Mitochondrial dysfunction and oxidative stress contribute to cross-generational toxicity of benzo(a)pyrene in Danio rerio.

The potential for polycyclic aromatic hydrocarbons (PAHs) to have adverse effects that persist across generations is an emerging concern for human and wildlife health. This study evaluated the role of mitochondria, which are maternally inherited, in the cross-generational toxicity of benzo(a)pyrene (BaP), a model PAH and known mitochondrial toxicant. Mature female zebrafish (F0) were fed diets containing 0, 12.5, 125, or 1250 µg BaP/g at a feed rate of 1% body weight twice/day for 21 days. These females were bred with unexposed males, and the embryos (F1) were collected for subsequent analyses. Maternally-exposed embryos exhibited altered mitochondrial function and metabolic partitioning (i.e. the portion of respiration attributable to different cellular processes), as evidenced by in vivo oxygen consumption rates (OCRs). F1 embryos had lower basal and mitochondrial respiration and ATP turnover-mediated OCR, and increased proton leak and reserve capacity. Reductions in mitochondrial DNA (mtDNA) copy number, increases in mtDNA damage, and alterations in biomarkers of oxidative stress were also found in maternally-exposed embryos. Notably, the mitochondrial effects in offspring occurred largely in the absence of effects in maternal ovaries, suggesting that PAH-induced mitochondrial dysfunction may manifest in subsequent generations. Maternally-exposed larvae also displayed swimming hypoactivity. The lowest observed effect level (LOEL) for maternal BaP exposure causing mitochondrial effects in offspring was 12.5 µg BaP/g diet (nominally equivalent to 250 ng BaP/g fish). It was concluded that maternal BaP exposure can cause significant mitochondrial impairments in offspring.

Polycyclic aromatic hydrocarbon and hypoxia exposures result in mitochondrial dysfunction in zebrafish.

Organisms are routinely subjected to a variety of environmental and chemical perturbations simultaneously. Often, multi-stressor exposures result in unpredictable toxicity that occurs through unidentified mechanisms. Here, we focus on polycyclic aromatic hydrocarbons (PAHs) and hypoxia, two environmental and physiological stressors that are known to co-occur in the environment. The aim of this study was to assess whether interactive mitochondrial dysfunction resulted from co-exposures of PAHs and hypoxia. Zebrafish embryos were co-exposed to non-teratogenic concentrations of an environmental PAH mixture and hypoxia beginning at 6 hpf for an acute period of 24 h and afterwards were given either no recovery period, 45 min, 5 -hs, or 18 -hs of recovery time in clean conditions. Mitochondrial function and integrity were assessed through the use of both in ovo and in vitro assays. Hypoxia exposures resulted in drastic reductions in parameters relating to mitochondrial respiration, ATP turnover, and mitochondrial DNA integrity. PAH exposures affected ATP production and content, as well as mitochondrial membrane dynamics and lactate content. While PAH and hypoxia exposures caused a broad range of effects, there appeared to be very little interaction between the two stressors in the co-exposure group. However, because hypoxia significantly altered mitochondrial function, the possibility remains that these effects may limit an individual's ability to respond to PAH toxicity and therefore could cause downstream interactive effects.

High-content screening in zebrafish embryos identifies butafenacil as a potent inducer of anemia.

Using transgenic zebrafish (fli1:egfp) that stably express enhanced green fluorescent protein (eGFP) within vascular endothelial cells, we recently developed and optimized a 384-well high-content screening (HCS) assay that enables us to screen and identify chemicals affecting cardiovascular development and function at non-teratogenic concentrations. Within this assay, automated image acquisition procedures and custom image analysis protocols are used to quantify body length, heart rate, circulation, pericardial area, and intersegmental vessel area within individual live embryos exposed from 5 to 72 hours post-fertilization. After ranking developmental toxicity data generated from the U.S. Environmental Protection Agency's (EPA's) zebrafish teratogenesis assay, we screened 26 of the most acutely toxic chemicals within EPA's ToxCast Phase-I library in concentration-response format (0.05-50 µM) using this HCS assay. Based on this screen, we identified butafenacil as a potent inducer of anemia, as exposure from 0.39 to 3.125 µM butafenacil completely abolished arterial circulation in the absence of effects on all other endpoints evaluated. Butafenacil is an herbicide that inhibits protoporphyrinogen oxidase (PPO)--an enzyme necessary for heme production in vertebrates. Using o-dianisidine staining, we then revealed that severe butafenacil-induced anemia in zebrafish was due to a complete loss of hemoglobin following exposure during early development. Therefore, six additional PPO inhibitors within the ToxCast Phase-I library were screened to determine whether anemia represents a common adverse outcome for these herbicides. Embryonic exposure to only one of these PPO inhibitors--flumioxazin--resulted in a similar phenotype as butafenacil, albeit not as severe as butafenacil. Overall, this study highlights the potential utility of this assay for (1) screening chemicals for cardiovascular toxicity and (2) prioritizing chemicals for future hypothesis-driven and mechanism-focused investigations within zebrafish and mammalian models.