Induction of cytochrome P450 1 genes and stress response genes in developing zebrafish exposed to ultraviolet radiation

Abstract

Ultraviolet (UV) radiation damages cell molecules, and has been suggested to up-regulate mammalian cytochrome P4501 (CYP1) genes through an aryl hydrocarbon receptor (AHR) mediated mechanism. In this study, embryos and larvae of zebrafish (Danio rerio) were exposed to UV to determine the effects on expression of CYP1 and stress response genes in vivo in these fish. Zebrafish embryos were exposed for varying times to UV on two consecutive days, with exposure beginning at 24 and 48h post-fertilization (hpf). Embryos exposed for 2, 4 or 6h twice over 2 days to UVB (0.62 W/m(2); 8.9-26.7 kJ/m(2)) plus UVA (2.05 W/m(2); 29.5-144.6 kJ/m(2)) had moderately (2.4+/-0.8-fold) but significantly up-regulated levels of CYP1A. UVA alone had no effect on CYP1A expression. Proliferating cellular nuclear antigen (PCNA) and Cu-Zn superoxide dismutase (SOD1) transcript levels were induced (2.1+/-0.2 and 2.3+/-0.5-fold, respectively) in embryos exposed to two 6-h pulses of 0.62 W/m(2) UVB (26.8 kJ/m(2)). CYP1A was induced also in embryos exposed to higher intensity UVB (0.93 W/m(2)) for two 3-h or two 4-h pulses (20.1 or 26.8 kJ/m(2)). CYP1B1, SOD1 and PCNA expression was induced by the two 3-h pulses of the higher intensity UVB, but not after two 4-h pulses of the higher intensity UVB, possibly due to impaired condition of surviving embryos, reflected in a mortality of 34% at that UVB dose. A single 8-h long exposure of zebrafish larvae (8dpf) to UVB at 0.93 W/m(2) (26.8 kJ/m(2)) significantly induced CYP1A and CYP1B1 expression, but other CYP1 genes (CYP1C1, CYP1C2 and CYP1D1) showed no significant increase. The results show that UVB can induce expression of CYP1 genes as well stress response genes in developing zebrafish, and that UVB intensity and duration influence the responses.

Figure 1

Levels of CYP1A gene expression after 4, 8 and 12 hours of cumulative UVA+ UVB radiation exposure over two days. (A) Embryos were exposed to 0.62 W/m² UVB and 2.05 W/m² UVA. Embryos in each biological replicate (n=4) were pooled and sampled at 72 hours post fertilization (arrows). (B) CYP1A gene expression. Error bars represent one standard deviation. Differences from control values were determined by one-way ANOVA followed by Dunnett’s- Post-hoc test (* p< 0.05, ** p< 0.01).

Figure 2

Levels of CYP1A, SOD1 and PCNA gene expression after 12h of cumulative UVB exposure. (A) Embryos were exposed to UVB at 0.62 W/m² and were sampled from each biological replicate immediately after a second pulse of UVB and 18 hours later, i.e., at 54 and 72 hours post fertilization (arrows), respectively (n=4). (B) Levels of CYP1A expression, (C) SOD1 expression, and (C) PCNA expression. Error bars represent one standard deviation. Differences from control values were determined by unpaired t-tests. (*p< 0.05, ** p< 0.01).

Figure 3

Levels of CYP1A, CYP1B1, PCNA, and SOD1 gene expression after UVB exposure. (A) Embryos were exposed to 0.93 W/m² for two periods during development. Embryos treated with 6 hours of UVB were sampled at 51 hours post fertilization and 8 hour treatments were sampled at 52 hpf (arrows). The embryos from each biological treatment were pooled (n=4). (B) Levels of CYP1A expression, (C) CYP1B1 expression, (C) PCNA expression, and (D) SOD1 expression. Error bars represent one standard deviation. Significant differences from control values were determined by one-way ANOVA followed by Dunnett’s- Post-hoc test (* p< 0.05, ** p< 0.01, *** p<0.001).

Figure 4

Levels of CYP1 gene expression in 8 day old zebrafish larvae after UVB exposure. Larvae were irradiated for 8 hours with 0.93 W/m² UVB, resulting in a cumulative dose of 26.8 kJ/m2 UVB. Sampling occurred directly after irradiation and each biological replicate contained eighteen larvae (n=6). Error bars represent one standard deviation. Significant differences from control values were determined by unpaired t-test (* p< 0.05, ** p< 0.01).