N-ALKYL-N-NITROSOUREA INDUCED SECONDARY STRUCTURAL-CHANGES IN DNA FROM RAT EMBRYOS AND FETAL BRAINS INVIVO

N-methyl-N-nitrosourea (MNU) and N-ethyl-N-nitrosourea (ENU) are gestational stage dependent teratogens and transplacental carcinogens capable of inducing neurogenic tumours in rats. Intravenous treatment of gravid Wistar rats showed that MNU is teratogenic but ENU is a transplacental carcinogen and may be a teratogen when administered on day 12 of gestation. Twenty-four hours after single doses of 2, 5, or 10 mg MNU/kg on day 12, dose dependent decreases in embryonic wet weight and total embryonic DNA were observed. Rats similarly treated with 2 and 5 mg MNU/kg showed dose dependent decreases in fetal brain DNA synthesis, DNA content, and wet weight 9 days later. Administration of single ENU doses of 1. 5, 3, 6, 12, 48, and 80 mg/kg to day 12 embryos resulted in a dose dependent reduction in [methyl-C-14]-thymidine (C-14-TdR) incorporation into DNA after 24 h although total DNA amounts and embryonic wet weights were unaffected. Benzoylated DEAE-cellulose (BD-cellulose) chromatography fractionates DNA on the basis of secondary structure by stepwise elution of double-stranded DNA with 1.0 M NaCl solution (SE-DNA) followed by elution of DNA containing single-stranded regions with caffeine solution (CE-DNA). Day 13 embryonic and day 21 fetal brain DNA was monitored by in vivo labelling with [methyl-H-3]-thymidine on days 6 and 7 of gestation. Significant reduction in percentages of CE-DNA (%CE-DNA) 24 h after treatment of day 12 embryos with 2, 5, or 10 mg MNU/kg were attributed to the necrotic effect of MNU. Day 12 treatment with MNU produced no change in %CE-DNA values of day 21 fetal brains. A teratogenic dose of 80 mg ENU/kg to day 12 embryos resulted in significantly increased %CE-DNA values compared to controls but no changes were observed after 1.5 to 48 mg/kg. Analysis of the distribution of %CE-DNA values from the 80 mg ENU/kg treated litter showed that the increase in %CE-DNA was due to a second distinct population of embryos with higher %CE-DNA values than controls. Incorporation of C-14-TdR into embryonic and fetal brain DNA demonstrated the effects of treatment with these compounds on DNA synthesis in vivo. The relative %CE-DNA is expressed as the ratio of the percentage of caffeine-eluted C-14-labelled DNA to %CE-DNA(i.e., %CE-C-14-DNA:%CE-H-3-DNA). In the majority of control embryos the C-14-specific activity of CE-DNA was higher than the C-14-specific activity of SE-DNA. Treatment with ENU doses between 1.5 and 48 mg/kg on day 12 of gestation resulted in a dose dependent increase in the percentage of embryos from each litter with higher concentrations of newly synthesized DNA in SE-DNA than in CE-DNA. The results of this study suggest that mechanisms of ENU induced teratogenesis and transplacental carcinogenesis may be different although alkylation of embryonic DNA may initiate both phenomena. BD-cellulose fractionation of embryonic DNA showed differences in the DNA secondary structural changes produced by MNU and ENU, suggesting they may not share the same mechanism of teratogenesis. The pertinence of relative %CE-DNA to transplacental carcinogenesis is also discussed.