AhR, ARNT, and CYP1A1 mRNA quantitation in cultured human embryonic palates exposed to TCDD and comparison with mouse palate in vivo and in culture.
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is developmentally toxic in many species and induces cleft palate in the C57BL/6N mouse embryo. Palatogenesis in mouse and human embryos involves homologous processes at the morphological, cellular, and molecular levels. In organ culture, mouse and human palates respond similarly to TCDD. The present study quantitates the expression of AhR, ARNT, and CYP1A1 mRNA in human embryonic palates in organ culture. Palatal tissues were exposed to 1 x 10(-10), 1 x 10(-9), or 1 x 10(-8) M TCDD or control medium and sampled at 0, 2, 4, and 6 hours for quantitative RT-PCR using a synthetic RNA internal standard. Similar measurements of CYP1A1 gene expression were collected for mouse palates cultured in this model. In human palates, AhR expression correlated with ARNT and CYP1A1 mRNA expression. TCDD induction of CYP1A1 was time- and concentration-dependent. The expression of these genes presented a uniform and continuous distribution across the group of embryos, with no subset of either high or low expressors/responders. The ratio of AhR to ARNT was approximately 4:1. AhR mRNA increased during the culture period in both treated and control subjects; however, ARNT expression was relatively constant. TCDD did not alter either AhR or ARNT expression in a consistent dose- or time-related manner. Comparison of human and mouse data showed a high correlation across species for the induction of CYP1A1. Human embryos expressed approximately 350 times less AhR mRNA than the mouse, and in earlier studies it was shown that human palates required 200 times more TCDD to produce the same effects. When the morphological, cellular, and molecular responses to TCDD between mouse and human are compared, it seems highly unlikely that human embryos could be exposed to sufficient TCDD to achieve changes in palatal differentiation that would lead to cleft palate.
Reproductive Toxicology Division, National Health Effects and Environmental Research Laboratory, Environmental Protection Agency, Research Triangle Park, North Carolina 27711, USA.
B D
BD
Abbott
G A
GA
Held
C R
CR
Wood
A R
AR
Buckalew
J G
JG
Brown
J
J
Schmid
D000818:Animals; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator; D001483:Base Sequence; D019363:Cytochrome P-450 CYP1A1 / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins; D004305:Dose-Response Relationship, Drug; D004785:Environmental Pollutants / Q000633:toxicity*; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D009924:Organ Culture Techniques; D010159:Palate / Q000187:drug effects* / Q000196:embryology / Q000378:metabolism; D000072317:Polychlorinated Dibenzodioxins / Q000633:toxicity*; D012333:RNA, Messenger / Q000378:metabolism; D018336:Receptors, Aryl Hydrocarbon / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013723:Teratogens / Q000633:toxicity*; D013997:Time Factors; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
C497396:ARNT protein, human; C497397:Arnt protein, mouse; D004268:DNA-Binding Proteins; D004785:Environmental Pollutants; D000072317:Polychlorinated Dibenzodioxins; D012333:RNA, Messenger; D018336:Receptors, Aryl Hydrocarbon; D013723:Teratogens; D014157:Transcription Factors; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator; D019363:Cytochrome P-450 CYP1A1