Chapter 36
DNA Pyridyloxobutylation 4-(Acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone the Repair of O -Methylguanine
Inhibits
Nitrosamines and Related N-Nitroso Compounds Downloaded from pubs.acs.org by SWINBURNE UNIV OF TECHNOLOGY on 11/27/18. For personal use only.
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Lisa A. Peterson, Xiao-Keng Liu, and Stephen S. Hecht Division of Chemical Carcinogenesis, American Health Foundation, 1 Dana Road, Valhalla, NY 10595
4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) both methylates and pyridyloxobutylates DNA (Figure 1). Mechanistic studies in the A/J mouse demonstrated that O -methylguanine (O -mG) persistence was correlated to NNK-induced lung tumor activity (1). While only weakly carcinogenic, the pyridyloxobutylating agent, 4-(acetoxymethylnitrosammo)-1-(3-pyridyl)-1-butanone (NNKOAc), enhanced the tumorigenicity of the methylating agent, acetoxymethylmethylnitrosamine (AMMN). This increase in tumorigenic activity was associated with an increase in O -mG levels. One mechanism by which pyridyloxobutylation may increase O -mG levels is through inhibition of the repair protein, O -alkylguanine-DNA alkyltransferase (AGT). We tested this hypothesis by determining whether NNKOAc could inactivate AGT. Transferase activity was determined by measuring the amount of [ H]methyl groups transferredfrom[ H]Me-DNA to the protein (2). Co-incubation of NNKOAc (0-5 mM) with semipurified rat liver AGT and [ H]Me-DNA led to a concentration-dependent decrease in transferase activity (Figure 2). Pre-incubation of NNKOAc with AGT prior to the addition of the [ H]Me-DNA substrate led to a modest increase in inhibitory activity (Figure 2), suggesting that NNKOAc may react directly with the protein. 6
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Pre-incubation of AGT with NNKOAc-treated DNA also resulted in AGT inactivation. The extent of AGT inhibition was related to the concentration of NNKOAc (0-5 mM) used to alkylate the DNA (Figure 3). Little or no inhibition was observed when AGT was incubated with DNA which had been reacted with the hydrolysis products of NNKO7Ac, including formaldehyde, 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB), or 1-(3-pyridyl)but-2-en-1-one. Furthermore, DNA incubated with NNKOAc in the absence of esterase did not significantly affect transferase activity. The inhibitory activity of NNKOAc-treated DNA was lost when the DNA was subjected to neutral thermal hydrolysis. This treatment releases the unstable pyridyloxobutyl DNA adductsfromDNA as HPB (3). These data indicate that pyridyloxobutyl adducts are capable of inactivating rat liver AGT. When the ratio of pmol AGT inactivated to pmol of HPB-releasing adducts present was determined, 2-4% of the HPB-releasing adducts were responsible for the inhibition reaction. These inhibitory adducts were stable for at least 4 days in pH 7 buffer at 37°C, suggesting that they have sufficient lifetime in DNA under physiological conditions. The ability of pyridyloxobutyl adducts to compete with O -mG was determined by co-incubating NNKOAc-treated DNA with [ H]MeDNA and AGT. The presence of pyridyloxobutyl DNA adducts diminished the amount of [ H]methyl groups transferred to AGT (Table I). Therefore these adducts compete well with O -mG for reaction with AGT. 6
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0097-6156/94/0553-0343$08.00/0 © 1994 American Chemical Society
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NITROSAMINES AND RELATED JV-NITROSO COMPOUNDS N=0
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DNA Methylation DNA Pyridyloxobutylation
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ι 7-mG, 0*-mG, 0 4 -mT
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Figure 1. Bioactivation pathways of NNK. Reproduced with permissionfromReference 4. Copyright 1993 American Association for Cancer Research.
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2.0 3!θ 4.0 5.0 NNKOAc concentration (mM)
Figure 2. Inhibition of AGT by NNKOAc; · , co-incubation; o, pre-incubation.
Figure 3. Inhibition of AGT by NNKOAc-Treated DNA. Reproduced with permission from Reference 4. Copyright 1993 American Association for Cancer Research.
36. PETERSON ET AL.
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DNA Pyridyloxobutylation
Table I. Competition of NNKOAc-Treated DNA with pHJMeDNA for Reaction with AGT pmol HPB-releasing pmol [H]methyl % inhibition of pmol 0 -mG in adducts in DNA [H]MeDNA transferred to AGT transfer 0 1.5 0.85 ± 0.07 0 5.5 (0.17) 1.5 0.79 ± 0.03 7 11.0 (0.33) 1.5 0.55 ±0.01 35 16.5 (0.50) 1.5 0.45 ± 0.06 47 27.5 (0.83) 1.5 0.37 ±0.01 56 NNKOAc-treated DNA and [H]MeDNA were incubated with AGT for 30 min at 37°C Number in parentheses is estimated level of inhibitory adducts (3% of total). a
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Our results demonstrate that the pyridyloxobutylation pathway can inactivate AGT either directly or via a DNA adduct. Furthermore these adducts can compete with 0 -mG for reaction with AGT. If this competition occurs in vivo, more 0 -mG adducts will persist to potentially initiate tumors when both DNA alkylation pathways are operative. Therefore we propose a co-carcinogenic role for pyridyloxobutylation in which a pyridyloxobutyl DNA adduct is capable of increasing the levels of 0 -mG through competition for reaction with AGT. These studies are supported by CA-44377fromthe National Cancer Institute. 6
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References 1. Peterson, L.A.; Hecht, S.S. Cancer Res. 1991, 51, 5557-5564. 2. Myrnes, B., Norstrand, K., Giercksky, K.-E., Sjunneskog, C., and Krokan, H. Carcinogenesis 1984, 5, 1061-1064. 3. Hecht, S.S., Spratt, T.E., and Trushin, N. Carcinogenesis 1988, 9, 161-165. 4. Peterson, L.A.; Liu, X.-K.; and Hecht, S.S. Cancer Res. 1993, 53, 2780-2785. RECEIVED January 26, 1994
