonist), fluoranthene (50 µg l-1; weak AhR agonist and CYP1a inhibitor) and their binary mixture for 1, 3, 7 and 14 days, we aimed to uncover novel mechanisms of cardiotoxicity using a targeted microarray approach. At the end of the exposure, standard length, yolk area, blue sac disease (BSD) index and PAH body burden were measured, while the hearts were prepared for microarray analysis. Each exposure produced a unique toxicity profile. We observed that retene and the mixture, but not fluoranthene, significantly reduced growth by Day 14 compared to the control, while exposure to the mixture increased the BSD-index significantly from Day 3 onward. Body burden profiles were PAH-specific and correlated well with the exposure-specific upregulations of genes encoding for phase I and II enzymes. Exposure to the mixture over-represented pathways related to growth, amino acid and xenobiotic metabolism and oxidative stress responses. Alevins exposed to the individual PAHs displayed over-represented pathways involved in receptor signaling: retene downregulated genes with a role in G-protein signaling, while fluoranthene upregulated those involved in GABA signaling. Furthermore, exposure to retene and fluoranthene altered the expression of genes encoding for proteins involved in calcium- and potassium ion channels, which suggests affected heart structure and function. This study provides deeper understanding of the complexity of PAH toxicity and the necessity of investigating PAHs as mixtures and not as individual components.