Expression and activity of the DNA repair enzyme uracil DNA glycosylase during organogenesis in the rat conceptus and following methotrexate exposure in vitro.
Uracil incorporation into DNA occurs under conditions that limit thymidine biosynthesis; uracil is removed by two isoforms of uracil DNA glycosylase (UNG; EC 3.2.2.3), UNG1 and UNG2. We hypothesize that UNG is important in protecting the mid-organogenesis stage [gestational day (GD) 10-12] rat conceptus against conditions that limit thymidine biosynthesis. Transcripts for both UNG isoforms were expressed highly in the yolk sac and embryo, increasing over 400% in the embryo between GD11 and 12. GD10 and 11 yolk sacs showed the highest levels of putatively active UNG2 protein, with little UNG1 protein. Moderate levels of UNG2 and UNG1 proteins were found in the embryo on GDs 10 through 12; no significant increase in either protein occurred on GD12. UNG activity was higher in yolk sac than embryo on GDs 10 and 11, mirroring protein levels. Exposure to the teratogen methotrexate (MTX) leads to nucleotide pool imbalance, uracil incorporation into DNA, and genotoxic stress-induced cell death. Concentration-dependent decreases in developmental growth parameters, decreased yolk sac vasculature, and malformations such as kinked tail and retarded limb development were observed in embryos exposed to MTX (0.5, 2.5, or 5 microM). UNG transcripts were elevated 30-40% in both yolk sac and embryo after a 6-hr culture with 0.5 microM MTX; however, protein expression and activity were unaffected. Thus, MTX exposure caused malformations but did not modify UNG protein expression or activity, indicating an inability to increase the removal of MTX-induced genotoxic damage. Furthermore, UNG expression was developmental stage- and tissue-specific; the discrepancy between transcript and protein levels suggests post-transcriptional regulation.
Vinson
Robert K
RK
Department of Pharmacology and Therapeutics, McGill University, 3655 Promenade Sir-William-Osler, Montréal, Que., Canada H3G-1Y6.
Hales
Barbara F
BF
D000818:Animals; D018929:Cell Culture Techniques; D045647:DNA Glycosylases; D011088:DNA Ligases / Q000235:genetics / Q000378:metabolism; D005314:Embryonic and Fetal Development / Q000187:drug effects*; D005260:Female; D005333:Fetus / Q000187:drug effects* / Q000201:enzymology; D015870:Gene Expression / Q000187:drug effects; D008727:Methotrexate / Q000494:pharmacology*; D009699:N-Glycosyl Hydrolases / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013723:Teratogens / Q000494:pharmacology; D051981:Uracil-DNA Glycosidase
D013723:Teratogens; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D051981:Uracil-DNA Glycosidase; D011088:DNA Ligases; D008727:Methotrexate