Early sublethal TCDD exposure in Zebrafish: Toxicity in adults and subsequent generations

Dioxin, an aryl hydrocarbon receptor (AhR) agonist, is a known endocrine disrupter and an extremely potent pollutant: zebrafish exposed to 1 part per billion dioxin die by 7 days post fertilization (pf). Effects of dioxin at lethal levels have been extensively studied but the effects of dioxin exposure in the sublethal range are poorly understood. Zebrafish (Danio rerio) are an established vertebrate model for studying human health and development. To thoroughly explore the adverse effects of sublethal exposure we divided newly fertilized zebrafish eggs into two sublethal (50 pg/ml; 1hour) exposure groups: one that was exposed during embryogenesis (4 hours pf) and the second that was exposed during sexual differentiation and gonad maturation (3 and 7 weeks pf). These fish were examined as adults (21-40 weeks pf). We observed the most abnormalities, including craniofacial and skeletal abnormalities, in the fish that were exposed during sexual differentiation. In addition, we found significant changes in egg production, fertility, mating behaviors, and sex ratios. Surprisingly, on postmortem histological evaluation, some fish that had been identified as female based on secondary sex characteristics had testes. Next, we evaluated the next two generations of offspring (F1 and F2) for dioxin-induced transgenerational changes in sex ratios, skeletal abnormalities, and reproductive capacity. The F1 generation may have been exposed as germ cells, but the F2 generation was never exposed to TCDD. In the F1 and F2 generations, the sex ratios showed a significantly higher percentage of females compared to controls. The incidence of skeletal abnormalities was also significantly higher than controls in the F1 and F2 generations with a scoliosis-like kink in the spinal column most commonly observed. Decreased reproductive capacity was observed in the F1 and F2 generations with both egg release and percentage of eggs fertilized significantly decreased compared to controls. Spawning of the F2 generation of the TCDD exposed founders with control fish revealed that decreased egg release and egg fertilization were due to the F2 males. We developed a model for studying transgenerational effects and our results show that TCDD exposure in zebrafish leads to persistent effects in the F1 and F2 generations.