Glutathione transferase pi class 2 (GSTp2) protects against the cardiac deformities caused by exposure to PAHs but not PCB-126 in zebrafish embryos

Abstract

Glutathione transferases (GSTs) are phase II enzymes that detoxify a wide range of toxicants and reactive intermediates. One such class of toxicants is the ubiquitous polycyclic aromatic hydrocarbons (PAHs). Certain PAHs are known to cause developmental cardiac toxicity in fish. Herein, we explored the role of GST pi class 2 (GSTp2) in PAH- and PCB-induced cardiac toxicity in zebrafish (Danio rerio) embryos. We measured expression of GSTp2 in embryos exposed to individual and co-exposures of the PAHs benzo[k]fluoranthene (BkF), benzo[a]pyrene (BaP), and fluoranthene (FL) as well as 3,3',4,4',5-pentachlorobiphenyl (PCB-126). GSTp2 mRNA expression was induced by exposure to BkF, BaP, PCB-126, and BaP+FL and BkF+FL co-exposure. A splice junction morpholino was then used to knockdown GSTp2 in developing zebrafish. GSTp2 knockdown exacerbated the toxicity caused by co-exposures to BkF+FL and BaP+FL. However, GSTp2 knockdown did not affect PCB-126 toxicity. These results further suggest that pi class GSTs serve a protective function against the synergistic toxicity caused by PAHs in developing zebrafish.

Fig. 1

Effect of PAHs and PCB-126 on GSTp2 gene expression. A) 10 mg/L BkF and 200 mg/L FL; B) 100 mg/L BaP and 500 mg/L FL; and C) 1 mg/L PCB-126. Expression is shown as fold induction compared to DMSO controls. n = 9 per treatment; each n represents 10 pooled embryos. Groups not sharing a common letter are significantly different (p ≤ 0.05; ANOVA, Tukey’s post-hoc test).

Fig. 2

Effective knockdown of GSTp2 via splice junction morpholino in embryos exposed to DMSO or 10 mg/L BkF + 200 mg/L FL at 24 hpf. Representative image is from RT-PCR amplification of 10 pooled embryos per each treatment. Ld = 25 bp DNA ladder (lanes 1 and 10). NI embryos exposed to BkF + FL show strong amplification of the GSTp2 exon 1 –3 cDNA fragment (lanes 6 and 7). BkF + FL-exposed GSTp2-mo embryos exhibit weaker amplification of the GSTp2 exon 1–3 cDNA fragment and exhibit weak amplification of a smaller band, indicating deletion of exon 2 by the GSTp2-mo (8 and 9).

Fig. 3

PAH-induced deformities in NI and GSTp2-mo injected embryos. A) 100 mg/L BkF and 100 mg/L FL; B) 10 mg/L BkF and 200 mg/L FL; C) 100 mg/L BaP and 500 mg/L FL. Embryos were dosed at 24 hpf and scored at 96 hpf. NI embryos are represented by black bars, 100 mM GSTp2-mo embryos are represented by grey bars, and 250 mM GSTp2-mo embryos are represented by white bars. Deformities are expressed as percent NI control (DMSO) pericardial effusion ± SEM (n = 12 per treatment; each n represents the average of five embryos). Groups not sharing a common letter are significantly different (p ≤ 0.05; ANOVA, Tukey adjusted LSMeans).

Fig. 4

PCB-126-induced deformities in NI and GSTp2-mo injected embryos. Embryos were dosed with DMSO, 1 mg/L PCB-126, or 2 mg/L PCB-126 at 24 hpf and scored at 96 hpf. NI embryos are represented by black bars, 100 mM GSTp2-mo embryos are represented by grey bars, and 250 mM GSTp2-mo embryos are represented by white bars. Deformities are expressed as percent NI control (DMSO) pericardial effusion ± SEM (n = 12 per treatment; each n represents the average of five embryos). Groups not sharing a common letter are significantly different (p ≤ 0.05; ANOVA, Tukey adjusted LSMeans).