No abstract

The toxic effects of sulfur mustard have been attributed to DNA modification with the formation of 7-hydroxyethylthioethyl guanine, 3-hydroxyethylthioethyl adenine and the cross-link, di-(2-guanin-7-yl-ethyl)sulfide. To investigate the action of bacterial 3-methyladenine DNA glycosylase II (Gly II) on these adducts, calf thymus DNA was modified with [14C]sulfur mustard and used as a substrate for Gly II. Gly II releases both 3-hydroxyethylthioethyl adenine and 7-hydroxyethylthioethyl guanine from this substrate. In comparison with the activity of Gly II towards methylated DNA, 3-hydroxyethylthioethyl adenine is released somewhat more slowly than 3-methyladenine, while 7-hydroxyethylthioethyl guanine is released much more readily than 7-methylguanine. Glycosylase action may play a role in protecting cells from the toxic effects of sulfur mustard.

No abstract

Many anttuor agents, including the mustards, form N-7 deoxyguanouine adducts in DNA thd are difficult to quantitate by the 32posabelng pre e because of their Inabili. We 32P-postlabeling has been used successfully to measure a range of DNA adducts encountered in environmental exposure, many of which involve modifications at the 12 or C-8 positions of guanine. However, there are relatively few reports of its successful use to measure the N-7 adducts of guanine that are formed by many antitumor agents. An exception to this is the successful measurement of 7-methylguanine, an adduct formed by procarbazine and dacarbazine as well as by many methylating carcinogens (9-12).Previous investigators have commented on the instability and low labeling efficiency of N-7 guanine adducts (10, 13, 14). Our studies of the haloethylnitrosoureas indicated that there would be an even more serious problem with 32p-postlabeling of these adducts: the two major N-7 guanine adducts produced by the haloethylnitrosoureas, 7-hydroxyethyldeoxyguanosine and 7-chloroethyldeoxyguanosine, depurinate almost completely during enzymatic digestion and would, therefore, not be substrates for T4 kinase (15).Because many environmental agents as well as many important antitumor agents modify the N-7 position of guanine, we undertook an investigation of DNA modification by the prototype mustard, bis(2-chloroethyl)sulfide, or sulfur mustard (SM), which extensively attacks the N-7 position of guanine (16). In this communication, we show that depurination of 7-hydroxyethylthioethyldeoxyguanosine (HETEdG), an N-7 guanine adduct formed by SM, is nearly complete during enzymatic digestion under standard conditions. However, this problem has been solved by performing the digestion at 100C, where 50%o ofthe adduct is released as the 3'-deoxynucleotide, making 32P-postlabeling possible. We have also introduced the use of an internal standard with a similar structure and with similar stability and HPLC characteristics to correct for variations in the recovery of the substituted nucleotide. Finally, we have made use of disposable anion columns to remove the bulk of unmodified nucleotides and most of the [32P]ATP to increase both the safety and sensitivity of this method. With these modifications, we are able to detect 1 unstable N-7 deoxyguanosine adduct in 107 normal nucleotides with good precision. MATERIALS AND METHODSMaterials. Calf thymus DNA and all nucleosides and nucleotides were purchased from Sigma.