Chem. Res. Toxicol. 1992,5, 528-531
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Differential Effects of Thiols on DNA Modifications via Alkylation and Michael Addition by a-Acetoxy-N-nitrosopyrrolidine Mingyao Wang, Akiyoshi Nishikawa,? and Fung-Lung Chung' Section of Nucleic Acid Chemistry, Division of Chemical Carcinogenesis, American Health Foundation, Valhalla, New York 10595 Received March 4,1992
The hepatocarcinogen NPYR is metabolically activated by a-hydroxylation mediated by cytochrome P-450 enzymes to yield a 4-oxobutylating agent and 2-butenal (crotonaldehyde). Both are reactive intermediates capable of modifying DNA with guanine either by simple alkylation or by Michael type addition, respectively. In order to assess the roles of these pathways in NPYR tumorigenesis, we are interested in identifying agents which can selectively modify one of these two pathways. In this study, we examined the effects of three thiols-(mesna), glutathione (Glu), and N-acetylcysteine (Nac)-on DNA adduct formation by a-acetoxyNPYR, a stable precursor of a-hydroxyNPYR. Calf thymus DNA isolated from incubation of a-acetoxyNPYR with or without thiol was hydrolyzed and analyzed for the adducts formed by alkylation (adducts 1and 2) and Michael addition (adducts 3-5). The results showed that the addition of mesna completely blocked the formation of the crotonaldehyde-derived adducts 3-5, whereas it exerted little effect on the formation of the alkylated adducts 1and 2. These results indicate the preferential conjugation of mesna with crotonaldehyde. In contrast, Nac had little selectivity on adduct formation; levels of adducts 1 to 5 were reduced by 36-75%. These results suggest that Nac conjugated with both alkylating agent and crotonaldehyde. Similar to mesna, Glu blocked the formation of the crotonaldehyde-derived adducts (adducts 3-5) efficiently. However, unlike mesna, Glu inhibited the formation of adduct 1,while it did not inhibit the formation of adduct 2, although both adducts are presumably derived from the 4-oxobutylating agent. The reaction rates of thiols with crotonaldehyde were determined to be in the following order: mesna > Glu > Nac. These results indicate that the differential effects of thiols on DNA modification by alkylation and Michael addition are determined largely by their rates of reaction with crotonaldehyde.
Introduction It is well established that, upon metabolic activation by a-hydroxylation, nitrosamines yield alkane diazonium ions which alkylate cellular DNA (1). The formation of the alkylated adducts such as @-methylguanine and @-methylthymine is considered to be an important step in carcinogenesis by nitrosamines (2, 3). In addition to the alkylating species, however, some nitrosamines such as N-nitrosopyrrolidine (NPYR) could also be metabolized to release the reactive a,fl-unsaturatedaldehydes or enals. Acrolein and crotonaldehyde, the simplest enals, are mutagenic, and crotonaldehyde is carcinogenic in rats (4,6). We have shown that these enals readily react with DNA, yielding 1,Wpropanodeoxyguanosineadducts in vitro (7). Using an ELISA method, we detected these cyclic adducts in DNA of Salmonella typhimurium tester strains incubated with acrolein and in DNA of Chinese hamster ovary cells treated with crotonaldehyde (8,9). A 32P-postlabeling method enabled us to detect these adducts in skin DNA of mouse treated topically with crotonaldehyde and in liver DNA of rats treated with NPYR, a hepatocarcinogen which yields, besides a 4-oxybutylating agent, crotonaldehyde upon a-hydroxylation(10). A site-specific + Present address: Divisionof Pathology,National Institute of Hygienic Sciences, Tokyo 158, Japan. Abbreviations: NPYR, N-nitrosopyrrolidine;mesna, 2-mercaptoethanesulfonate; Glu, glutathione;Nac, N-acetylcysteine; HLPC,highperformance liquid chromatography.
mutagenesisstudy with Escherichia coli and simian kidney cells using a 1,Wpropanodeoxyguanosinemodel adduct showed that this adduct induced primarily G to T transversions (11). These results suggest that, together with alkylating agents, enals may contribute to the carcinogenicity of NPYR. To better define the relative role of these two pathways in nitrosamine carcinogenesis,we are interested in identifying agents capable of differentially conjugating with these reactive intermediates. Because thiols are known to conjugate readily with enals, in this study we examined the effects of thiols, mesna, Glu, and Nac (Figure 1)on the formation of the alkylated and the crotonaldehyde-derivedadducts in DNA incubated with a-acetoxyNPYR. In addition, we examined the relative rates of reaction of the thiols with crotonaldehyde.
Materials and Methods Chemicals. a-AcetoxyNF'YRwas synthesizedby a previously described method (12). Because of ita reactivity toward DNA, it is considered to be hazardous and should be handled carefully. Mesna, Glu,Nac, calf thymus DNA, and esterase were purchased from Sigma Chemical Co. (St. Louis, MO). Crotonaldehydewas purchasedfrom Aldrich ChemicalCo. (Milwaukee,WI). Guanine adduct standards (adducts 1-5) were prepared and identified by published methods (13). High-Performance Liquid Chromatography. HPLC was performed with a Waters Associates System (Millipore,Milford, MA) equipped with a Model 990 photodiode array detector or a Perkin-Elmer Model 650-10sfluorescence detector (Perkin0 1992 American Chemical Society
Effects of Thiokr on DNA Modifications HSCH2CH2SOgNat Mesna
HSCH2CHCOOH
I
NHCCH3
II
0 Nac
0
II
y 2
HSCH2CHNHCCH2CH2CHCOOH
I
C-NHCH2COOH
II
0 Glu Figure 1. Structures of mesna, Nac, and Glu.
Elmer Corp., Norwalk, CT). The followingsolvent systems were used: System 1: two Whatman 4.6-mm X 25-cm Partisill0 SCX strong cation-exchange columns (Clifton, NJ) in series eluted isocratically with 50 mM ammonium phosphate buffer, pH 2.0, at a flow rate of 1 mL/min using fluorescence detection with excitation wavelength at 290 nm and emission at 380 nm. System 2 one 4.6-mm X 25-cm Partisil 5-pm B and J OD5 octadecyl column (Burdick and Jackson, Baxter, Muskegon, MI) eluted isocratically with 10% methanol in H2O at a flow rate of 1mL/ min with UV detection at 225 nm. Quantitation of Guanine Adducts i n DNA. Calf thymus DNA (10 mg) and a-acetoxyNPYR (30 mg, 0.19 mmol) in 2 mL of 0.1 M phosphate buffer at pH 7 were incubated in the presence of esterase (28 units) with and without the addition of equal molar of thiol (0.19 mmol) in 0.5 mL of H2O (mesna, 31 mg; Glu, 58.4 mg; and Nac, 31 mg) at 37 OC for 18 h. After incubation, the reaction mixture was added to 2.5 mL of H2O and extracted with 5 mL of chloroform/isoamyl alcohol (2411, followed by centrifugation at 14000g for 20 min. The aqueous layer was removed, and 10 mL of cold ethanol was added to precipitate DNA. DNA was collected and dissolved in 1mL of 10mM sodium cacodylate buffer at pH 7.4. This solution was then heated at 100 OC for 1 h to release thermally labile adducts (adducts 1-4 in Figure 2). After cooling 100 pL of cold 1 N HCI was added to precipitate the DNA. The DNA was collected by centrifugation, redissolved in 0.1 N HCI, and subjected to heating at 75 OC for 45 min to release acid-labile adduct (adduct 5). Adducts were analyzed by HPLC using system 1. The amount of unreacted crotonaldehyde in the incubation mixture was determined as follows: an aliquot of 0.5 mL was removed from the reaction mixture and kept frozen at -20 OC until analysis. The aliquot was thawed in an ice bath and filtered through a Centri-Free filter (Amicon Division, W. R. Grace and Co., Danvers, MA). The filtrate was analyzed for crotonaldehyde using HPLC system 2. Determination of t h e Reaction Rates of Thiols with Crotonaldehyde. Crotonaldehyde (5pmol) with and without thiols (100 pmol) in 4 mL of 0.1 M phosphate buffer, pH 7.0, was incubated at 37 OC. Aliquots (100-300 pL) were taken at time intervals of 0, 3, 5, 10, 15, 20, 25, and 30 min and cooled in an ice bath before analysis by HPLC for the unreacted croton. aldehyde using system 2.
Chem. Res. Toxicol., Vol. 5, No. 4, 1992 529
guanine adduct corresponding to adduct 2 was also detected; however, it was not quantitatively measured due to poor separation on HPLC. Adducts 1-5 were quantitated by a HPLC-fluorescence method. The detection limits were 20,84,4,4, and 1.6 pmol for adducts 1, 2, 3, 4, and 5, respectively. Our previous studies showed that adduct 1is presumably formed by a concerted alkylation and ring closure reaction at the 7- and 8-positions of guanine, adduct 2 from 4oxybutylation at the 7-position, and adducts 3-5 by Michael addition with crotonaldehyde followed by ring closure. Table I shows the levels of guanine adducts in DNA upon incubation of a-acetoxyNPYRwith equal molar of thiol or without thiol and summarizes the yields of each adduct as compared with those from reactions without thiol. Addition of mesna almost totally blocked the formation of adducts 3-5, whereas it exerted little effect on the formation of adducts 1and 2. These results indicate a high selectivity of mesna toward conjugation with crotonaldehyde. In contrast, Nac exhibited nonspecific effects on the formation of all 5 adducts, reducing the yields to 25-64%, as compared with the control (without thiol). Analogous to mesna, Glu effectively conjugated with crotonaldehyde as indicated by its facile blockage of the formation of adducts 3-5. Interestingly, the addition of Glu had little effect on the levels of adduct 2, but it blocked 71% of adduct 1 formation. The amount of crotonaldehyde in the reaction mixture after incubation was quantitated by HPLC with UV detection. Incubation of a-acetyoxyNPYR for 18 h without thiol yielded 8262 nmol of crotonaldehyde, representing an approximate 4-5 7% yield. Coincubation with mesna or Glu under identical conditions resulted in nondetectable levels of crotonaldehyde, whereas coincubation with Nac yielded 7 nmol of crotonaldehyde. These results agreed with those from the adduct study, indicating the more efficient scavenging of crotonaldehyde by mesna and Glu than Nac. It is possible that the differential effects of the thiols on the adduct formation are due to the different rates of reaction with crotonaldehyde. To obtain direct evidence, we compared the rates of reaction of the thiols with crotonaldehyde under conditionssimilar to those used in DNA adduct studies. A ratio of crotonaldehyde to thiol of 1to 20 was used in these reactions, since approximately 5% of a-acetoxyNPYR was hydrolyzed to crotonaldehyde. Figure 3 shows the rates of reaction between thiols and crotonaldehyde. Crotonaldehyde was apparently stable during the entire period of reaction without thiol. At any given time point examined, the relative rates of reaction were consistent with the following order: mesna > Glu > Nac. Crotonaldehyde was completely depleted by mesna within 10 min of incubation, within 20 min by Glu. However, there was still a substantial amount of crotonaldehyde in the reaction mixture after 30 min of incubation with Nac.
Rssults Upon metabolic a-hydroxylation, NPYR generates a 4-oxobutanediazonium ion, an alkylating species, and crotonaldehyde. cy-AcetoxyNPYR,a stable precursor of cyhydroxyNPYR, also releases these reactive intermediates in the presence of esterase (13). Incubation of cy-acetoxyNPYR with calf thymus DNA in the presence of esterase yielded 5 guanine adducts shown in Figure 2 (14). The previously reported 7-substituted (3-carboxypropy1)-
Discussion Our observation that mesna selectively blocked the formation of adducts 3-5, but had no effect on the formation of adducts 1and 2, clearly indicates that mesna preferentially reacted with crotonaldehyde released from the hydrolysis of a-acetoxyNPYR. These results further confirm the involvement of crotonaldehyde in the formation of adducts 3-5. The selectivity observed with me-
630 Chem. Res. Toxicol., Vol. 5, No.4, 1992
Wang et al.
I
N=O n.acelory-NPYR
1
2
Figure 2. Formation of five guanine adducts upon incubation of a-acetoxyNPYR with DNA. Table I. Levels of Guanine Adducts Formed in Calf Thymus DNA upon Reaction with a-AcetoxyNPYR in the Absence or Presence of Thiols adduct in mmol/mol of guanine thiol 1 2 3 4 5 6.14 f 2.35 0.16 f 0.03 none 11.89 f 2.32O 0.18 f 0.02 3.22 f 0.70 mesna 12.55 f 2.22 (106)b 5.10 f 0.42 (83) NDc (0) ND (0) ND (0) Glu 3.48 f 0.32 (29) ND (0) 0.04 f 0.02 (22) 0.75 f 0.41 (23) 6.20 f 0.15 (101) Nac 2.93 f 0.30 (25) 3.95 f 0.65 (64) 0.08 f 0.01 (51) 0.10 f 0.03 (56) 1.30 f 0.37 (40) Mean f SD from 3 separate experiments. Percentage of adduct as control (without thiol). e Not detectable.
- 104 -- 103 PI
-n > x 0
E
-E
102
0
F
;101
E 0
100 0
5
10 15 20 25
30 35
Time (min)
Figure 3. Relative rates of reaction of the thiols with crotonaldehyde. Reactions were carried out by incubating crotonaldehyde with or without thiol, and an aliquot was analyzed at the indicated time intervals for the unreacted crotonaldehyde using HPLC system 2 (see Materialsand Methods). ( 0 )No thiol added; ( 0 )Nac; (A) Glu; and (A)mesna.
ana, however, was not seen with Nac. A broad spectrum of inhibition of alkylated and crotonaldehyde-derived adducts by Nac indicated a nonspecific conjugation of this thiol with alkylating agents and enals. Glu, like mesna, blocked effectively the formation of adducts 3-5. However, Glu exhibited a profound differential effect on the formation of adducts 1and 2, although both adducts are presumably formed with the 4-oxobutylating agent. These results provide some insights for the formation of adducts 1and 2. One could envisage that the difference in Glu's effects on adducts 1and 2 may reside in the structure, as Glu possesses an amino group while mesna does not. Since our previous study showed that adduct 1was not formed by a subsequent ring closure of adduct 2 in DNA (14), a plausible mechanism could involve the formation of a Schiff base intermediate with the 4-oxobutylating agent which prohibits the ring closure to occur. This mechanism is, however, somewhat discounted by the fact that levels of adduct 2 were not increased as a result of the decrease in the formation of adduct 1. An alternative, perhaps more probable, mechanism is that the formation
of adducts 1and 2 may involve two alkylating species, an alkanediazonium ion and an alkanecarbonium ion, and these intermediates exhibited different rates of conjugation with Glu. The facile blockage of the crotonaldehydederived adducts 3-5 by mesna and Glu paralleled their facile reaction with crotonaldehyde. These results suggest that the differential effects of the thiols in blocking DNA adduction by alkylation or enal addition is largely dictated by its rate of conjugation with crotonaldehyde. Ends are ubiquitous compounds which are produced by metabolism of drugs, nitrosamines, or from lipid peroxidation (13, 15, 16). Our results suggest that cellular Glu could effectively scavenge enals generated endogenously and, thus, play an important role in protecting DNA from the enal-derived adduction. We have previously shown that the level of adduct 1 was lo4 times that of adduct 5 in liver of rats treated with NPYR (10). Hepatic glutathione could contribute to this large difference. Since we also observed a similar inhibition of adduct 1by Glu, these results suggest that other factors such as metabolism of crotonaldehyde should be considered in order to fully explain the pronounced difference in the formation of adducts 1 and 5 in vivo. Furthermore, our results suggest that by decreasing Glu levels in the liver, one should expect an increase in the formation of adducts 1and 3-5. Indeed, Hunt and Shank reported recently that the levels of adduct 1were elevated in liver DNA of NPYR-treated rats upon administration of buthionine sulfoxime, a Glu depletor (17).Therefore, tissue Glulevels could be important in modulating the formation of DNA adducts by both enals and some alkylating agents. Several studies have shown that mesna is capable of inhibiting tumorigenicity. Mesna treatment diminished the bladder toxicity and tumorigenicity of cyclophosphamide, presumably by conjugating with acrolein, a major metabolite of cyclohosphamide (15, 18). Mesna also inhibited N-n-butyl-N-(4-hydroxybutyl)nitrosamine-in-
Effects of Thiols on DNA Modifications
duced bladder tumors in rats2 (18). The structure of this nitrosamine and others, such as N-nitrosodiallylamine, N-nitrosomethylallyamine,and the areca alkaloid-derived 3-(methylnitrosamino)propionaldehyde, permit formation of ends upon metabolic activation by a-hydroxylation. It would be interesting to see whether mesna or other thiols could differentially block the formation of adducts by enah in vivo. The present study provides a basis for the design of in vivo studies in which thiols could be used to specifically alter the levels of DNA adducts by alkylation or enal addition. These results, when compared with those from tumor bioassays, may be useful to better delineate the roles of ends in tumorigenesis. Acknowledgment. This work was supported by Grants CA-51830 and CA-44377 from the National Cancer Institute.
References (1) Preussmann, R., and Steward, B. W. (1984) N-nitroso carcinogens. In Chemical Carcinogens (Searle, C. E., Ed.) 2nd ed., pp 643-828, American Chemical Society, Washington, DC. (2) Pegg, A. E. (1984)Methylation of the 06 position of guanine in DNA is the most likely initiating event in carcinogenesis by methylating agents. Cancer Invest. 2, 223-231. (3) Singer, B. (1984) Alkylation of the 06 of guanine is only one of the many chemical events that may initiate carcinogenesis. Cancer Invest. 2, 233-238. (4) Lutz,D., Eder,T.,Neudecker,T.,andHemschler,D., (1982)Structure mutagenicity relationship in a,&unsaturated carbonyliccompounds and their corresponding allylic alcohols. Mutat. Res. 93,305-315. (5) Marnett, L. J., Hurd, H. K., Hollstein, M. C., Levin, D. E., Esterbauer, H., and Ames, B. N. (1985) Naturally occurring carbonyl compounds are mutagens in Salmonella tester strain TA 104. Mutat. Res. 148, 25-34. (6) Chung, F.-L., Tanaka, T., and Hecht, S. S. (1986) Induction of liver tumors in F344 rats by crotonaldehyde. Cancer Res. 46,1285-1289.
* Fa-L.Chung and A. Nishikawa, unpublished results.
Chem. Res. Toxicol., Vol. 5, No. 4, 1992 531 (7) Chung, F.-L., Young, R., and Hecht, S. S. (1984) Formation of cyclic 1.W-propanodeoxyguanosine adducts in DNA upon reactions with acrolein or crotonaldehyde. Cancer Res. 44,990-995. (8) Foiles, P. G., Akekar, S. A., and Chung, F.-L. (1989) Application of an immunoassay for cyclicacroleindeoxyguanosineadducteto asma their formation in DNA of Samonellatyphrimuriumunder conditions of mutation induction by acrolein. Carcinogenesis 10, 87-90. (9) Foiles, P. G.,Akekar, S. A., Miglietta, L. M., and Chung, F.-L. (1990) Formation of cyclic deoxyguanosine adducts in Chinese hamster ovary cellsby acrolein andcrotonaldehyde. Carcinogenesis11,18191823. (10) Chung, F.-L., Young, R., and Hecht, S. S. (1989) Detection of cyclic 1.W-propanodeoxyguanosine adducts in DNA of rats treated with N-nitrosopyrrolidine and mice treated with crotonaldehyde. Carcinogenesis 10, 1291-1297. (11) Moriya, M., Marinelli, E., Shibutani, S., and Joseph, J. (1989) Sitespecific mutagenesis using the model exocyclic DNA adduct, 1,Wpropanodeoxyguanosine. h o c . Am. Assoc. Cancer Res. 30, A665. (12) Saavedra, J. E. (1979) Oxidation of nitrosamines, 1. Formation of N-nitrosoproline, N-nitrosopipecolic acid, and N-nitrososarcosine. J. Org. Chem. 44,4511-4516. (13) Wang, M., Chung, F.-L., and Hecht, S. S. (1988) Identification of crotonaldehyde as a hepatic microsomal metabolite formed by ahydroxylation of the carcinogen N-nitrosopyrrolidine. Chem. Res. Toxicol. 1, 28-31. (14) Wang, M., Chung, F.-L., and Hecht, S. S. (1989) Formation of acyclic and cyclic guanine adducts in DNA reacted with a-acetoxy-Nnitrosopyrrolidine. Chem. Res. Toxicol. 2, 423-428. (15) Cox, P. J. (1979) Cyclophosphamide cystitis: identification of acrolein as the causative agent. Biochem. Pharmacol. 28,2045-2049. (16) Benedetti, A,, Pompella, A., Fulceri, R., Roamine, A., and Comproti, M. (1986)Detection of 4-hydroxynonenal and other lipid peroxidation products in the liver of bromobenzene-poisoned mice. Biochim. J. 214,479-487. (17) Hunt, E. J., and Shank, R. C. (1991) Formation and persistence of a DNA adduct in rodents treated with N-nitrosopyrrolidine. Carcinogenesis 12, 571-575. (18) Schmihl., D., Habs, M., and Tacchi, A. M. (1984) Prophylaze der tumorentstehung inder Harnblase durch Natrium-g-mercaptoethansulfonate (mesna). Urologe 23,291-296.
Registry No. adduct 1, 122413-77-8;adduct 2, 123752-07-8; adduct 3,123752-08-9;adduct 4,123752-09-0;adduct 5,14163593-0; mesna, 19767-45-4;Glu, 70-18-8; a-acetoxy-NPYR, 5943585-7; N-acetylcysteine, 616-91-1; crotonaldehyde, 4170-30-3.
