Cerebral Vascular Toxicity after Developmental Exposure to Arsenic (As) and Lead (Pb) Mixtures

Abstract

Arsenic (As) and lead (Pb) are environmental pollutants found in common sites linked to similar adverse health effects. This study determined driving factors of neurotoxicity on the developing cerebral vasculature with As and Pb mixture exposures. Cerebral vascular toxicity was evaluated at mixture concentrations of As and Pb representing human exposures levels (10 or 100 parts per billion; ppb; µg/L) in developing zebrafish by assessing behavior, morphology, and gene expression. In the visual motor response assay, hyperactivity was observed in all three outcomes in dark phases in larvae with exposure (1-120 h post fertilization, hpf) to 10 ppb As, 10 ppb Pb, or 10 ppb mix treatment. Time spent moving exhibited hyperactivity in dark phases for 100 ppb As and 100 ppb mix treatment groups only. A decreased brain length and ratio of brain length to total length in the 10 ppb mix group was measured with no alterations in other treatment groups or other endpoints (i.e., total larval length, head length, or head width). Alternatively, measurements of cerebral vasculature in the midbrain and cerebellum uncovered decreased total vascularization at 72 hpf in all treatment groups in the mesencephalon and in all treatment groups, except the 100 ppb Pb and 10 ppb As groups, in the cerebellum. In addition, decreased sprouting and branching occurred in the mesencephalon, while only decreased branching was measured in the cerebellum. The 10 ppb Pb group showed several cerebral vasculature modifications that were aligned with a specific gene expression alteration pattern different from other treatment groups. Additionally, the 100 ppb As group drove gene alterations, along with several other endpoints, for changes observed in the 100 ppb mix treatment group. Perturbations assessed in this study displayed non-linear concentration-responses, which are important to consider in environmental health outcomes for As and Pb neurotoxicity.

Keywords: arsenic; behavior; cerebral vasculature; lead; metal; neurotoxicity; zebrafish.

Figure 1

Neurobehavioral endpoints after developmental exposure to As, Pb, or a mixture of both from 1 to 120 hpf. Zebrafish larvae behavior was measured using the visual motor response assay and distance traveled (A), velocity (B), and time spent moving (C) measured among the dark and light phases. n = 6 with 12 subsamples per treatment per replicate to total 72 fish per treatment group. Error bars represent standard deviation. * p < 0.05.

Figure 2

Alterations in morphology after developmental exposure to As, Pb, or a mixture of both from 1 to 120 hpf. A decrease in brain length in the 10 ppb mix treatment group was observed (A). No differences in total length were measured (B), but a decreased ratio of brain length to total length in the 10 ppb mix treatment group was also seen (C). n = 7 with 10 subsamples per treatment per replicate to total 70 fish per treatment group. Error bars represent standard deviation. * p < 0.05. Different letters indicate different statistical groupings in (A).

Figure 3

Decreases in mesencephalon (midbrain, MB) and cerebellum (hindbrain, HB) cerebral vasculature at 72 hpf after embryonic exposure to As, Pb, or a mixture of As and Pb. To evaluate cerebral vasculature development, several endpoints were assessed in the mesencephalon (midbrain, MB) including the number of sprouting blood vessels (A), the number of branches (B), and total vasculature (C). Similar measurements were ascertained in the cerebellum (hindbrain, HB) including number of sprouting blood vessels (D), the number of branches (E), and total vasculature (F). n = 5 with 10 subsamples per treatment per replicate for a total of 50 fish analyzed per treatment group. Error bars represent standard deviation. * p < 0.05.

Figure 4

Examples of decreases observed in the mesencephalon (midbrain) and cerebellum (hindbrain) vasculature at 72 hpf. A representative brain of a fish in the control treatment group (A), of the mesencephalon of a fish in the 100 ppb As treatment group (B), and of the cerebellum of a fish in the 10 ppb Pb treatment group (C). Scale bar is 100 µM.

Figure 5

Expression of cerebral vasculature developmental and functional genes at 72 hpf after embryonic exposure (1–72 hpf) to As, Pb, or a mixture of As and Pb. There was a significant decrease in relative expression of vegfaa in 100 ppb As and 100 ppb mix treatment groups (A). A significant increase in cldn5a (B) and wnt7aa (D) expression occurred in the 10 ppb Pb treatment group. All treatment groups had an increase in cldn5b expression (C). There was no change in expression of lrp1aa for any treatment group (E). n = 6 (pools of 48–50 fish from each treatment in each biological replicate). Error bars represent standard deviation, * p < 0.05.