Toxicological Sciences, Vol 47, 76-85, Copyright © 1999 by Society of Toxicology
BD Abbott, JE Schmid, JG Brown, CR Wood, RD White, AR Buckalew and GA Held
C57BL/6N mouse embryos exposed to hydrocortisone (HC) or 2,3,7,8-
tetrachlorodibenzo-p-dioxin (TCDD) develop cleft palate. An interaction
between these agents produces clefts at doses which alone are not
teratogenic. The glucocorticoid 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 hybridization. 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 micrograms/kg), HC (100 mg/kg) or HC (25 mg/kg) + TCDD (3
micrograms/kg). The induction of CYP1A1 mRNA was also quantified at 2, 4,
6, 12, 24, and 48 h for control and TCDD-exposed samples. 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 synthesized, which included
the gene's primer sequences separated by a pUC19 plasmid sequence. Reverse
transcription-polymerase chain reaction (RT-PCR) was performed 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 samples at
all time points examined (p = 0.005 at 2 h and 0.001 after 2 h). During
palatal shelf outgrowth on GD12, AhR mRNA levels increased significantly
and this was not affected by treatment with TCDD or HC + TCDD. A
significant 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); however, 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 with controls (p < 0.001). The relative overall
expression level of the genes was AhR > ARNT > GR. Within
individuals, expression of AhR and/or ARNT was 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 not on the same schedule. The
TCDD-induced decrease in ARNT at 24 h after dosing and the HC and HC +
TCDD-induced delay in upregulation of ARNT may affect the dynamics of
heterodimer formation between AhR and ARNT. The changes in ARNT mRNA level
could also affect availability of this transcriptional regulator to
interact with other potential partners, and these effects, separately or in
combination, may be involved in disruption of normal embryonic development.
ARTICLES
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
Reproductive Toxicology Division, National Health Effects and Environmental Research Laboratory, Environmental Protection Agency, Research Triangle Park, North Carolina 27711, USA.
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