흰쥐의 수태 및 이유기간 중 TCDD의 노출에 의한 Cytochrome P450 동위효소의 발현양상에 관한 분자생물학적 연구
Molecular Biological Study on the Expression of Cytochrome P450 Isoforms in the Offspring Rats Exposed to 2,3,7,8-Tetrachlorodibenzo-p-dioxin Throughout Pregnancy and Lactation
흰쥐 수태 이유기간 TCDD Cytochrome P450 동위효소 발현양상 분자생물학;
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We investigated the effects of gestational and lactational exposures to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the differential induction of CYP1A1 in the levels of protein and gene expression in the liver and brain regions of offspring rats. For this study, pregnant Sprague Dawley rats were orally exposed to TCDD (1 or 10 ng/kg body weight/day) starting at Day 1 of gestation up to Day 20 of postpartum. The activity of ethoxyresorufin O-deethylase (EROD), induction of CYP1A1, and expression of CYP1A1 mRNA were measured in the liver and brain of fetuses and dams from Day 20 of gestation, of male and female neonates from Day 1 of postpartum, and of lactational offsprings 20 days after birth. EROD activity in the liver microsomes of fetal, postnatal, and lactational group increased 0, 5, and 15-fold compared to control group, respectively. Results of immunoblot analysis showed that CYP1A1 was strongly induced in the liver of lactational group, but not in the fetal group. These results suggest, even though fetuses are somewhat protected from the TCDD exposure by the placenta, that TCDD exposed to dams is directly transferred to their offsprings via lactation. However, there were not detectable in the activity of EROD and induction of CYP1A1 in the brains of all groups. Induction of CYP1A1 mRNA expression, quantitatively analyzed by the competitive reverse transcription-polymerase chain reaction (QC RT-PCR) method was detectable at extremely low amounts in the liver of control fetuses, neonates, and lactational offsprings. The mRNA in the livers of offsprings after birth from dams pre-exposed to TCDD was age-dependently increased. Expressed level of mRNA was 1, 5, and 10 pg/3㎍ total RNA in the liver of fetal, postnatal, and lactational offsprings, respectively. Induction of CYP1A1 mRNA expression in the brains of offspring rats by TCDD exposure was also detectable, but at extremely low levels compared to those in the livers of corresponding groups. Ethidium bromide-stained gel, in which the amplified mRNA bands were electrophoretically separated, showed that RT-PCR product for CYP1A1 mRNA in the brain of lactational offspring rats exposed to TCDD throughout pregnancy and lactation was 1161 bp, which was 462 bp longer in size than in the liver of offspring rats. DNA sequencing analysis unraveled this difference, i.e., RT-PCR product for CYP1A1 in the brain of offspring rat includes 4 introns, suggesting incomplete splicing out by TCDD by unknown mechanism(s), which remains for further study. The incomplete splicing out of introns in the CYP1A1 cDNA transcript in the brain of lactational offspring rats pre-exposed to TCDD throughout gestation might be related to the no response of EROD activity in the same tissue.