Imprinting of cerebral cytochrome P450s in offsprings prenatally exposed to cypermethrin augments toxicity on rechallenge

Abstract

Epigenetic studies were carried in the rat offsprings, born to dams treated with cypermethrin (orally; 5.0 mg/kg) from gestation day (GD) 5 to 21 and rechallenged with cypermethrin (orally; 10 mg/kg for 6 days), at adulthood (12 weeks) to understand the mechanism underlying the overexpression of cerebral cytochrome P450s (CYPs) in exposed offsprings. The data revealed alterations in histone H3 acetylation and DNA methylation in promoter regions of CYP1A- and 2B- isoenzymes in the brain isolated from rechallenged animals. Further, bisulphite sequencing revealed critical CpG methylation changes in BARBIE BOX (Barbiturate response element) and BTE (Basal transcription element) in promoter of CYP2B1 in the brain isolated from rechallenged animals. Western blotting and DNA laddering/fragmentation studies revealed a greater magnitude of increase in the signalling pathways associated with apoptosis in the rechallenged animals. The data have indicated that overexpression of cerebral CYPs could be due to the imprinting of CYPs. Further, increased apoptosis observed in the rechallenged offsprings has suggested that these epigenetic changes in CYPs may predispose the prenatally exposed offsprings to the neurotoxic effects of other centrally acting drugs and chemicals when subsequently rechallenged later at life.

Figure 1. Histone H3 acetylation ChIP and Methylated DNA immunoprecipitation assay for analysis of acetylation and methylation in the promoters of CYP1A- and 2B- genes in cypermethrin exposed offsprings.

(a) Histone H3 acetylation ChIP assay. a - compared to control group; b - compared to prenatally exposed offsprings group; c - compared to offsprings exposed postnatally with cypermethrin. (b) Methylated DNA immunoprecipitation assay. a - compared to control group; b - compared to prenatally exposed offsprings group; c - compared to offsprings exposed postnatally with cypermethrin. All the values represent mean ± S.E.M. of three experiments in each set (n = 3). Significant difference have been considered up to *p < 0.05, **p < 0.01 and ***p < 0.001.

Figure 2. MSP, UMSP analysis and Bisulphite sequencing of CYP1A1 and CYP2B1.

(a) Analysis of CYP1A1 promoter methylation through methylation specific-PCR inbrain of exposed offsprings. Bisulphite converted DNA was used for PCR with primers specific for both M (methylated CpG) and U (un-methylated CpG). (b) Densitometric analysis of UMSP (un-methylated DNA specific PCR) for CYP1A1 promoter in the brain samples. a - compared to control group; b - compared to prenatally exposed offsprings group; c - compared to offsprings exposed postnatally with cypermethrin. All the values represent mean × 10⁻⁴ ± S. E. M. × 10⁻⁴ of three experiments in each group. Significant difference have been considered up to *p < 0.05, **p < 0.01 and ***p < 0.001. (c) CYP2B1 promoter methylation in the brain of offspring which were either exposed only at adulthood (Postnatal) or exposed both during gestation period and at adulthood (Rechallenged) with cypermethrin. Bisulphite converted DNA was sequenced with promoter specific primers and analyzed using BIQ analyzer. The differentially methylated CGs is highlighted. (d) CYP2B1 proximal promoter sequence showing regulatory elements which could be affected by CG demethylation (dotted box). The CG elements showing demethylation in rechallenged group are highlighted. Single C in bold is the transcription start site of CYP2B1 promoter. Sites for barbiturate responsive element (BARBIE box), basal transcription element (BTE) binding and CCAAT-enhancer-binding proteins (C/EBP) are depicted.

Figure 3. Western blots of anti- and pro-apoptoic proteins and DNA laddering/fragmentation.

(a) Western blots of total protein isolated from brain of offsprings with anti-bcl2/bax/bad/caspase9/p53. Lane 1 contains protein from brain (50 μg) of rat offsprings raised on control mothers. Lane 2 contains protein prepared from brain (50 μg) of rat offsprings exposed prenatally to 5 mg/kg of cypermethrin. Lane 3 contains protein prepared from brain (50 μg) of rat offsprings exposed postnatally to 10 mg/kg of cypermethrin. Lane 4 contains protein prepared from brain (50 μg) of rat offsprings exposed prenatally to 5 mg/kg of cypermethrin and subsequently rechallenged with cypermethrin (10 mg/kg) at adulthood. (b) Representative image of DNA laddering/fragmentation.