Effect of Cations on the Formation of DNA ... - ACS Publications

In 9L cells treated with CNU, the same alkylation products were formed as in purified DNA; however, the product distribution was different. We interpr...
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Chem. Res. Toxicol. 1999, 12, 965-970

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Effect of Cations on the Formation of DNA Alkylation Products in DNA Reacted with 1-(2-Chloroethyl)-1-nitrosourea William J. Bodell* Brain Tumor Research Center of the Department of Neurological Surgery, University of California, San Francisco, California 94143-0555 Received August 20, 1998

The purpose of this study was to examine the influence of cations on the formation of the individual DNA alkylation products derived from 1-(2-chloroethyl)-1-nitrosourea (CNU). Reaction of calf-thymus DNA with [3H]CNU in 10 mM triethanolamine buffer produced 13 DNA adducts. Seven of these adducts were identified as N7-(2-hydroxyethyl)guanine, N7-(2chloroethyl)guanine, 1,2-(diguan-7-yl)ethane, N1-(2-hydroxyethyl)-2-deoxyguanosine, 1-(N12-deoxyguanosinyl)-2-(N3-2-deoxycytidyl)ethane, O6-(2-hydroxyethyl)-2-deoxyguanosine, and phosphotriesters. The ratios of the individual products indicated that the chloroethyl and hydroxyethyl adducts are derived from different alkylating intermediates. The influence of cations on the formation of these DNA alkylation products was investigated by the addition of either NaCl, MgCl2, or spermine. The results demonstrated that (1) the levels of DNA alkylation were inversely proportional to ionic strength, (2) the extent of inhibition was dependent on the alkylation product, and (3) the order of relative effectiveness of inhibition of DNA alkylation by these cations was as follows: spermine > Mg > Na. These results support a model whereby reactions which proceed via an SN2 mechanism are more sensitive to the effects of ionic strength than reactions which proceed via an SN1 mechanism. In 9L cells treated with CNU, the same alkylation products were formed as in purified DNA; however, the product distribution was different. We interpret this to indicate that within cells, cations modify the reaction of intermediates derived from CNU with DNA.

Introduction 1

The chloroethyl nitrosoureas (CENUs) are an important group of compounds for the treatment of a variety of tumors, including brain, breast, and melanoma. CENUs decompose to chloroethyl and hydroxyethyl intermediates (1, 2). These intermediates react with nucleophiles within the cell to form both DNA and protein adducts (Scheme 1). Studies by Ludlum have identified the DNA adducts that are produced by CENUs. These adducts include N7- and O6-alkylated guanines, intraand interstrand-cross-linked bases, and ethano derivatives (3). Treatment of cells with CENUs elicits a variety of responses, including cytotoxicity (4, 5), apoptosis (6, 7), increases in mutation frequency (8, 9), induction of sister chromatid exchanges (10, 11), and chromosomal aberrations (12). The induction of these biological responses may be determined by one or more of the individual DNA alkylation products. For example, the formation of the dG-dC cross-link is recognized as an important determinant in the induction of cytotoxicity (13-16) and sister * To whom correspondence should be addressed: University of California, Box-0555, San Francisco, CA 94143-0555. Phone: (415) 4764899. Fax: (415) 476-5799. E-mail: [email protected]. 1 Abbreviations: CENU, chloroethyl nitrosourea; CNU, 1-(2-chloroethyl)-1-nitrosourea; N7-ClEtG, N7-(2-chloroethyl)guanine; N7-HOEtG, N7-(2-hydroxyethyl)guanine; N7-bis-G, 1,2-(diguan-7-yl)ethane; N1-HOEtdG, N1-(2-hydroxyethyl)-2-deoxyguanosine; O6-HOEtdG, O6(2-hydroxyethyl)-2-deoxyguanosine; O6-ClEtdG, O6-(2-chloroethyl)-2deoxyguanosine; dG-dC, 1-[N1-(2-deoxyguanosinyl)]-2-[N3-(2-deoxycytidyl)]ethane; PTEs, phosphotriesters.

Scheme 1. Proposed Decomposition Pathway of CNU Leading to the Formation of N7-ClEtdG and N7-HOEtdG from ref 31

chromatid exchanges (10, 11), while the formation and repair of O6-HOEtdG may be critical to the induction of mutations (8, 9). Knowledge of the processes influencing

10.1021/tx980200c CCC: $18.00 © 1999 American Chemical Society Published on Web 09/18/1999

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the formation of the individual alkylation products is critical to achieving a better understanding of these biological responses. The reaction of CENUs with DNA is influenced by a number of factors, including base sequence (17, 18), cation concentration (19), and buffer composition (20). The influence of these factors on the formation of DNA adducts has been obtained primarily from analysis of alkylated DNA by sequencing gels. Although these studies have been very insightful, they have not elucidated the influence of these factors on the formation of the individual DNA alkylation products. To achieve a better understanding of the role of cellular cations in the formation of DNA adducts, we have investigated the influence of NaCl, MgCl2, and spermine on the formation of individual DNA alkylation products following treatment with [3H]CNU. In addition, we have compared the DNA alkylation products formed in purified DNA under these treatment conditions with those formed in 9L cells treated with CNU.

Materials and Methods Reaction with DNA. Calf-thymus DNA (250 µg) was reacted with 0.25 mCi of [3H]CNU (specific activity of 7.14 Ci/mmol, 3H label in the ethyl group; Moravek Biochemicals Inc., Brea, CA) in 10 mM triethanolamine buffer (pH 7.0). Various concentrations of NaCl, MgCl2, and spermine in 10 mM triethanolamine buffer were added to the reaction mixture to give a final volume of 1.0 mL. The reaction mixture was incubated at 37 °C for 6 h and the reaction stopped by precipitating the DNA with ethanol. The DNA was repeatedly precipitated with ethanol to obtain a constant specific activity (3H counts per minute per milligram). Treatment of 9L Cells. Rat 9L brain tumor cells were grown in four 850 cm2 roller bottles containing Eagle’s minimal essential medium, 10% newborn calf serum, and 50 µg/mL gentamycin. At confluence, the cells were washed with Hanks’ basal salt solution and trypsinized, with a solution of 0.5% trypsin and 0.02% versene in saline A. The cells were collected by centrifugation at 1500 rpm for 3 min and resuspended in 5 mL of minimum essential medium containing 10% newborn calf serum and 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer (pH 7.0). The cells were then treated with [3H]CNU for 1 h at 37 °C. For each treatment, 500 µCi of [3H]CNU (7.14 Ci/mmol) was diluted with unlabeled CNU to give a final specific activity of 4.0 Ci/mmol and a final concentration of 25 µM. The cells were collected by centrifugation, resuspended in Hanks’ balanced salt solution, centrifuged again, resuspended in 45 mL of medium, and incubated at 37 °C for 5 h. The cells were collected by centrifugation and frozen. The DNA was isolated and purified from the cellular pellet as previously described (21). Aliquots of the samples were used to measure the DNA concentration on the basis of the assumption that 1 absorbance unit at 260 nm equals 50 µg/mL DNA. The samples were subsequently transferred to a scintillation vial, and liquid scintillation cocktail was added. The levels of radioactivity in the samples were determined. From these results and using the specific activity of the [3H]CNU, the extent of DNA modification was expressed as micromoles of alkylation product per mole of DNA. Enzyme Digestion. Purified DNA isolated from 9L cells or DNA reacted with [3H]CNU was enzymatically digested overnight with pancreatic DNase, snake venom phosphodiesterase, and alkaline phosphatase (22). The mixture was then heated at 100 °C for 90 s to inactivate the enzymes and to ensure that the N7 products were converted completely to the free base form (23). The heated digest was passed through a centrifugal filter (0.2 µm pore size; Rainin Instrument Co., Emeryville, CA). N7ClEtG, N1-HOEtdG, O6-HOEtdG, and dG-dC, prepared as

Figure 1. (A) Radiochromatogram profile of DNA alkylation products formed by reaction of calf-thymus DNA in 10 mM triethanolamine buffer with [3H]CNU. (B) Same profile shown in panel A but redrawn to show the presence of the minor alkylation products. The identities of peaks A-G are given in Table 1. previously described (22), were added to the digest and used to identify the radiolabeled products. HPLC Analysis of Products. HPLC was performed with a Perkin-Elmer 250 pump (Norwalk, CT) and a 5 µm C-18 reversephase Sperisorb column (Alltech, Deerfield, IL). UV absorbance at 260 and 280 nm was monitored with a Perkin-Elmer LC-235 diode array detector. The mobile phase consisted of 10 mM NH4H2PO4 (pH 5.1) (solvent A) and various amounts of methanol (solvent B). The flow rate was 1 mL/min. The gradient used was as follows: 95% A and 5% B for 15 min, 85% A and 15% B in 30 min, 85% A and 15% B for 10 min, 30% A and 70% B in 20 min, and 30% A and 70% B for 10 min. Under these conditions, the retention times (minutes) of the standards were as follows: N7-HOEtG, 11.0; N7-bis-G, 26.1; N1-HOEtdG, 28.3; N7-ClEtG, 34.8; dG-dC, 41.0; and O6-HOEtdG, 47.6. Fractions (30 s) were collected for 85 min. The fractions were mixed with 5 mL of liquid scintillation cocktail, and their radioactivity was measured by scintillation counting. Effect of Cations on Individual Alkylation Products. The specific activity of DNA (micromoles of alkylation per mole of DNA) times the percent distribution in the HPLC profile gives the number of micromoles of alkylation product per mole of DNA. The effect of the cations on the extent of DNA alkylation was calculated as the ratio of the alkylation product (micromoles) in the treatment group to that in the control group.

Results Studies in Purified DNA. (1) Control Experiments. Calf-thymus DNA was reacted with [3H]CNU in 10 mM triethanolamine buffer at pH 7.0 and 37 °C. The level of modification produced was 179.1 ( 7.8 µmol of alkylation/mol of DNA. The radioactive profile of the alkylation products separated by HPLC exhibited at least 13 distinct alkylation products (Figure 1). The principal product (peak A) was N7-HOEtG; peaks B-G were identified as N1-HOEtdG, N7-bis-G, N7-ClEtG, dG-dC

Effect of Cations on DNA Alkylation

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Table 1. Distribution of CNU-DNA Alkylation Products in Calf-Thymus DNA Treated with CNU product

peak

µmol of alkylation/mol of DNA

N7-HOEtG N1-HOEtdG N7-bis-G N7-ClEtG dG-dC O6-HOEtdG PTE total

A B C D E F G

103.6 (57.8 ( 1.8)a 3.4 (1.9 ( 0.5) 8.7 (4.9 ( 0.4) 14.1 (7.9 ( 0.5) 4.0 (2.2 ( 0.2) 2.4 (1.3 ( 0.2) 24.1 (13.5 ( 1.3) 179.1 ( 7.8

a Values in parentheses represent the percentage of total alkylation.

Table 2. Effect of NaCl on the Formation of DNA Alkylation Products by CNU µmol of alkylation/mol of DNA product N7-HOEtG N1-HOEtdG N7-bis-G N7-ClEtG dG-dC O6-HOEtdG PTE total

Table 3. Effect of MgCl2 on the Formation of DNA Alkylation Products by CNU µmol of alkylation/mol of DNA product

peak

1 mM MgCl2

10 mM MgCl2

N7-HOEtG N1-HOEtdG N7-bis-G N7-ClEtG dG-dC O6-HOEtdG PTE total

A B C D E F G

5.0 (5)a 0.8 (22) 0.4 (5) 3.0 (21) 0.7 (18) 0.6 (25) 10.7 (44) 25.6 (14)

1.0 (1) 0.5 (13) 0.1 (1) 1.7 (12) 0.3 (8) 0.2 (8) 6.1 (25) 12.4 (7)

a Values in parentheses represent the percentage of control levels found in triethanolamine.

Table 4. Effect of Spermine on the Formation of DNA Alkylation Products by CNU

0.75 7.5 37.5 75 peak mM NaCl mM NaCl mM NaCl mM NaCl A B C D E F G

86.7 (84)a 58.9 (57) 13.8 (13) 4.1 (121) 2.1 (62) 1.6 (47) 6.9 (79) 4.2 (48) 1.1 (13) 16.1 (114) 8.7 (62) 6.6 (47) 4.1 (103) 2.3 (58) 1.6 (40) 2.6 (108) 1.5 (63) 0.8 (33) 30.0 (124) 15.5 (64) 12.2 (51) 172.7 (96) 106.2 (59) 42.9 (24)

6.4 (6) 1.3 (38) 0.6 (7) 5.3 (38) 1.2 (30) 0.7 (29) 10.1 (42) 31.0 (17)

a Values in parentheses represent the percentage of control levels found in triethanolamine.

cross-link, O6-HOEtdG, and PTEs, respectively. The relative extent of formation of these products is given in Table 1. Several minor peaks in Figure 1, each representing 0.5-2.0% of the total extent of alkylation, have not been identified. The coefficient of variation (cv) for the measurement of the adducts was dependent on the individual adducts. It was lowest for N7-HOEtG (3.1%) and highest for N1-HOEtdG (25%). The cv for the remaining products varied from 6.8 to 11.1% with a overall mean of 10.5%. On the basis of these results, we interpreted a 20% change in the adduct level to represent a significant change. (2) Effect of NaCl. Addition of NaCl to the reaction mixture caused a concentration-dependent inhibition of DNA alkylation by [3H]CNU. The influence of NaCl concentration on the relative distribution and levels of the individual DNA alkylation products is presented in Table 2. With 75 mM NaCl, the extent of DNA alkylation was inhibited by 83% and a 94% decrease in the amount of N7-HOEtG was observed. A similar level of inhibition was also measured with the formation of N7-bis-G. The production of the other DNA alkylation products was also inhibited but to a lesser extent (35-54%). As the concentration of NaCl decreased from 75 to 0.75 mM, the overall level of DNA alkylation increased to 96% of that formed in triethanolamine buffer. Parallel increases in the level of formation of N7-HOEtG were observed. At 0.75 mM NaCl, the level of formation of certain products such as the dG-dC cross-link exceeded that detected in triethanolamine buffer. (3) Effect of MgCl2. MgCl2 (1 and 10 mM) inhibited the overall level of DNA alkylation by 75 and 93%, respectively (Table 3). The levels of the DNA of the individual alkylation products formed in reaction mixtures containing are MgCl2 are shown in Table 3. With 10 mM MgCl2, the formation of N7-HOEtG was inhibited by 99% and that of the other alkylation products ap-

µmol of alkylation/mol of DNA product N7-HOEtG N1-HOEtdG N7-bis-G N7-ClEtG dG-dC O6-HOEtdG PTE total

37.5 µM 25 µM 7.5 µM peak spermine spermine spermine A B C D E F G

3.75 µM spermine

49.7 (48)a 60.2 (58) 87.7 (84) 93.8 (91) 2.4 (71) 2.9 (85) 3.1 (91) 3.3 (97) 4.3 (49) 4.9 (56) 6.0 (69) 7.4 (85) 9.5 (67) 10.8 (76) 12.8 (91) 13.0 (92) 2.7 (68) 3.6 (90) 3.8 (95) 3.6 (90) 1.8 (75) 2.0 (83) 1.9 (79) 2.1 (88) 21.6 (90) 22.2 (92) 27.0 (112) 25.9 (107) 104.5 (58) 120.7 (67) 163.4 (91) 169.3 (95)

a Values in parentheses represent the percentage of control levels found in triethanolamine.

proximately 90%. Under these conditions, the formation of PTEs was inhibited the least. (4) Effect of Spermine. Spermine (37.5 µM) inhibited the total DNA alkylation by [3H]CNU by approximately 40% (Table 4). Higher concentrations of spermine caused the DNA to precipitate from solution and could not be used. The radioactive profile of the adducts formed by the reaction of CNU with DNA in buffer containing 37.5 µM spermine was very similar to that of control reactions. N7-HOEtG accounted for ∼50% of the total alkylation, and the relative distribution of the other products was similar to that produced under the control reaction conditions (Table 4). The extent of inhibition of DNA adduct formation was dependent upon the spermine concentration, and as with the other cations that were studied, the addition of spermine inhibited the formation of N7-HOEtG the most and that of PTEs the least. Studies in Rat 9L Cells. In 9L cells treated with 25 µM [3H]CNU, the total DNA modification was 1.76 µmol of alkylation/mol of DNA. The DNA alkylation profile (Figure 2) showed that multiple products are formed in 9L cells treated with CNU. The relative extent of formation of the individual alkylation products is given in Table 5. In contrast to the results with purified DNA, the principal product formed in 9L cells was PTEs, followed by N7-HOEtG and N7-ClEtG.

Discussion The purpose of these studies was to investigate the influence of cations on the formation of the individual DNA alkylation products formed by CNU. In control reactions, the principal DNA alkylation products that formed were N7-HOEtG and PTEs. Five minor products (N7-ClEtG, N7-bis-G, N1-HOEtdG, dG-dC cross-link, and O6-HOEtdG) were identified and quantified. Both the

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Figure 2. Radiochromatogram profile of DNA alkylation products formed by treatment of 9L cells with 25 µM [3H]CNU. The identities of peaks A-G are given in Table 5. Table 5. DNA Alkylation Products in 9L Cells Treated with CNU product

peak

µmol of alkylation/mol of DNA

N7-HOEtG N7-bis-G and N1-HOEtdG N7-ClEtG dG-dC O6-HOEtdG PTE

A B and C D E F G

0.23 (13.6)a 0.06 (3.5) 0.38 (22.0) 0.05 (3.3) 0.01 (0.6) 0.67 (38.1)

a Values in parentheses represent the percentage of total alkylation.

dG-dC cross-link and N1-HOEtdG are formed from O6ClEtdG (24, 25). Therefore, the sum of the amounts of the dG-dC cross-link and N1-HOEtdG can be used as an estimate of the amount of O6-ClEtdG initially formed. From Table 1, we estimate that this value would be ∼4.0% of the total reaction. Using this estimate, the calculated ratio of O6-ClEtdG to N7-ClEtG would be 0.51. This value is very similar to the O6/N7 ratio of 0.5-0.72 produced by ethylnitrosourea (26, 27), 2-hydroxyethylnitrosourea (28, 29), and 1-(2-hydroxyethyl)-3-methyl-3carbethoxytriazene (30) and suggests that these alkylation products are derived from chloroethyldiazohydroxide or a related intermediate (3, 20, 30, 31) (Scheme 1). The O6-HOEtdG/N7-HOEtG product ratio is 0.02 which is in agreement with the value of 0.03 reported by Tong et al. (32) for CNU treatment of DNA. This O6-HOEtdG/ N7-HOEtG product ratio is similar to that observed with ethylene oxide (29) and methylhydroxyethylnitrosoamine (33). Comparison of the O6-HOEtdG/N7-HOEtG ratio with the O6-ClEtdG/N7-ClEtG ratio shows that the former is ∼25-fold lower. The electrophiles leading to the formation of the hydroxyethyl adducts have not been identified. Investigations of the DNA adducts formed by 3-methyl-1,2,3-oxadiazolinium suggest that the corresponding oxadiazolinium does not produce the hydroxyethyl adducts (34). The observed product ratios suggest that ethylene oxide formed from the oxadiazole intermediate (Scheme 1) is responsible for the formation of these adducts (1, 3, 20, 33, 35, 36). The ratio of O6/N7 alkylation products is dependent on the nature of the alkylating intermediate. Those

Bodell

Figure 3. Effect of ionic strength on the formation of the individual DNA alkylation products. The ionic strength was adjusted by the addition of NaCl to the reaction medium: N7HOEtG (bsb), PTE (b-‚‚-b), N7-ClEtG (1), N7-bis-G (2), dGdC ([), N1-HOEtdG (9), and O6-HOEtdG (-‚‚-).

intermediates which produce a high O6/N7 product ratio are described as reacting by more of an SN1 mechanism and being harder electrophiles which react with harder nucleophiles such as the exocylic oxygens (37-41). In contrast, those intermediates which produce low O6/N7 product ratios react by an SN2 mechanism and are weaker electrophiles which react with the softer nucleophiles such as N7 of guanine. Our results indicate that the intermediates leading to the formation of the chloroethyl products are reacting by an SN1 mechanism while those intermediates leading to the formation of hydroxyethyl adducts have an SN2 character. Addition of either NaCl or MgCl2 to the incubation mixture had a significant influence on DNA alkylation with the level of DNA alkylation inhibited up to 93%. The influence of these cations on the formation of the individual alkylation products varied considerably. We have plotted the levels of the individual alkylation products versus ionic strength (Figure 3). As previously observed by Kroger-Koepke et al. (33), the levels of the alkylation products were inversely related to ionic strength and the effect of ionic strength was dependent on the individual alkylation product. The formation of N7HOEtG was the most dependent on the ionic strength of the reaction medium, and the formation of PTEs was the least influenced. These results indicate that the hydroxylation reactions proceeding via an SN2 mechanism are more sensitive to the effects of ionic strength then are the corresponding chloroethylation reactions. Similar interpretations have been provided by Kroger-Koepke et al. (33) in their investigation of the effects salts on DNA alkylation. Inhibition of alkylation product formation was also dependent on the cation. For example, if we compare the formation of N7-HOEtG in 7.5 mM NaCl versus that in 1 mM MgCl2, we find that although the ionic strengths of the solutions are similar (-2.1 vs -2.5, respectively) the formation of N7-HOEtG is inhibited by 43 and 95%, respectively. The addition of spermine resulted in a concentration-dependent inhibition of DNA alkylation.

Effect of Cations on DNA Alkylation

Comparison of the different cations demonstrates that the order of efficiency of inhibition of DNA alkylation was as follows: spermine > Mg > Na. These differences are consistent with the efficiencies of binding of these cations to DNA (42, 43). 9L cells were treated with [3H]CNU, and the DNA alkylation products that formed were analyzed. Although the same products were produced in both 9L cells and purified DNA, the relative extent of formation of the individual products was different. In 9L cells, the principal alkylation product that formed was the PTEs. This is similar to what has been observed for treatment of cells with ethylnitrosourea (26). N7-HOEtG and N7-ClEtG were also significant alkylation products; however, the relative percentage of N7-HOEtG was considerably reduced in 9L cells compared to that in DNA. If we compare the distribution of the alkylation products, the profile of 9L cells treated with CNU most closely resembles the profile of DNA reacted in the presence of 75 mM NaCl. Taken together, these results demonstrate that within the cell the reaction of CNU-derived intermediates with cellular DNA has been modified compared to that with purified DNA. We interpret this result to indicate that cellular chromosomal proteins, including histones and polyamines, in addition to cellular cations such as Na+ and Mg2+ act to modify the reaction of cellular DNA with the reactive intermediates formed by CNU.

Acknowledgment. These studies were supported by NIH Grants CA 13525 and CA 80685.

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