Abstract
Dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin) induces cleft palate and hydronephrosis in mice, when exposed in utero; these effects are mediated by the aryl hydrocarbon receptor. The Cyp1a1, Cyp1a2, and Cyp1b1 genes are up-regulated by the aryl hydrocarbon receptor. To elucidate their roles in dioxin-induced teratogenesis, we compared Cyp1a1(-/-), Cyp1a2(-/-), and Cyp1b1(-/-) knock-out mice with Cyp1(+/+) wild-type mice. Dioxin was administered (25 microg/kg, gavage) on gestational day 10, and embryos were examined on gestational day 18. The incidence of cleft palate and hydronephrosis was not significantly different in fetuses from Cyp1a1(-/-), Cyp1b1(-/-), and Cyp1(+/+) wild-type mice. To fetuses carried by Cyp1a2(-/-) dams, however, this dose of dioxin was lethal; this effect was absolutely dependent on the maternal Cyp1a2 genotype and independent of the embryonic Cyp1a2 genotype. Dioxin levels were highest in adipose tissue, mammary gland, and circulating blood of Cyp1a2(-/-) mothers, compared with that in the Cyp1(+/+) mothers, who showed highest dioxin levels in liver. More dioxin reached the embryos from Cyp1a2(-/-) dams, compared with that from Cyp1(+/+) dams. Fetuses from Cyp1a2(-/-) dams exhibited a approximately 6-fold increased sensitivity to cleft palate, hydronephrosis, and lethality. Using the humanized hCYP1A1_1A2 transgenic mouse (expressing the human CYP1A1 and CYP1A2 genes in the absence of mouse Cyp1a2 gene), the teratogenic effects of dioxin reverted to the wild-type phenotype. These data indicate that maternal mouse hepatic CYP1A2, by sequestering dioxin and thus altering the pharmacokinetics, protects the embryos from toxicity and birth defects; substitution of the human CYP1A2 trans-gene provides the same protection. In contrast, neither CYP1A1 nor CYP1B1 appears to play a role in dioxin-mediated teratogenesis.
Highlights
Suppression [5], wasting syndrome [6, 7], and tumor promotion (8 –10)
All three Cyp1 knock-out lines have been bred into the C57BL6/J (B6) inbred mouse strain (Ͼ99.9%) background; B6 inbred mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and used as the wild-type Cyp1(ϩ/ϩ) control
Many studies have suggested that CYP1 metabolism is required for mediating TCDD-induced pathology: TCDD is the prototype of several environmentally ubiquitous planar teratogenic, carcinogenic, and toxic polyhalogenated aromatic hydrocarbon (PHAH) contaminants
Summary
Chemicals—TCDD was purchased from Accustandard (New Haven, CT). [3H]TCDD (10 Ci/mmol) was a gift from Alvaro Puga (Cincinnati, OH). TCDD-pretreated mice were housed separately, and their carcasses were treated as contaminated biological materials. Real-time PCR Analysis—RT-PCR to quantify CYP1A1, CYP1A2, and CYP1B1 mRNA, was conducted on total RNA isolated from liver of corn oil- or TCDD-pretreated pregnant dams of the Cyp1(ϩ/ϩ), Cyp1a1(Ϫ/Ϫ), Cyp1a2(Ϫ/Ϫ), and Cyp1b1(Ϫ/Ϫ) genotypes. Data were normalized to RT-PCR detection for -actin, and the accumulation of -actin mRNA did not differ significantly between untreated and treated liver. Tissue Distribution of TCDD—The amount of [3H]TCDD (10 Ci/mmol) used to spike the usual dose (TCDD, 25 g/kg) was 20 Ci. For the Cyp1(ϩ/ϩ) and Cyp1a2(Ϫ/Ϫ) genotypes, three pregnant female mice of each were given an intraperitoneal injection of the spiked TCDD on GD15. The fetuses, placentas, and maternal liver, thymus, kidney, spleen, heart, brain, adipose tissue, mammary gland, and lung were weighed and added to hyamine hydroxide (1 ml/100 mg) in glass scintillation vials.
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