Reevaluation of the effect of ellagic acid on N-methyl-N-nitrosourea

ACS eBooks; C&EN Global Enterprise. A; Accounts of Chemical .... Heather L. Lord, P. David Josephy, and Victor A. Snieckus. Chem. Res. Toxicol. , 1990...
0 downloads 0 Views 453KB Size
Chem. Res. Toxicol. 1990, 3, 195-198

195

Articles Reevaluation of the Effect of Ellagic Acid on N-Methyl-N-nitrosourea DNA Alkylation and Mutagenicity Heather L. Lord,+ P. David Josephy,**t and Victor A. Snieckud Guelph- Waterloo Centre for Graduate Work in Chemistry, Department of Chemistry and Biochemistry, University of Guelph, Guelph, Ontario, Canada N1G 2W1, and Department of Chemistry, University of Waterloo, Waterloo, Ontario, Canada N 2 L 3G1 Received December 19, 1989

N-Methyl-N-nitrosourea (MNU) is a reactive, mutagenic methylating agent. MNU methylates DNA a t various sites, including guanine N7, guanine 06,and adenine N3. Dixit and Gold [(1986) R o c . Natl. Acad. Sci. U.S.A.83,8039-80431 reported that ellagic acid, a phenolic natural product, inhibited the mutagenicity of MNU in Salmonella typhimurium strain TA 100,inhibited salmon sperm DNA alkylation by [3H]MNU, and also greatly reduced the ratio of guanine O6to guanine N7 alkylation. We have examined the MNU-induced alkylation of calf thymus DNA and evaluated the effect of ellagic acid on this binding. Ellagic acid had only a slight effect on total alkylation and did not alter the ratio of methylation a t guanine-06 and -N7 positions. In further experiments, ellagic acid did not significantly inhibit MNU mutagenicity. These findings do not support the potential use of ellagic acid as an inhibitor of biological damage induced by nitrosoureas.

Introduction N-Alkyl-N-nitrosoureas [RCH2N(NO)CONH2]are DNA-reactive chemicals which have been extensively studied as mutagens, carcinogens, and antineoplastic agents ( I , 2 ) . N-Methyl-N-nitrosourea (MNU) alkylates DNA a t various positions; guanine N’ alkylation is the most frequent event, but guanine O6and thymine O4 alkylations are probably the more important lesions for mutagenesis and tumor induction (reviewed in ref 3). Most proposed mechanisms for the biological activity of these compounds postulate that hydrolysis of the bond between the nitrosated N atom and the urea C atom generates an unstable cation, such as RCH2N2+or RCH2+. Buckley has proposed an alternative mechanism for DNA alkylation, involving the initial addition of the imidourea tautomer [RCH,N(NO)C(OH)=NH] to the base ( 4 ) , which may better account for the remarkable sequence specificity of DNA alkylation (5). However, this proposal has been disputed (6). Ellagic acid, a natural product found in strawberries and other soft fruits, is under investigation in several laboratories as an inhibitor of mutagenesis and carcinogenesis induced by polycyclic hydrocarbons and nitrosamines. Ellagic acid abolished the mutagenic effect of benzo[a]pyrene dihydrodiol epoxide (BPDE) in the Ames test system (7). This action of ellagic acid is mediated by a direct interaction between ellagic acid and the BPDE molecule; Sayer et al. (8) isolated covalent adducts of ellagic acid and BPDE. We have shown that, contrary to

* Author to whom correspondence should be addressed.

t University f

of Guelph. University of Waterloo.

0893-228x/90/2703-0195$02.50/0

a previous report ( 9 ) , ellagic acid does not inhibit dimethylnitrosamine mutagenicity in the Ames assay (10). Dixit and Gold studied the effect of ellagic acid on DNA alkylation by, and mutagenicity of, MNU (9). Salmon sperm DNA was incubated with [3H]MNUin the presence of various concentrations of ellagic acid. The DNA was then precipitated, washed, and acid-hydrolyzed to release purine bases, which were separated by high-performance liquid chromatography (HPLC). 06-Methylguanine and N-methylguanine were quantitated by scintillation counting of HPLC fractions. The authors reported that ellagic acid “inhibits the MNU-mediated methylation of guanine in DNA at the O6 position without significantly altering methylation at the N-7 position ...” (9). In experiments using the Ames assay, ellagic acid reduced MNU mutagenicity by up to 60% (9). The present paper is a reexamination of these reported effects of ellagic acid.

Materials and Methods Chemicals were obtained as follows: ellagic acid, Aldrich Chemical Co., Milwaukee, WI; MNU (Isopac containers), calf thymus DNA, and N‘-methylguanine, Sigma Chemical Co., St. Louis, MO; dimethyl sulfoxide, Fisher Scientific Ltd., Unionville, Ontario. @-Methylguanine and Nj-methyladenine were generous gifts of Dr. D. B. Yarosh, Applied Genetics, Freeport, NY. [3H]MNU (sp act. 15 Ci/mmol) was purchased from Amersham Canada Ltd., Oakville, Ontario, dissolved in chloroform, stored at -15 “C, and used within 2 months. Silica gel TLC analysis (solvent, hexane/ethyl ether, 1:3) revealed that the [3H]MNU was 86% radiochemically pure when received; the impurities were polar materials a t low Rp Teflon conical screw cap vials ( 5 mL, Norton Performance Plastics) were purchased from Cole-Parmer, Chicago, IL. Filtering units (Ultrafree-MC, 0.45 pm) were obtained from Millipore, Mississauga, Ontario.

0 1990 American Chemical Society

196 Chem. Res. Toxicol., Vol. 3, No. 3, 1990

Lord et al.

For the mutagenicity assays, the methods of Maron and Ames (11) were followed. MNU (unlabeled) was dissolved in DMSO, stored at 4 "C, and used within a month. Overnight cultures of

Salmonella typhimurium strain TAl00 were grown a t 37 "C with shaking a t 120 rpm, for about 10 h, until the optical density a t 650 nm reached 1.0. Phosphate buffer (0.2 M sodium phosphate buffer, pH 7.4), 0.5 mL, bacterial culture, 0.1 mL, and ellagic acid in DMSO, or DMSO alone (30 pL, 0-lo00 nmol), were incubated at 37 "C for 10 min. Then, MNU (in 10 pL of DMSO) was added and the tubes were allowed to sit for a further 30 min a t room temperature. (In preliminary experiments, we found that incorporation of this delay between addition of MNU and plating increased MNU mutagenicity almost 2-fold.) Top agar (2 mL) was added and the mixtures were plated. Plates were incubated a t 37 "C for 48 h. Revertant colonies were scored by using a 3M automatic colony counter (size threshold 0.3 mm), and plates were routinely monitored for toxicity by microscopic examination of the background lawn. For the DNA alkylation studies, incubation mixtures (total volume 300 pL) contained Tris buffer, 0.05 M, pH 7.4; calf thymus DNA, 83.5 pg (0.27 pmol of nucleotide); ellagic acid in dimethyl sulfoxide (DMSO concentration was 30% by volume in all incubations); and MNU (0.2 pmol; [SHIMNU, 30-50 pCi), in a Teflon vial. For preincubation tubes, addition of MNU was delayed 30 min. The mixture was incubated overnight a t 37 OC with gentle shaking. At the end of the incubation, the liquid was transferred to a filtering unit, chilled for 3 h at 4 "C, and centrifuged for 5 min in a microfuge, to remove precipitate. (The amount of radioactivity left on the filter was less than 0.5% of the total and did not appear to vary with the ellagic acid concentration.) DNA was then isolated (9). DNA was hydrolyzed by treatment with HCl (0.5 mL, 0.1 N) for 60 h a t 37 "C. The acid hydrolysate was neutralized by the addition of NHIOH (1.0 M, approximately 35 bL). A sample of hydrolysate (250 pL) was mixed with phosphate-buffered saline (150 mM NaC1, 5 mM potassium phosphate buffer, pH 7.0; 600 pL). The DNA concentration was calculated from the absorbance at 260 nm. The hydrolysate was analyzed by cation-exchange HPLC, on a Partisil SCX Radial-PAK cartridge column (Waters Scientific, Mississauga, Ontario). A sample of the hydrolysate was mixed with a solution of authentic standards (N-methylguanine, 06methylguanine, and P-methyladenine) as UV markers. An aliquot of 200 pL (190 pL of hydrolysate plus 10 pL of standards solution) was injected into the HPLC. The solvents used in the gradient were as follows: (solvent A) 0.02 M ammonium formate buffer, pH 4.0,94%; methanol, 6%; (solvent B) 0.2 M ammonium formate buffer, pH 4.0, 92%; methanol, 8%. The flow rate was 3.0 mL/min. Initial conditions: 100% A; gradient to 20% A a t 6 min, using Waters curve 3; gradient to 100% B a t 12 min, using Waters curve 9; return to initial conditions at 16 min, using Waters curve 6. Fractions (15 s) were collected, mixed with 4.25 mL of scintillation cocktail (Beckman Ready-Safe), and counted in a Packard scintillation counter, Model 4430. Activity was calculated in disintegrations per minute (dpm) by automatic quench correction; typical efficiencies were 35-40%. Adduct levels are reported as dpm per absorbance unit at 260 nm of the hydrolysate (dpm/A,,), following the method of Rajalakshmi et al. (12).

Results In t h e alkylation experiments, a b o u t 50% of t h e initial radioactivity was recovered following t h e incubation a n d filtration. A small amount (less than 0.5%)was lost during filtration. T h e remainder was apparently lost during t h e incubation, perhaps as volatile decomposition products of N-methyl-N-nitrosourea. Following isolation of DNA and acid hydrolysis, 0.13-0.19'70 of t h e initial activity was recovered as putative DNA adducts. T h i s percentage d i d n o t a p p e a r t o vary with t h e ellagic acid concentration. Figure 1reproduces a typical chromatogram of t h e DNA acid hydrolysate and demonstrates t h e resolution and quantitation of t h e methylated purine peaks. In Figure 2, we show t h e levels of N7-methylguanine, 06-methylguanine, a n d M - m e t h y l a d e n i n e a n d t h e 06:N7ratios, as

15000 r

Figure 1. Separation of methylated purines by HPLC. See text for details of HPLC procedures. A typical separation is shown (1.0 mM ellagic acid).

-0 . I

4.

r.,, Z ID

0

._

401

20

o'20/-g---q 0.10

I

0.00 1

0.0

1 .o

2.0

3.0

Ellagic Acid (mM)

Figure 2. (A) Methylated DNA adduct formation: ellagic acid dependence. Open diamonds, W-methylguanine; open triangles, 06-methylguanine; open squares, P-methyladenine. Closed symbols to the right of "3.0 mM" points correspond to 30-min preincubation at room temperature with 3.0 mM ellagic acid before MNU was added. For both panels A and B, each point is the average of three determinations. Error bars represent standard errors, and, where an error bar is not present, the error is smaller than the size of the symbol. Lines in both panels A and B were fitted by linear regression. (B)Ratio of 06-to W-methylguanine adduct levels: ellagic acid dependence. Open inverse triangles, ratio of @-methylguanine to W-methylguanine. Closed symbol to the right of "3.0 mM" points corresponds to 30-min preincubation at room temperature with 3.0 mM ellagic acid before MNU was added.

a function of t h e concentration of ellagic acid in t h e incubation. W e observed a slight decrease in total binding (about 20%), b u t n o significant change in the ratio (R)of 06-methylguanine t o W-methylguanine, with increasing ellagic acid concentration over t h e range of 0-3 m M (Figure 3). A t ellagic acid concentrations of 3 mM and above, some of t h e ellagic acid precipitated o u t in t h e initial incubation and was removed during t h e filtration s t e p immediately before DNA isolation. If t h i s step was omitted, t h e precipitate interfered with t h e subsequent

Chem. Res. Toxicol., Vol. 3, No. 3, 1990 197

Ellagic Acid and MNU

-5

‘Ooo

3000

i

n /

\

0-9

0

4-0’ 0.0

1 .o

0.5

1.5

2.0

MNU (pmol)

Figure 3. MNU mutagenicity dose-response curve. The procedure described for the inhibition experiments (Figure 4) was employed here also. Toxicity was not observed at any dose. Each point is the average of two experiments performed on separate days, each with three plates per dose point. Error bars (here and Figure 4) represent standard errors, and, where an error bar is not present, the error is smaller than the size of the symbol. Spontaneous revertant values were not subtracted from the results

(here and Figure 4).

+ 0

3000

4

5 L1:

iL

--u---.A.........a .......................0..........................................

1000

0 ‘ 0.0

0.2

0.4

0.6

0.8

P

1 .o

Ellagic A c i d (pmol)

Figure 4. MNU mutagenicity in the presence of ellagic acid. Squares, 0.5 pmol of MNU, circles, 1.0 rmol of MNU. The

experimental procedure followed may be found under Materials and Methods. Toxicity was not observed at any dose; however, precipitate was observed on the plates at loo0 m o l of ellagic acid. Each point is the average of two experiments performed on separate days, each with three plates per dose point. Lines were fitted by linear regression. DNA isolation step. Because of its limiting solubility in the incubation mixture, the alkylation experiment was not conducted with ellagic acid concentrations above 3 mM. On the basis of the observed dose-response for MNU mutagenicity (Figure 3), we selected doses of 0.5 and 1.0 pmol per plate for mutagenicity inhibition experiments. The inhibitory effect observed with ellagic acid concentrations up to 1000 nmol (Figure 4) was slight (no greater than 20% inhibition). Higher doses were not studied, since, at or above 1000 nmol, ellagic acid precipitated out of solution during the incubation and remained as flecks of precipitate on the plates.

Discussion MNU-DNA binding has been studied in many systems. Lawley and Shah reported a binding level of about 5 nmol of adduct/rmol of nucleotide, following incubation of 4 mM MNU with salmon sperm DNA in vitro (13) a t pH 8. They found a ratio of 06-methylguanine to IVmethylguanine of about R = 0.10. Rajalakshmi et al. (12) reported R = 0.094 for rat hepatic chromatin DNA treated in vitro. Richardson et al. found R = 0.12 in Escherichia coli exposed to 7 mM MNU for 5 min (6). A ratio R = 0.10 was also found for DNA from the organs of mice injected with MNU intraperitoneally (14), at short times after in-

jection, although the effects of repair alter this ratio with time. Dixit and Gold (9) reported a ratio R = 0.19 in the absence of ellagic acid, decreasing in a dose-dependent manner to R = 0.04 at the highest concentration tested (6.60 mM). Other agents, such as spermine, which inhibit MNU-DNA binding (12,15), do not alter R (12). We have used calf thymus, rather than salmon sperm DNA, in our experiments. However, the spectrum of DNA modifications induced by MNU appears not to vary with the source of the nucleic acid, as judged from the above reports. The previous experiments (9) were performed with incubations containing 10 pCi of [3H]MNU, 0.66 mM, and 0.9 mM DNA (nucleotide) in a volume of 300 pL. Therefore, each incubation contained 0.27 pmol of nucleotide. The reported level of 06-methylguanine varied from 20 to 3.3 pmol/pmol of nucleotide, with increasing ellagic acid. At 3.3 pmol of adductlpmol of nucleotide, the yield of adduct per incubation would be 0.89 pmol. The amount of radioactivity associated with this adduct, at the specific activity given (10 pCi/0.198 pmol, or 50.5 pCi/Kmol) would be 45 pCi, or about 90 disintegrations per minute (dpm). The DNA was hydrolyzed in a volume of 0.5 mL of acid, and 100 pL was injected into the HPLC (9). Such an injection appears to correspond to fewer than 20 dpm in the 06-methylguanine peak. Accurate quantitation of this level of tritium is difficult. Ellagic acid binds noncovalently to DNA in vitro (9),and covalently to DNA of organ explants in vitro (16). Mechanisms for selective inhibition of P-methylguanine formation have been proposed, based on postulated stereochemical specificity of the DNA-ellagic acid interaction (9). However, we are unable to confirm the reported ability of ellagic acid to reduce the ratio of 06-methylguanine to W-methylguanine formation in vitro following MNU treatment, and therefore, we do not believe that such mechanisms need to be invoked. We did not observe substantial inhibition of MNU mutagenicity by ellagic acid, as had been reported previously (9). Dixit and Gold found substantial inhibition of MNU mutagenicity at a molar ratio (ellagic acid to MNU) of 1.25. We examined ratios between 0.05 and 2.0. Our experimental protocol differed somewhat from that of the earlier report: we used slightly higher doses of MNU (0.5 and 1.0 pmol in 0.64 mL, versus 0.4 pmol in 1mL) and 0.1 M (final concentration) phosphate buffer versus 2.5 mM buffer. We employed a 10-min incubation at 37 “C before addition of MNU and then a further 30-min incubation at room temperature after MNU addition (before plating). Our conditions were established on the basis of preliminary experiments and previously reported Ames test procedures. In conclusion, we have been unable to confirm any of the reported inhibitory effects of ellagic acid on the toxicity of nitrosamines (10) or nitrosoureas.

Acknowledgment. This research was supported by a grant from the National Cancer Institute of Canada. Registry No. N-Methyl-N-nitrosourea,684-93-5;ellagic acid, 476-66-4; M-methylguanine, 578-76-7; 06-methylguanine, 20535-83-5;W-methyladenine, 5142-23-4; purine, 120-73-0.

References (1) Lawley, P. D. (1984) Carcinogenesis by alkylating agents. In Chemical Carcinogens, 2nd ed. (Searle, C. E., Ed) pp 326-484, American Chemical Society, Washington, DC. (2) Singer, B., and KuBmierek, J. T. (1982) Chemical mutagenesis. Annu. Reu. Biochem. 52, 655-693. (3) Yarosh, D. B. (1985) The role of OB-methylguanine-DNA methyltransferase in cell survival, mutagenesis and carcinogenesis. Mutat. Res. 145, 1-16.

198 Chem. Res. Toxicol., Vol. 3, No. 3, 1990 (4) Buckley, N. (1987) A regioselective mechanism for mutagenesis

and oncogenesis caused by alkylnitrosourea sequence-specific DNA alkylation. J. Am. Chem. SOC. 109, 7918-7920. (5) Richardson, K. K., Richardson, F. C., Crosby, R. M., Swenberg, J. A., and Skopek, T. R. (1987) DNA base changes and alkylation following in vivo exposure of Escherichia coli to N-methyl-Nnitrosourea or N-ethyl-N-nitrosourea. Proc. Natl. Acad. Sci. U.S.A. 84, 344-348. (6) Wurdeman, R. L., Church, K. M., and Gold, B. (1989) DNA Methylation by N-methyl-N-nitrosourea, N-methyl-N’-nitro-Nnitrosoguanidine, N-nitroso(1-acetoxyethyl)methylamine,and diazomethane: mechanism for the formation of N7-methylguanine in sequence-characterized 5’-32P-end-labeledDNA. J . Am. Chem. SOC. 111, 6408-6412. (7) Wood, A. W., Huang, M. T., Chang, R. L., Newmark, H. L., Lehr, R. E., Yagi, H., Sayer, J. M., Jerina, D. M., and Conney, A. H. (1982) Inhibition of the mutagenicity of bay-region diol epoxides of polycyclic aromatic hydrocarbons by naturally occurring plant phenols: exceptiocal activity of ellagic acid. Proc. Natl. Acad. Sci. U.S.A. 79, 5513-5517. (8) Sayer, J. M., Yagi, H., Wood, A. W., Conney, A. H., and Jerina, D. M. (1982) Extremely facile reaction between the ultimate and elcarcinogen benzo[a]pyrene-7,8-dihydrodiol-9,1O-epoxide lagic acid. J . Am. Chem. SOC. 104, 5562-5564. (9) Dixit, R., and Gold, B. (1986) Inhibition of N-methyl-Nnitrosourea-induced mutagenicity and DNA methylation by el-

Lord et al. lagic acid. Proc. Natl. Acad. Sci. U.S.A. 83, 8039-8043. (10) Lord, H. L., Snieckus, V. A., and Josephy, P. D. (1989) Reevaluation of the effect of ellagic acid on dimethylnitrosamine mutagenicity. Mutagenesis 4, 453-455. (11) Maron, D. M., and Ames, B. N. (1983) Revised methods for the Salmonella mutagenicity test. Mutat. Res. 113, 173-215. (12) Rajalakshmi, S., Rao, P. M., and Sarma, D. S. R. (1978) Studies on carcinogen chromatin-DNA interaction: inhibition of Nmethyl-N-nitrosourea-induced methylation of chromatin-DNA by spermine and distamycin A. Biochemistry 17, 4515-4518. (13) Lawley, P. D., and Shah, S. A. (1973) Methylation of DNA by 3H-14C-methyl-labelled N-methyl-N-nitrosourea-evidence for transfer of the intact methyl group. Chem.-Biol. Interact. 7, 115-120. (14) Frei, J. V., Swenson, D. H., Warren, W., and Lawley, P. D. (1978) Alkylation of DNA in uiuo in various organs of C57BL mice

by the carcinogens N-methyl-N-nitrosourea, N-ethyl-N-nitrosourea and ethyl methanesulfonate in relation to induction of thymic lymphoma. Biochem. J . 174, 1031-1044. (15) Wurdeman, R. L., and Gold, B. (1988) The effect of DNA sequence, ionic strength, and cationic DNA affinity binders on the methylation of DNA by N-methyl-N-nitrosourea. Chem. Res. T O X ~ C1,O146-147. ~. (16) Teel, R. W. (1986) Ellagic acid binding to DNA as possible mechanism for its antimutagenic and anticarcinogenic action. Cancer Lett. 30, 329-336.