Elevated 8-hydroxy-2'-deoxyguanosine levels in lung DNA of A/J mice and F344 rats treated with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and inhibition by dietary 1,4-phenylenebis(methylene)selenocyanate.

Jose G.V.Rosa 0 Bogdan Prokopczyk 0 Dhimant H.Desai 0 Shantu G.Amin 0 Karam El-Bayoumy 0 0 American Health Foundation , 1 Dana Road, Valhalla, NY 10595, USA 1To whom correspondence should be addressed Email: - 1,4-Phenylenebis(methylene)selenocyanate (p-XSC) is an effective chemopreventive agent against 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung adenoma in female A/J mice. While p-XSC can effectively inhibit NNK-induced DNA methylation in female A/J mice and in male F344 rats, its effect on NNK-induced oxidative DNA damage had not been determined. Thus, the effect of p-XSC on the levels of 8-hydroxy-29-deoxyguanosine (8-OHdG) in lung DNA from A/J mice and F344 rats treated with NNK was examined. Mice were given NNK by gavage (0.5 mg/mouse in 0.2 ml corn oil, three times per week for 3 weeks) or by a single i.p. injection (2 mg/mouse in 0.1 ml saline) while maintained on a control diet (AIN-76A) or control diet containing p-XSC at 10 or 15 p.p.m. (as Se) starting 1 week before NNK administration and continuing until termination. Mice were killed 2 h after the last NNK gavage in the multiple administration protocol or 2 h after the single i.p. injection. Treatment with NNK by gavage significantly elevated the levels of 8-OH-dG in lung DNA of A/J mice from 0.7 K 0.1 to 1.6 K 0.2 adducts/105 29deoxyguanosine (dG) (P < 0.001), while dietary p-XSC (at 10 p.p.m. Se) prevented significant elevation of the levels of this lesion caused by NNK, keeping them at 0.9 K 0.1 adducts/105 dG (P < 0.003). Injection of NNK in saline also significantly increased the levels of 8-OH-dG in lung DNA of A/J mice from 1.2 K 0.6 to 3.6 K 0.8/105 dG adducts (P < 0.01), while dietary p-XSC (at 15 p.p.m. Se) kept these levels at 1.9 K 0.5 adducts/105 dG (P < 0.03). Rats were given a single i.p. injection of NNK (100 mg/kg body wt) in saline while being maintained on control diet (AIN-76A) or control diet containing p-XSC (15 p.p.m. as Se) starting 1 week before NNK administration and continuing until termination. The rats were killed 2 h after injection. Treatment with NNK using this protocol significantly elevated the levels of 8-OH-dG in lung DNA of F344 rats from 2.6 K 0.5 to 3.5 K 0.5 adducts/105 dG (P < 0.03), while dietary p-XSC (at 15 p.p.m. Se) kept the levels of this lesion at 2.2 K 0.6 adducts/105 dG (P < 0.01). Our findings suggest that the chemopreventive efficacy of p-XSC against NNK-induced lung tumorigenesis in A/J mice and F344 rats may be due in part to inhibition of oxidative DNA damage. The tobacco-specific N-nitrosamines (TSNA) are a unique class of alkaloid-derived compounds found in tobacco smoke and tobacco products (1). 4-(Methylnitrosamino)-1-(3-pyridyl)1-butanone (NNK) (Figure 1), as a representative TSNA, induces lung adenocarcinoma in rats, mice and hamsters independent of the route of administration (2). Levels of NNK comparable with those encountered by smokers during their lifetime induce lung adenocarcinoma in laboratory animals (3,4). Thus, it is thought that NNK may be partly responsible for the induction of tobacco-related lung cancer in humans as well. NNK requires metabolic activation to exhibit its carcinogenic properties (Figure 2; 5,6). In rodents and primates, a -hydroxylation of the methylene carbon of NNK leads to the formation of electrophilic intermediates which can ultimately alkylate DNA bases. DNA adducts identified from this pathway include 7-methylguanine (7-MeG), O6-methylguanine (O6-MeG) and O4-methylthymidine (6). In addition to O6MeG, a potent mutagenic lesion, unknown DNA adducts derived from a -hydroxylation of the methyl group of NNK appear to play important roles in NNK-induced lung tumorigenesis (710). Elevated levels of 8-hydroxy-29-deoxyguanosine (8-OH-dG), a weak mutagenic lesion and biomarker of oxidative DNA damage, in lung DNA of A/J mice, F344 rats and Swiss mice treated with NNK have also been demonstrated (1114). However, the role of 8-OH-dG and of other types of oxidative DNA damage in NNK-induced lung tumorigenesis have not been defined. While DNA methylation plays an important role in the initiation phase of NNK-induced lung tumorigenesis, oxidative DNA damage also appears to be important in initiation, but perhaps even more so in the postinitiation phase of NNK-induced lung tumorigenesis (6). Among several compounds tested as chemopreventive agents against NNK-induced lung tumorigenesis (15), dietary Fig. 1. Chemical structures of NNK, p-XSC and 8-OH-dG. supplementation with the synthetic organoselenium compound 1,4-phenylenebis(methylene)selenocyanate (p-XSC) (Figure 1) inhibits NNK-induced lung tumor multiplicity in A/J mice whether administered during initiation, post-initiation or during both phases of NNK-induced lung tumorigenesis (16,17). Moreover, dietary p-XSC significantly suppresses the levels of 7-MeG and O6-MeG in lung DNA of A/J mice and F344 rats treated with NNK (18). Collectively, these studies indicate that the chemopreventive efficacy of p-XSC against NNKinduced lung tumorigenesis in A/J mice is partly due to inhibition of DNA methylation. In the present study, we tested the hypothesis that the chemopreventive efficacy of p-XSC against NNK-induced lung tumorigenesis in A/J mice may also be due in part to inhibition of oxidative DNA damage measured as 8-OH-dG. Although the efficacy of p-XSC against NNK-induced lung tumorigenesis in F344 rats remains to be examined, our observations in A/J mice encouraged us to test its effect on the levels of 8-OH-dG in lung DNA of F344 rats treated with NNK. Materials and methods Citric acid (.99.5%), sodium acetate (.99%) and sodium hydroxide (99.99%) were obtained from Aldrich Chemical (Milwaukee, WI). Glacial acetic acid, isoamyl alcohol and HPLC grade methanol and chloroform were bought from Fisher Scientific (Fairlawn, NJ). Absolute ethanol was purchased from Pharmco Products (Brookfield, CT). Trizma base (99.9%), EDTA (99.7%), sodium chloride (99.5%), SDS (99%), 29-deoxyguanosine (dG) (99100%), 8-OHdG, proteinase K, RNase A (type II-A), RNase T1, nuclease P1 and alkaline phosphatase (type III) were from Sigma (St Louis, MO). p-XSC was synthesized and purified (.99.9%) as described (19). Diet ingredients were obtained from Dyets (Bethlehem, PA). Control diet (AIN-76A-5% corn oil) was prepared by mixing AIN-76A diet with corn oil. Control diets with p-XSC (AIN-76A-5% corn oil containing 10 or 15 p.p.m. Se) were prepared by adding a solution of p-XSC in ethanol to the control diet and removing the solvent under high vacuum. The doses of p-XSC were chosen on the basis of our previous studies, which had shown that p-XSC at selenium levels between 7.5 and 15 p.p.m. lacked toxicity in A/J mice and F344 rats (1618). A/J mice multiple NNK dosing protocol. For the multiple dose experiment, 36 5-week-old female A/J mice (Jackson Laboratories, Bar Harbor, ME) were divided into four groups of nine animals each. The first and second groups were fed (...truncated)