RT-PCR quantification of AHR, ARNT, GR, and CYP1A1 mRNA in craniofacial tissues of embryonic mice exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin and hydrocortisone

C57BL/6N mouse embryos exposed to hydrocortisone (HC) or 2,3,7,8-tetrachlor odibenzo-p-dioxin (TCDD) develop cleft palate. An interaction between these agents produces clefts at doses which alone are not teratogenic. The gluco corticoid receptor (GR) and dioxin receptor (AhR) mediated these responses and their gene expression was altered by TCDD and/or HC in palates examined on gestation day (GD) 14 by Northern blot analysis and in situ hybridizati on. The present study quantifies AhR, AhR nuclear translocator (ARNT), and GR mRNA at 4, 12, 24, and 48 h after exposure (time 0 = dose administration at 8 A.M. On gestation day 12) on GD12 to TCDD (24 mu g/kg), HC (100 mg/kg ) or HC (25 mg/kg) + TCDD (3 mu g/kg). The induction of CYP1A1 mRNA was als o quantified at 2, 4, 6, 12, 24, and 48 h for control and TCDD-exposed samp les. Total RNA was prepared from midfacial tissue of 4-6 embryos/litter at each time and dose. An RNA internal standard (IS) for each gene was synthes ized, which included the gene's primer sequences separated by a pUC19 plasm id sequence. Reverse transcription-polymerase chain reaction (RT-PCR) was p erformed on total RNA + IS using a range of 5-7 IS concentrations across a constant level of total RNA. PCR products were separated in gels (mRNA and IS-amplified sequences differed by 30-50 bases), ethidium bromide-stained, imaged (Hamamatsu Photonics Systems, Bridgewater, NJ), and quantified with NIH Image. CYP1A1 mRNA was significantly induced in the TCDD-exposed sample s at all time points examined (p = 0.005 at 2 h and 0.001 after 2 h). Durin g palatal shelf outgrowth on GD12, AhR mRNA levels increased significantly and this was not affected by treatment with TCDD or HC + TCDD. A significan t increase in GR was detected at 24 h (p < 0.05) and this was unaffected by any of the exposures. Expression of ARNT increased at 12 h (p < 0.001); ho wever, treatment with HC or HC + TCDD blocked this increase (p < 0.05). At 24 h, the TCDD-treated embryos had significantly lower ARNT mRNA compared w ith controls (p < 0.001). The relative overall expression level of the gene s was AhR > ARNT > GR. Within individuals, expression of AhR and/or ARNT wa s highly correlated with GR level. In conclusion, CYP1A1 mRNA was expressed in developing craniofacial tissue and was highly induced by TCDD exposure. AhR, ARNT, and GR mRNA are upregulated in early palatogenesis, although no t on the same schedule. The TCDD-induced decrease in ARNT at 24 h after dos ing and the HC and HC + TCDD-induced delay in upregulation of ARNT may affe ct the dynamics of heterodimer formation between AhR and ARNT. The changes in ARNT mRNA level could also affect availability of this transcriptional r egulator to interact with other potential partners, and these effects, sepa rately or in combination, may be involved in disruption of normal embryonic development.