DNA hydroxyethylation by hydroxyethylnitrosoureas in relation to their organ specific carcinogenicity in rats

Abstract N -Hydroxyethylnitroso- N ′-ethylurea (HEENU) and N -hydroxyethylnitroso- N ′-chloroethylurea (HCNU) are two of the few nitrosoureas which induce hepatocellular tumours in rats without further treatment. In the present study we have investigated whether this is due to selectively elevated levels of DNA hydroxyethylation in the target tissue. Formation of the promutagenic base O 6 -hydroxyethyldeoxyguanosine ( O 6 -HEdG) in various rat tissues was determined by immuno-slot-blot assay. After a single dose by gavage (0.36 mmol/kg body wt) of HEENU, initial levels of O 6 -HEdG in liver and brain were close to the detection limit of 1.5 μmol/mol deoxyguanosine. In liver, steady state concentrations of 3.5 μmol/mol were reached after 6 h and maintained for at least 18 h. In brain, O 6 -HEdG levels were 1.7 μmol/mol after 6 h and 3.0 μmol/mol after 24 h. In a second experiment, the formation of O 6 -HEdG was assessed in target and non-target tissues 6 h after a single dose by gavage (0.36 mmol/kg) of HEENU, HCNU or hydroxyethylnitrosourea (HENU), which is not hepatocarcinogenic. The extent of DNA hydroxyethylation was greatest with HENU in all tissues examined. Concentrations of O 6 -HEdG were highest in liver (37.2 μmol/mol), followed by kidney (23.3 μmol/mol), lung (18.9 μmol/mol), brain (6.8 μmol/mol) and testes (3.8 μmol/mol). With HEENU and HCNU, levels of 1.4–3.3 μmol O 6 -HEdG/mol dG were observed in all tissues. In vitro, the alkylation reactions for all three compounds were nearly complete within 6 h. On a molar basis, yields of O 6 -HEdG in vitro were similar for HENU and HCNU and 3.7 times lower for HEENU. This suggests that the in vivo reactions of the dialkylnitrosoureas are by pathways other than or in addition to those occurring in vitro. We conclude that the hepatocarcinogenicity of HCNU and HEENU cannot be explained on the basis of their reaction with cellular DNA.