Transgenerational inheritance of neurobehavioral and physiological deficits from developmental exposure to benzo[a]pyrene in zebrafish

Abstract

Benzo[a]pyrene (B[a]P) is a well-known genotoxic polycylic aromatic compound whose toxicity is dependent on signaling via the aryl hydrocarbon receptor (AHR). It is unclear to what extent detrimental effects of B[a]P exposures might impact future generations and whether transgenerational effects might be AHR-dependent. This study examined the effects of developmental B[a]P exposure on 3 generations of zebrafish. Zebrafish embryos were exposed from 6 to 120h post fertilization (hpf) to 5 and 10μM B[a]P and raised in chemical-free water until adulthood (F0). Two generations were raised from F0 fish to evaluate transgenerational inheritance. Morphological, physiological and neurobehavioral parameters were measured at two life stages. Juveniles of the F0 and F2 exhibited hyper locomotor activity, decreased heartbeat and mitochondrial function. B[a]P exposure during development resulted in decreased global DNA methylation levels and generally reduced expression of DNA methyltransferases in wild type zebrafish, with the latter effect largely reversed in an AHR2-null background. Adults from the F0 B[a]P exposed lineage displayed social anxiety-like behavior. Adults in the F2 transgeneration manifested gender-specific increased body mass index (BMI), increased oxygen consumption and hyper-avoidance behavior. Exposure to benzo[a]pyrene during development resulted in transgenerational inheritance of neurobehavioral and physiological deficiencies. Indirect evidence suggested the potential for an AHR2-dependent epigenetic route.

Keywords: Aryl hydrocarbon receptor; Benzo[a]pyrene; Developmental toxicity; Neurobehavioral; Physiological deficits; Transgenerational; Zebrafish.

Figure 1. Overview of epigenetic study

Embryonic zebrafish were developmentally exposed to 0, 5 and 10 μM Benzo[a]pyrene (B[a]P) from 6 - 120 hours post fertilization, then raised until adulthood (>90 days post fertilization; dpf) in chemical- free water [F0]. The F0 cohort was spawned to create 2 subsequent generations. Epigenetic inheritance was evaluated in F2.

Figure 2. Larval behavioral, heartbeat and mitochondrial function in F0 and epigenetic generations F2

The inset (top right) illustrates the generation(s) discussed. (a) Heartrate differences for the F0 and F2 5 and 10 μM B[a]P and controls groups larvae. Note that while the relative treatments effects are conserved across generations, baseline heartrates differ because F2 animals were assayed 3°C cooler than the F0 animals (see Methods for details) (b) The oxygen consumption rate was measured in 24 hpf larvae in F0, and F2. * denotes statistical significance based on a one-way ANOVA (p < 0.05). (c) 5 day LPR response in the F2 larvae assessed in the light-dark assay.

Figure 3. Global DNA methylation and Dnmt expression at 24 and 120 hpf in F0 embryos exposed to B[a]P

The inset (top right) illustrates the generation(s) discussed. (a) Global DNA methylation for F0 5 and 10 μM B[a]P and F0 control groups. (b) Dnmt expression measured at 24 and 120 hpf. Beta-actin was used as an internal standard. All values represent mean + standard error of mean (S.E.M; n=3) of fold change relative to control. Two-way ANOVA was used to determine the effect of treatment and time on Dnmt expression. A Tukey’s post-hoc test was used to determine statistical significance denoted by asterisks from the vehicle lineage (p<0.05).

Figure 4. Dnmt expression in wild type (WT) and ahr2^hu2334 embryos exposed to 0 and 10 μM B[a]P

Gene expression changes were evaluated in WT and ahr2-null embryos at 24 (left panel) and 120 hpf (right panel). Beta-actin was used as an internal standard, and all values represent fold change relative to the controls. Two-way ANOVA with a Tukey Post Hoc was used to determine statistical significance. Points filled with white represent statistical significance (p<0.05). The gray shaded polygon represents 1-fold change. Any points within the gray represent repression of the gene.

Figure 5. Physiological characteristics and fitness in F0 and F2 cohort

The inset (top right) illustrates the generation(s) discussed. (a) The BMI for adult zebrafish from F0 and F2 were assessed to identify potential gender differences, and (b) the oxygen consumption rate over 5 speeds were measured. * denotes statistical significance relative to vehicle (p < 0.05, two-way ANOVA).

Figure 6. Persistent neurobehavioral deficits in F0, F1 and F2 cohort

The inset (top right) illustrates the generation(s) discussed. F0 were evaluated in 1 neurobehavioral assay with 2 endpoints (nnd and iid) using 24 males and 24 females per treatment group. The N/A values denote assays that were not available at the time the F0 generation was tested. The cumulative distribution function (cdf) is illustrated for each lineage, with red text denoting statistically significant differences from the referent group (see Methods). The assays measured nearest neighbor distance (nnd), inter-individual distance (iid), the swimming velocity, and the preference for avoidance (% Time Near Predator). The gray line in each panel represents the vehicle lineage, with colored treatment lines indicating increases (right shift) or decreases (left shift) along the cumulative distribution for each assay.