In vivo assessment of respiratory burst inhibition by xenobiotic exposure using larval zebrafish

Abstract

Currently, assessment of the potential immunotoxicity of a given agent involves a tiered approach for hazard identification and mechanistic studies, including observational studies, evaluation of immune function, and measurement of susceptibility to infectious and neoplastic diseases. These studies generally use costly low-throughput mammalian models. Zebrafish, however, offer an excellent alternative due to their rapid development, ease of maintenance, and homology to mammalian immune system function and development. Larval zebrafish also are a convenient model to study the innate immune system with no interference from the adaptive immune system. In this study, a respiratory burst assay (RBA) was utilized to measure reactive oxygen species (ROS) production after developmental xenobiotic exposure. Embryos were exposed to non-teratogenic doses of chemicals and at 96 h post-fertilization, the ability to produce ROS was measured. Using the RBA, 12 compounds with varying immune-suppressive properties were screened. Seven compounds neither suppressed nor enhanced the respiratory burst; five reproducibly suppressed global ROS production, but with varying potencies: benzo[a]pyrene, 17β-estradiol, lead acetate, methoxychlor, and phenanthrene. These five compounds have all previously been reported as immunosuppressive in mammalian innate immunity assays. To evaluate whether the suppression of ROS by these compounds was a result of decreased immune cell numbers, flow cytometry with transgenic zebrafish larvae was used to count the numbers of neutrophils and macrophages after chemical exposure. With this assay, benzo[a]pyrene was found to be the only chemical that induced a change in the number of immune cells by increasing macrophage but not neutrophil numbers. Taken together, this work demonstrates the utility of zebrafish larvae as a vertebrate model for identifying compounds that impact innate immune function at non-teratogenic levels and validates measuring ROS production and phagocyte numbers as metrics for monitoring how xenobiotic exposure alters the innate immune system.

Keywords: Chemical screen; endocrine disrupting compounds (EDC); high throughput; lead; phagocyte; polycyclic aromatic hydrocarbons (PAH); reactive oxygen species (ROS).

Figure 1.. Chemicals for zebrafish exposures.

Names and structures of chemicals used for zebrafish exposures. Chemicals were provided by (1) Battelle (Columbus, OH) or (2) MRI Global (Kansas City, MO). Chemical structures were generated with ChemDraw Professional (v16.0.1.4) using International Union of Pure and Applied Chemistry (IUPAC) nomenclature.

Figure 2.. Developmental toxicity of 12 structurally-different xenobiotics.

(A) Experimental design. Zebrafish embryos were exposed to several concentrations of xenobiotics from 6 hpf to 96 hpf and monitored for gross malformation and death. (B) Summary graphic of all twelve chemicals tested at all concentrations. Six embryos were exposed to each condition. Pie charts indicate the percentage of embryos that, by 96 hpf, were dead (red), appeared malformed (abnormal, blue), or appeared normal (green). The highest concentrations for each chemical at which no adverse effects were observed (no-observed-effect level, or NOEL) are indicated by asterisks and employed as the highest doses for respiratory burst experiments in Figure 3.

Figure 3.. ROS production in whole zebrafish larvae.

Zebrafish embryos were exposed to several concentrations of xenobiotics from 6 hpf to 96 hpf. At 96 hpf, larvae were plated into a 96 well plate, and ROS production was measured via H₂DCFDA after stimulation with PMA. Maximum fluorescence of each well was used for all analyses; results of two replicate experiments are reported. (A) Acenaphthenequinone, (B) azathioprine, (C) benzo[a[pyrene, (D) dexamethasone, (E) dichloroacetic acid, (F) 17-β-estradiol, (G) hydroquinone, (H) lead (II) acetate trihydrate, (I) methoxychlor, (J) phenanthrene, (K) tributyltin oxide and (L) trichloroethylene. Bis I (10 μM; selective protein kinase C inhibitor) was used as a positive control. Significance (*p < 0.05) was determined by a one-way ANOVA with Dunnett’s post-hoc test for pairwise comparisons to the DMSO control. Red data points denote significance for tested chemicals.

Figure 4.. Summary of zebrafish larval RBA results.

Data provided in Figure 3 are summarized to highlight the range of chemical doses employed in, and doses that suppressed the zebrafish RBA. Each circle indicates the dose (above) used for each chemical (left) in the RBA assay. Combinations that led to significant reproducible suppression of the zebrafish RBA are indicated by red circles with asterisks.

Figure 5.. Benzo[a]pyrene alters macrophage number, but not neutrophil number, in whole zebrafish larvae.

Tg(mpx:GFP) or Tg(mfap4:tdTomato-caax) transgenic zebrafish embryos were exposed to highest dose of one of the five compounds that suppressed ROS production in the RBA (8.19 μM benzo[a]pyrene, 839 nM 17-β-estradiol, 80 μM lead (II) acetate trihydrate, 268 nM methoxychlor, 2.62 μM phenanthrene), or exposed to DMSO from ~6 hpf to 96 hpf. At 96 hpf, larvae were mechanically homogenized into a single cell suspension and the numbers of GFP⁺ and tdTomato⁺ cells were quantified via flow cytometry. Wild-type unexposed larvae were included in all experiments in order to measure baseline autofluorescence. Data are presented as percentage of (A) GFP⁺ neutrophils or (B) tdTomato⁺ macrophages observed in individual experiments and include at least three biological replicates per compound. Significance (*p < 0.05) was determined by one-way ANOVA with a Dunnett’s post-hoc test for pairwise compare-sons to the DMSO control. Flow cytometry gating methods are outlined in Supplemental Figure S1.