Age-related studies on the removal of 7-methylguanine from DNA of mouse kidney tissue following N-methyl-N-nitrosourea treatment

Abstract

To investigate the effects of age on DNA repair of alkylation damage, C57BL/6NNia mice ranging from 9 months to 29 months of age were injected by the intraperitoneal route with single doses of N-methyl-N-nitrosourea (MNU). The rates of removal of 7-methylguanine (m⁷Gua) in nuclear DNA from kidney were determined at various intervals from 1 to 288 h after injection of either 25 mg or 50 mg MNU per kg body weight. Reversed phase HPLC with electrochemical detection was used to monitor adduct disappearance from DNA hydrolysates. The kinetics of m⁷Gua removal from DNA were at least biphasic. Evidence was obtained that there was a rapid removal of m⁷Gua occuring in the first 24 h after MNU administration, followed by a slow phase of removal with a $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ greater than 150 h. We assume that these two phases of m⁷Gua removal correspond to active repair of DNA by N-alkylgylcosylases and to passive elimination via sponteneous hydrolysis, respectively. Young and old kidney tissues all exhibited significant repair of m⁷Gua (55–73% of the induced adducts were removed in the first 24 h), but a substantial fraction of m⁷Gua was removed slowly, indicating that there are methylated bases which were refractory to repair processes. At both doses of MNU studied, old tissues showed active repair of m⁷Gua that, within the limits of detection, had similar initial rates of removal as young tissues. However, old kidney did not remove this adduct with the same overall efficiency as young kidney. Therefore, the amount of m⁷Gua in the repair-resistant fraction was greater in the senescent tissues. The biochemical mechanisms responsible for the less efficient DNA repair in senescent kidney are not known, but we suggest that such differences are due in part to structural alterations in the chromatin.