Changes in secondary structure of DNA of rat embryos following treatment with 1,2-diethylhydrazine and dimethylnitrosamine in vivo

Abstract

1,2-Diethylhydrazine (DEH) and dimethylnitrosamine (DMN) are indirect acting carcinogens that require metabolic activation to exert their potency. DEH is a transplacental carcinogen and teratogen in Wistar rats when administered by i.p. injection on day 12 of gestation. DMN is embryotoxic during this period. In this study, gravid Wistar rats were injected i.v. with DEH (10, 15, or 20 mg/kg) or i.p. with DMN (10 or 30 mg/kg) and the effects on the embryos 24 hours later were observed. Controls were similarly injected with saline vehicle. The incidence of resorptions increased after treatment with 20 mg DEH/kg. DEH treatment also resulted in decreases in embryo wet weights and total DNA that were not dose dependent. Treatment with DMN did not affect embryonic wet weights and total embryonic DNA amount when compared to the saline-treated controls. The effects of DEH and DMN on DNA synthesis in vivo were monitored by injecting [methyl-14C]-thymidine 1 hour prior to embryo death. DEH induced significant increases in thymidine incorporation into embryo DNA but the increases were not proportional to the doses administered. DNA synthesis was significantly decreased in embryos treated with 30 mg DMN/kg. The DNA of treated and control embryos was fractionated by benzoylated DEAE-cellulose (BD-cellulose) chromatography to determine differences in DNA secondary structure following treatment. BD-cellulose chromatography separates double-stranded DNA from DNA containing single-stranded regions by step elution with 1 M NaCl solution and caffeine solution, respectively. Embryonic DNA was monitored by in vivo labelling with [methyl-3H]-thymidine on days 6 and 7 of gestation. Significant dose dependent increases in percentages of caffeine-eluted DNA (%CE-DNA) compared to controls were detected after treatment with 10, 15, and 20 mg DEH/kg and 10 and 30 mg DMN/kg. The relative %CE-DNA is expressed as the ratio of %CE-14C-labeled DNA to %CE-3H-labeled DNA. Litters treated with 10, 15, and 20 mg DEH/kg had relative %CE-DNA values significantly lower than controls. The results support the hypothesis that initiation mechanisms of transplacental carcinogenesis and teratogenesis are different. The pertinence of %CE-DNA and relative %CE-DNA values to the study of transplacental carcinogenesis and teratogenesis is discussed.