Circadian clock regulates response to pesticides in Drosophila via conserved Pdp1 pathway

Abstract

Daily rhythms generated by the circadian clock regulate many life functions, including responses to xenobiotic compounds. In Drosophila melanogaster, the circadian clock consists of positive elements encoded by cycle (cyc) and Clock (Clk) and negative elements encoded by period (per) and timeless (tim) genes. The epsilon-isoform of the PAR-domain protein 1 (Pdp1epsilon) transcription factor is controlled by positive clock elements and regulates daily locomotor activity rhythms. Pdp1 target genes have not been identified, and its involvement in other clock output pathways is not known. Mammalian orthologs of Pdp1 have been implicated in the regulation of xenobiotic metabolism; therefore, we asked whether Pdp1 has a similar role in the fly. Using pesticides as model toxicants, we determined that disruption of Pdp1epsilon increased pesticide-induced mortality in flies. Flies deficient for cyc also showed increased mortality, while disruption of per and tim had no effect. Day/night and Pdp1-dependent differences in the expression of xenobiotic-metabolizing enzymes Cyp6a2, Cyp6g1, and alpha-Esterase-7 were observed and likely contribute to impaired detoxification. DHR96, a homolog of constitutive androstane receptor and pregnane X receptor, is involved in pesticide response, and DHR96 expression decreased when Pdp1 was suppressed. Taken together, our data uncover a pathway from the positive arm of the circadian clock through Pdp1 to detoxification effector genes, demonstrating a conserved role of the circadian system in modulating xenobiotic toxicity.

FIG. 1.

Disruption of Pdp1 or cyc expression results in increased sensitivity to pesticides. Flies were exposed to permethrin or malathion at various concentrations and data represent the average LC₅₀ (+ SEM) for each mutant and their matched control flies in three independent experiments unless otherwise noted. (A) Data represent five independent experiments for control flies, six for Pdp1i_a flies, and three for Pdp1i_b. (D) Four independent experiments were completed for cyc⁰¹ and control flies. Statistical significance was determined by a one-way ANOVA and a Tukey's multiple comparison post-test for (A) and unpaired t-test for (B–F) where *p < 0.05, **p < 0.01, and ***p < 0.001. See also Supplementary figure 1 and Supplementary table 1.

FIG. 2.

Pdp1 mRNA expression in cyc and tim mutants. Pdp1ϵ (left) and total Pdp1 (right) mRNA levels are lower in (A) cyc⁰¹ flies with increased susceptibility to permethrin but not in (B) tim⁰¹ flies that do not show this sensitivity. Data represent mean mRNA levels + SEM for three independent experiments. Significant difference from control ZT 4 values, where values **p < 0.01 and ***p < 0.001, as calculated by ANOVA and Tukey's multiple comparison post-test.

FIG. 3.

Influence of Pdp1 on DHR96 expression. (A–B) DHR96 mRNA expression levels in Pdp1 RNAi, Pdp1³¹³⁵, and their controls (control or iso¹³¹ flies, respectively) at ZT 4. Data represent mean relative expression (+ SEM) of three independent experiments. (C) Permethrin susceptibility is increased in the DHR96 mutant as compared to its respective control (CS^D). Data are average LC₅₀ (+ SEM) for four independent experiments. ANOVA and Tukey’s multiple comparison post-test (A) or an unpaired t-test (B–C) were used to determine statistical significance from control samples where *p < 0.05 and **p < 0.01.

FIG. 4.

Pdp1 regulates components of xenobiotic metabolism. (A–C) Time of day and Pdp1-dependent Cyp6g1, Cyp6a2, and α-Esterase-7 mRNA expression levels in control and Pdp1 RNAi flies. Data represent mean relative expression (+ SEM) for three independent experiments. ANOVA and Tukey's multiple comparison post-test were used to determine statistical significance from control ZT 4 samples where *p < 0.05, **p < 0.01, and ***p < 0.001. (D) Flies with disrupted Pdp1 expression have significantly lower general esterase activity for α-naphthyl acetate. Significant difference from control ZT 4 was determined by an ANOVA and Bonferonni post-test, p < 0.05. See also Supplementary figure 2.

FIG. 5.

The positive arm of the circadian clock regulates xenobiotic-metabolizing genes and the response of flies to a toxic exposure.