Embryonic exposures to cadmium and PAHs cause long-term and interacting neurobehavioral effects in zebrafish

Abstract

Developmental exposure to either polycyclic aromatic hydrocarbons (PAHs) or heavy metals has been shown to cause persisting and overlapping neurobehavioral effects in animal models. However, interactions between these compounds have not been well characterized, despite their co-occurrence in a variety of environmental media. In two companion studies, we examined the effects of developmental exposure to cadmium (Cd) with or without co-exposure to prototypic PAHs benzo[a]pyrene (BaP, Exp. 1) or fluoranthene (FA, Exp. 2) using a developing zebrafish model. Zebrafish embryos were exposed to Cd (0-0.3 μM), BaP (0-3 μM), FA (0-1.0 μM), or binary Cd-PAH mixtures from 5 to 122 h post fertilization (hpf). In Exp. 1, Cd and BaP produced independent effects on an array of outcomes and interacting effects on specific outcomes. Notably, Cd-induced deficits in dark-induced locomotor stimulation were attenuated by BaP co-exposure in the larval motility test and BaP-induced hyperactivity was attenuated by Cd co-exposure in the adolescent novel tank test. Likewise, in Exp. 2, Cd and FA produced both independent and interacting effects. FA-induced increases on adult post-tap activity in the tap startle test were attenuated by co-exposure with Cd. On the predator avoidance test, FA- and 0.3 μM Cd-induced hyperactivity effects were attenuated by their co-exposure. Taken together, these data indicate that while the effects of Cd and these representative PAHs on zebrafish behavior were largely independent of one another, binary mixtures can produce sub-additive effects for some neurobehavioral outcomes and at certain ages. This research emphasizes the need for detailed risk assessments of mixtures containing contaminants of differing classes, and for clarity on the mechanisms which allow cross-class toxicant interactions to occur.

Keywords: Behavior; Benzo[a]pyrene; Cadmium; Developmental; Fluoranthene; Neurobehavioral; Zebrafish.

Figure 1:. Experiment 1: Larval and Juvenile Effects of Cd and BaP.

Motility testing measured locomotor activity as distance moved (cm/10 min), and time mobile (sec/10 min). (A) BaP treatment reduced distance moved in larvae regardless of Cd exposure. (B) In the light (left), BaP reduced time mobile in larvae, but only within 0.1 and 0.3μM Cd-exposed groups. In the dark (right), BaP, 0.1μM CD and 0.3μM Cd reduced time mobile in larvae compared to controls. Co-exposure had no additional effect. Dark-induced stimulation (right) was reduced in larvae by 0.1 and 0.3μM CD. BaP co-exposure attenuated the effect of 0.3μM Cd. (C) 0.3μM Cd treatment reduced juvenile distance moved relative to non-Cd and 0.1μM Cd treated groups, regardless of BAP exposure. (D) BaP treatment reduced juvenile distance moved under lit (left) but not dark (right) conditions. (E) 0.3μM Cd treatment reduced juvenile time mobile relative to non-CD and 0.1μM Cd treated groups, regardless of BaP exposure. Data expressed as mean ± SEM. Asterisk (*) indicates significance at p < 0.05. Ns = Larval 67–72/group; Juvenile 25–48/group.

Figure 2:. Experiment 1: Adolescent and Adult Effects of Cd and BaP.

Figures represent results of the adolescent and adult test batteries. (A) BaP treatment increased locomotor activity (cm/min) in the adolescent novel tank, but co-exposure to 0.1μM Cd attenuated this effect. (B) BaP reduced baseline (pre-tap) activity (cm/5 sec) in the adolescent tap startle test, regardless of CD exposure. (C) BaP impaired the diving response (distance to bottom in cm) in the adult novel tank, regardless of Cd exposure. (D) 0.3μM Cd treatment increased baseline (pre-tap) activity (cm/5 sec) in the adult tap startle test relative to non-Cd and 0.1μM Cd treated groups, regardless of BaP exposure. (E) BaP impaired the startle response (change in activity, cm/5 sec) in the adult tap startle test, regardless of Cd exposure. (F) BAP increased locomotor activity (cm/min) in the adult shoaling test, regardless of Cd exposure. (G) 0.3μM Cd enhanced fleeing responses (change in position, in cm) to the slow predator cue (left) in the adult predator avoidance test regardless of BaP exposure. (H) BaP reduced the average distance from the screen (in cm) in the adult predator avoidance test, regardless of Cd exposure. Data expressed as mean ± SEM. Asterisk (*) indicates significance at p < 0.05. Ns = Adolescent 19–31/group; Adult 22–36/group.

Figure 3:. Experiment 2: Larval Effects of Cd and FA.

Locomotor activity was measured as distance moved (cm/10 min), and time mobile (sec/10 min). (A) FA treatment reduced distance moved under both lit (left) and dark (right) conditions, regardless of Cd exposure. 0.1μM Cd treatment increased distance moved in larvae in the dark (right), but not the light (left), regardless of FA exposure. (B) In the light (left), FA reduced time mobile in larvae under both lit (left) and dark (right) conditions, regardless of Cd exposure. Data expressed as mean ± SEM. Asterisk (*) indicates significance at p < 0.05. Ns = Larval 38–44/group.

Figure 4:. Experiment 2: Adolescent and Adult Effects of Cd and FA.

Figures represent results of the adolescent and adult test batteries. (A) FA treatment impaired the adolescent diving response (distance to bottom in cm) in the novel tank diving test, regardless of Cd exposure. (B) FA exposure reduced baseline (pre-tap) activity (cm/5 sec) (left) and post-tap activity (right) in the adolescent tap startle test, regardless of Cd exposure. (C) 0.1μM Cd enhanced baseline (pre-tap) activity (cm/5 sec) (left) in the adult tap startle test when administered alone, but not in a Cd mixture. FA treatment enhanced post-tap activity (right) in the adult tap startle test, but co-exposure to 0.1μM Cd attenuated this effect. (D) FA exposure increased startle magnitude (change in activity, cm/5/sec) in the adult tap startle test, regardless of Cd exposure. (E) FA exposure reduced locomotor activity (cm) in the adult shoaling test, regardless of Cd exposure. (F) FA and 0.3μM Cd each increased locomotor activity (cm) in the adult predator avoidance test relative to controls. The FA effect was attenuated by co-exposure to either 0.1μM or 0.3μM Cd. The 0.3μM Cd effect was attenuated by co-exposure with FA. Data expressed as mean ± SEM. Asterisk (*) indicates significance at p < 0.05. Ns = Adolescent 28–35; Adult 25–28/group.