Chem. Res. Toxicol. 1995, 8, 356-362
356
DNA Base and Deoxyribose Modification by the Carbon-Centered Radical Generated from 4-(Hydroxymethy1)benzenediazoniumSalt, a Carcinogen in Mushroom Kazuyuki Hiramoto, Masae Kaku, Atsushi Sueyoshi, Mizuko Fujise, and Kiyomi Kikugawa" Tokyo College of Pharmacy, 1432-1 Horinouchi, Hachioji, Tokyo 192-03, Japan Received July 8, 1994@ Modification of the base and the sugar moieties of DNA with 4-(hydroxymethy1)benzenediazonium salt (HMBD), a carcinogen in the mushroom Agaricus bisporus, was investigated. When deoxyribonucleosides dGuo, dAdo, dThd, and dCyd were incubated with HMBD at pH 7.4and 37 "C, the levels of all the nucleosides were decreased. The decrease was inhibited by ethanol or Cys. When deoxyribose was incubated with HMBD, malonaldehyde was released a s assessed by the thiobarbituric acid reactivity. The release was inhibited by ethanol. Major products of the reaction of dGuo and dAdo with HMBD were isolated, and their structures were established to be 8-[4-(hydroxymethyl)pheny1ldGuo(8-HMP-dGuo) and 8-[4-(hydroxymethy1)phenylldAdo (8-HMP-dAdo1, respectively. Calf thymus DNA treated with HMBD was enzymatically digested into nucleosides, in which 8-HMP-dGuo and 8-HMP-dAdo were detected. Formation of the modified nucleosides in DNA was inhibited by ethanol or 2-mercaptoethanol. Malonaldehyde was released from DNA treated with HMBD, which indicated t h a t the deoxyribose moiety of DNA had been damaged. The results indicate that the 44hydroxymethyl)phenyl radical generated from HMBD can directly modify the base and the sugar moieties of DNA under the mild conditions. Inhibitory effect of ethanol was ascribable to its scavenging activity for the carbon-centered radical. The inhibitory effect of Cys and 2-mercaptoethanol was found to be due to the formation of the reversible adducts between HMBD and the SH compounds.
Introduction Mushroom Agaricus bisporus contains relatively large amounts of agaritine (1-6). Presence of two related compounds, 4-(hydroxymethy1)phenylhydrazine (7) and 4-(hydroxymethy1)benzenediazoniumion (HMBD)' (Chart 1) ( 3 , 8), in the fresh mushroom has been suggested. While agaritine shows negative results in the carcinogenicity test of mice ( 9 , I O ) , HMBD has been shown to induce mice subcutis and skin cancer (11)and also gastric cancer (12). Furthermore, HMBD was found to be mutagenic (I3,14). Hence, HMBD has received attention in relation to carcinogenicity of the mushroom. Our laboratory has demonstrated that HMBD induces DNA single strand breaks and suggested that the active species for the breaking is carbon-centered 4-(hydroxymethy1)phenyl radical generated by removal of molecular nitrogen from HMBD (25). However, a detailed profile of the reaction of HMBD with the base and the sugar moieties of DNA has not been demonstrated. In the present study, characteristics of the modification of the
* All correspondence should be addressed to this author at the Tokyo College of Pharmacy, 1432-1 Horinouchi, Hachioji, Tokyo 192-03, Japan. Phone 0426-76-4503. Fax 0426-76-4508. Abstract published in Advance ACS Abstracts, February 15,1995. Abbreviations: BHA, butylated hydroxyanisole;DEW, distortionless enhancement by polarization transfer; DMPO, 5,5-dimethyl-1pyrroline N-oxide; ESI, electrospray ionization; HMBD, 4-(hydroxymethy1)benzenediazonium salt; 8-HMP-dGu0, 8-[4-(hydroxymethyl)phenylIdGuo; 8-HMP-dAd0,8-[4-(hydroxymethyl)phenylldAdo; TBA, thiobarbituric acid. @
Chart 1
+ NiN
CH,OH HMBD base and the sugar moieties of DNA with HMBD were elucidated.
Experimental Procedures Materials. 4-(Hydroxymethy1)benzenediazoniumtetrafluoroborate (HMBD) was prepared as described previously (15) according to the method of Ross et aZ. (8). dGuo, dAdo, dCyd, and nuclease P1 (from Penicillium citrinum) were obtained from Wako Pure Chemical Industries (Osaka, Japan). dThd, calf thymus DNA, phosphodiesterase I (from Crotalus adamanteus crude dried venom), and alkaline phosphatase (type I11 from Escherichia coli) were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Cellulose powder C (above 300 mesh) for cellulose column chromatography was obtained from Advantec (Tokyo, Japan). 5,5-Dimethyl-l-pyrroline N-oxide (DMPO) was obtained from Labotec Co. (Tokyo, Japan). Analysis. UV absorption spectra were measured using a Hitachi U-2000 spectrophotometer. Fluorescence spectra were measured using a Hitachi 650-60 fluorescence spectrophotometer equipped with a xenon lamp. The instrument was standardized with a solution of 0.1 pM quinine sulfate in 0.1 N sulfuric acid to give a fluorescence intensity of 1.00 at 450 nm
0893-228xf95/2708-0356$09.00/00 1995 American Chemical Society
DNA Modification by a Carcinogen in Mushroom when excited a t 350 nm. The relative molar intensity against quinine sulfate was obtained. The concentration of DNA was determined by absorbance a t 260 nm: 1 absorbance unit corresponds to 50 pg of DNA. HPLC was carried out using a Hitachi 655 liquid chromatograph equipped with a column of YMC ODS A 303 (4.6 mm i.d. x 250 mm) (Yamamura Research Laboratories, Kyoto, Japan). The column was eluted with solvent A 20 mM ammonium formate/methanol(8:2 v/v) a t a flow rate of 1.0 mumin; solvent B: 20 mM ammonium formate/methanol(7:3.05 v/v) a t 1.0 mL/ min; solvent C: 20 mM ammonium formate/methanol(7:3.2 v/v) at 1.0 mumin; solvent D: 0.04 M acetate buffer (pH 5.5)/ methanol (6:4 v/v) at 0.8 mumin; solvent E: 10 mM sodium phosphate (pH 3.0)/methanol(8:2 v/v) at 1.0 mumin; or solvent F: 10 mM acetate buffer (pH 4.5)/methanol(8:2 v/v) a t 1.0 mL/ min. For preparative HPLC, a column of Inertsil ODS (20 mm i.d. x 250 mm) (GL Science Corp., Tokyo, Japan) was used, and the column was eluted with solvent G water/methanol(7:3v/v) or solvent H: water/methanol(6:4 v/v) a t a flow rate of 7.0 mL/ min. The peaks were monitored by UV or vis light using a Hitachi L-4200 UV-vis detector or by fluorescence using a Shimadzu RF-535 fluorescence HPLC monitor. Paper chromatography was performed using 1-butanol saturated with water as a developing solvent. Spots were visualized by irradiation at 254 nm. MS was obtained by a n electrospray ionization (ESI) technique on a TSQ 700 machine (Finnigan MAT). lH and 13CNMR spectra were obtained using a Bruker AM-400 spectrometer using MezSO&,j as a solvent and TMS as an internal standard. Signals in 13CNMR spectra were assigned by the distortionless enhancement by polarization transfer (DEPT) technique. ESR spectra were obtained on a Varian E-4 EPR spectrometer. The instrumental conditions were as follows: field setting a t 338.5 mT, scan range of 10 mT, microwave power of 10 mW, and modulation amplitude of 0.1 mT. Decrease of Deoxyribonucleosidesby Incubation with HMBD. Each nucleoside (1.0 mM) was incubated with 10 mM HMBD in 0.1 M sodium phosphate buffer (pH 7.4) a t 37 “C for 1 h in the absence and presence of 10% ethanol or 10 mM Cys. Each reaction mixture was subjected to HPLC using solvent A, and the peak due to the nucleoside was detected a t 260 nm. dCyd, dGuo, dThd, and dAdo were eluted a t retention times of 4.4,6.0,6.8, and 9.9 min, respectively. The amount of the intact nucleoside in the incubation mixture was determined by comparing the peak area with that of the standard 1.0 mM nucleoside solution. Determination of Malonaldehyde. The amount of malonaldehyde released in the reaction mixture was determined according to the method of Greenwald et al. (16).To a 0.8-mL reaction mixture were added 1.0 mL of 0.8% thiobarbituric acid (TBA) solution in water and 0.5 mL of 0.5% trichloroacetic acid, and the mixture was heated a t 100 “C for 15 min. Absorption spectrum of the mixture showed a maximum at 532 nm, and the absorbance was recorded (direct spectrometry). As a reference standard, a 0.8-mL solution of 30 pM tetramethoxypropane was similarly treated. By comparing the absorbance of the reaction mixture with that from the standard solution, the amount of malonaldehyde released was determined. For more accurate determination, a 10-pL aliquot of the reaction mixture was subjected to HPLC using solvent D, and the peak was detected at 532 nm (17)(HPLC determination). A single peak due to the red pigment derived from malonaldehyde appeared at a retention time of 12 min. Malonaldehyde was determined by comparing the peak area with that from the standard solution. 8-[4-(Hydroxymethyl)phenyl]dGuo(8-HMP-dGuo). A mixture of 0.71 g of dGuo (2.5 mmol) and 1.11g of HMBD (5 mmol) in 25 mL of water was adjusted at pH 10-11 with sodium hydroxide solution a t 0 “C and stirred a t room temperature for 24 h. Paper chromatography of the dark-brown reaction mixture revealed 6 W-absorbing spots, among which spots corresponding to dGuo (Rf0.23) and 8-HMP-dGuo (Rf0.44)were detected. The reaction mixture was evaporated to dryness and
Chem. Res. Toxicol., Vol. 8, No. 3, 1995 357 applied to a column of cellulose (20 mm i.d. x 870 mm), and the column was eluted with 1-butanol saturated with water. The fraction containing 8-HMP-dGuo as identified by paper chromatography was evaporated to dryness to afford 253 mg of solid. The solid was dissolved into 20 mL of methanol, and a 2.3-mL aliquot of the solution in 12 portions was applied to HPLC with a preparative Inertsil ODS column using solvent G. The product appeared as a single peak a t a retention time of 33 min when detected at 260 nm. The fractions containing the product were collected and evaporated to dryness to afford 30.6 mg of white amorphous solid. UV spectrum: A,, (0.1 N HC1) 309 ( E : 20 loo), A,, (0.1 M phosphate buffer (pH 7.4)) 278 (21 600), and Amax (0.1 N NaOH) 291 (16 300) nm. Fluorescence spectrum: maxima a t 297 (excitation) and 402 nm (emission) (0.1 M phosphate buffer (pH 7.4)) (relative molar intensity 1.45). ESVMS m/z: 396.4 (M Na)+, 412.4 (M K)+, 769.2 (2M Na)+, and 785.3 (2M K)+. lH NMR spectrum (6): 7.60 (2H, d, J = 8.1 Hz, benzyl aromatic), 7.47 (2H, d, J = 8.1 Hz, benzyl aromatic), 6.45 (2H, s, NHz), 6.06 ( l H , t, 1’1, 5.34 (lH, bs, 3’, 5’, or benzyl OH), 5.16 ( l H , bs, 3’, 5‘, or benzyl OH), 5.11 (2H, bs, 3‘, 5’, or benzyl OH), 4.58 (2H, s, benzyl CHz), 4.35 ( l H , m, 3’1, 3.80 ( l H , m, 4‘), 3.67 ( l H , m, 57, 3.16 (lH, m, 29, and 2.02 ( l H , m, 2’). l3C NMRspectrum (6): 157.1,153.2,151.9,147.0,143.88 (C, 2 , 4 , 6 or two benzyl C), 128.9,126.4 (CH, four benzyl CHI, 128.6 (C, 8), 117.0 (C, 51, 87.8 (CH, 47, 84.7 (CH, l’),71.2 (CH, 3’),62.5, 62.1 (CHz, 5’ or benzyl CHZ)and 36.5 (CHZ,~’).Anal. Calcd for C ~ ~ H ~ ~ N S O ~ ~C,/ ~52.65; H Z OH, : 5.35; N, 18.06. Found: C, 52.63; H, 5.25; N, 17.83. 8-[4-(Hydroxymethyl)phenyl]dAdo(8-HMP-dAdo). A mixture of 1.35 g (5 mmol) of dAdo and 5.55 g (25 mmol) of HMBD in 500 mL of water was adjusted a t pH 7-8 with a sodium hydroxide solution and kept a t 37 “C for 2 h. Paper chromatography of the dark-brown reaction mixture revealed 6 W-absorbing spots, among which the spots corresponding to dAdo (RfO.47) and 8-HMP-ado (RfO.7-0.8) were detected. The mixture was evaporated t o dryness, and a U6.5 amount of the residue was applied to a cellulose column as described above. Fractions containing 8-HMP-dAdo as analyzed by paper chromatography were collected and evaporated to dryness to afford 0.5 g of solid. The solid was dissolved in 5 mL of methanol and subjected in 15 portions t o HPLC using a preparative Inertsil ODS column and solvent H with detection at 260 nm. The fractions containing the product were collected and rechromatographed. The fractions were evaporated to dryness to afford 9.5 mg of white amorphous solid. UV spectrum: ,A (0.1 N HC1) 296 ( E : 25 800), A,, (0.1 M phosphate buffer (pH 7.4)) 274 (16 goo), and Am, (0.1 N NaOH) 275 (16 900) nm. Fluorescence spectrum: maxima at 294 (excitation) and 386 nm (emission) (0.1 M phosphate buffer (pH 7.4)) (relative molar intensity 1.56). ESI/MS m/z: 357.4 (M+). lH NMR spectrum (6): 8.15 ( l H , s, 2), 7.67 (2H, d, J = 8.1 Hz, benzyl aromatic), 7.54 (2H, d, J = 8.1 Hz, benzyl aromatic), 7.40 (2H, s, NHz), 6.15 ( l H , m, l’), 5.62 ( l H , bs, 3‘, 5’, or benzyl OH), 5.38 ( l H , bs, 3’, 5’, or benzyl OH), 5.28 ( l H , bs, 3’, 5‘, or benzyl OH), 4.62 (2H, s, benzyl CHz), 4.46 ( l H , m, 3’), 3.88 ( l H , m, 4‘), 3.69 ( l H , m, 5’1, 3.52 (lH, m, 5’), 3.30 ( l H , m, 27,and 2.15 ( l H , m, 2’). I3C NMR spectrum (6): 156.0, 150.5, 149.8, 144.7 (C, 4,6 or two benzyl C), 151.8 (CH, 2), 129.2, 126.5 (CH, four benzyl CHI, 127.8 (C, 81, 119.0 (C, 5), 88.3 (CH, 4‘),85.6 (CH, l’),71.4 (CH, 37, 62.4,62.3 (CHz, 5’ or benzyl CHZ),and 37.2 (CHz, 2’). Anal. Calcd for C17H19N504-9/&z0: C, 54.66; H, 5.61; N, 18.74. Found: C, 54.47; H, 5.13; N, 18.38. Reaction of Calf Thymus DNA with HMBD. A 40-mL mixture of 2.0 mg of calf thymus DNA and 0.10 M HMBD in 0.1 M sodium phosphate buffer (pH 7.4) was incubated a t 37 “C for 2 h in the absence and presence of 10% ethanol or 0.1 M 2-mercaptoethanol. The mixture was extracted 6 times with an equal volume of 1-butanol to be condensed into about 10 mL, then 5 times with an equal volume of a mixture of phenol saturated with 10 mM Tris-HClD mM EDTA buffer (pH 8.0)/ chloroform (1:1vh), and finally twice with an equal volume of a mixture of chlorofodisoamyl alcohol (24:l v/v). The aqueous layer was dialyzed against 2 L of physiological saline. As the
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358 Chem. Res. Toxicol., Vol. 8, No. 3, 1995 Table 1. Release of Malonaldehyde from Deoxyribose and DNA Treated with HMBW reaction mixture malonaldehyde released amt deoxyribose HMBD or DNA (mM) additives determination luM) deoxyribose 0 A 1.8 0.5 A 12.7 1 A 20.1 1 10% ethanol A 2.5 1 50 mM BHA A 2.4 1 B 19.8 1 minus 0 2 ' B 15.2 Fentonb A 49.8 DNA 0 A 0.7 A 5.7 0.5 A 6.7 1 Fentonb A 10.1 Deoxyribose (10 mM) or calf thymus DNA (3.2 mg/mL) was incubated with HMBD in 0.1 M phosphate buffer (pH 7.4) at 37 "C for 3 h. For comparison, they were incubated similarly with Fenton reagent (0.5 mM ferrous chloride and 0.5 mM hydrogen peroxide). Malonaldehyde released was determined by the TBA assay in the direct spectrometry (A) or the HPLC determination (B) (see Experimental Procedures section). In the case of the oxygen-free reaction; the TBA assay was performed in the absence of oxygen. Control reaction mixture with 1 mM HMBD alone yielded a negligible amount of malonaldehyde. The same experiments were repeated more than 3 times, and similar results were obtained. control, calf thymus DNA was similarly incubated in the absence of HMBD and extracted. The recoveries of the control DNA and the DNA treated with HMBD in the absence of ethanol or 2-mercaptoethanol were 60% and 40%, respectively, when estimated by absorbance at 260 nm. Enzymatic Digestion of DNA Treated with HMBD. The control and the modified DNAs were enzymatically digested into nucleosides. To a 1-mL solution of the control or the modified DNA (50 pg/mL) were added 0.11 mL of 0.2 M sodium acetate buffer (pH 4.8) and 1pL of a solution of nuclease PI (1 mg/50 pL) in water, and the mixture was incubated at 37 "C for 30 min. Then, 72 pL of 1 M Tris base solution was added to make the pH of the solution a t 8.8. To the mixture was added 10 p L of a solution of phosphodiesterase I ( 1 mg/mL) in 50 mM TrisHC1 buffer (pH 8.81, and the mixture was incubated at 37 "C for 60 min. After the incubation, 33 pL of 1 N HC1 was added to make the pH of the mixture 7.5. To the mixture were added 137 pL of 1M Tris-HC1 buffer (pH 7.5) and 12 pL (1.3 unit) of a solution of alkaline phosphatase (50 unitd0.45 mL of 2.5 M ammonium sulfate), and the mixture was incubated at 37 "C for 60 min. The digest was kept at -20 "C until use.
Results When a solution of 1mM each of deoxyribonucleosides dGuo, a d o , dThd, and dCyd was incubated with 10 mM HMBD a t pH 7.4 and 37 "C for 1 h, the level of each nucleoside estimated by HPLC was decreased to 70% (dGuo), 75% (dAdo), 75% (dThd), and 83% (dCyd). The decrease was markedly inhibited by addition of 10% ethanol or 10 mM Cys to the incubation mixture. Time course studies showed that the loss of dGuo reached a maximum after 20-min incubation. Deoxyribose was incubated with 0.5 or 1 mM HMBD a t pH 7.4 and 37 "C for 3 h, and the formation of malonaldehyde was assessed by the TBA reactivity (Table 1, upper part). The level of malonaldehyde released on treatment with 1 mM HMBD was higher than that released on treatment with 0.5 mM. The release of malonaldehyde was markedly inhibited by radical scavengers ethanol and butylated hydroxyanisole (BHA). The release was inhibited by removal of molecu-
Hiramoto et al. ~~~
A
HMBD Benzyl alcohol
.1
0
10
20
30
40
Retention time ( min )
Figure 1. HPLC of the incubation mixture of HMBD alone (A) and with deoxyribose (B). A solution of 1.0 mM HMBD (A) or 1.0 mM HMBD with 1 M deoxyribose (B) in 0.1 M sodium phosphate buffer (pH 7.4) was incubated at 37 "C for 1h. The mixture was applied to HPLC using solvent D with detection at 260 nm. The peak fraction corresponding to benzyl alcohol showed absorption maximum at 258 nm identical with that of authentic benzyl alcohol. The amount of benzyl alcohol was determined by comparing the peak area with that obtained from standard 1 mM benzyl alcohol solution.
lar oxygen from the incubation mixture, indicating that oxygen was necessary for the release of malonaldehyde. In the control experiment, hydroxyl radical generated from Fenton reaction similarly produced malonaldehyde as has been previously demonstrated (16). The results suggest that HMBD damaged deoxyribose by a radical mechanism. While the incubation of HMBD alone produced only 10% benzyl alcohol (Figure lA), the incubation with a high concentration of deoxyribose produced 57% benzyl alcohol (Figure 1B). The result suggests that deoxyribose donated hydrogen atom to carbon-centered 44hydroxymethy1)phenyl radical to produce benzyl alcohol. From the reaction mixture of dGuo and HMBD a major fluorescent product was isolated. MS of the product revealed a molecular ion peak (M+)a t mlz 373. 'H NMR spectrum of the product showed that the signal of the proton due to the 8-position of dGuo disappeared and those of the protons due to 4-(hydroxymethyl)phenyl group appeared. Signals of the carbons due to the 4-(hydroxymethyl)phenylgroup appeared in the 13CNMR spectrum. Elemental analysis indicated the molecular formula of the product t o be C1,H19N504. Hence, the structure of the product was established to be 8-[4(hydroxymethyl)phenyl]dGuo (8-HMP-dGuo) (Chart 2). From the reaction mixture of dAdo and HMBD a major fluorescent product was isolated. MS of the product showed a molecular ion peak (M+)at mlz 357. The signal of the proton due to the 8-position of dAdo disappeared, and those of the protons due to the 4-(hydroxymethyl)phenyl group appeared in the lH NMR spectrum. Signals of the carbons due to the 4-(hydroxymethyl)phenyl group appeared in the 13CNMR spectrum. Elemental analysis indicated the formula of the product to be C17H19N504. Hence, the structure of the product was established to be 8-[4-(hydroxymethyl)phenylldAdo(8-HMP-&do) (Chart
Chem. Res. Toxicol., Vol. 8, No. 3, 1995 359
DNA Modification by a Carcinogen in Mushroom
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B I
l
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Figure 4. HPLC of the reaction mixture of dAdo and HMBD. A mixture of 1.0 mM dAdo and 10 mM HMBD in 0.1 M sodium phosphate buffer (pH 7.4) was incubated at 37 "C for 2 h. (A) It was applied t o HPLC using solvent C with detection at 260 nm. The sample (-1 and the incubation mixture of HMBD alone
(- - -) are shown. The yield of 8-HMP-dAdo was determined by
comparing the peak area with that of the standard 30 pM 8-HMP-dAdo solution. (B) It was applied to HPLC using solvent B with the fluorometric detection at 300/400 nm.
300
400
500
Wavelength ( nm )
Figure 2. Fluorescence spectra of 8-HMP-dGuo (A) and 8-HMP-dAdo (B). Spectra were taken in 0.1 M sodium phosphate buffer (pH 7.4). Relative molar intensity indicates the intensity against quinine sulfate.
3 sCI
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8 2
-E
D
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0
10 20 30 40 50
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Retention time ( min )
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Figure 3. HPLC of the reaction mixture of dGuo and HMBD. A mixture of 0.1 mm dGuo and 10 mM HMBD in 0.1 M sodium phosphate buffer (pH 7.4) was incubated at 37 "C for 2 h. (A) It was applied to HPLC using solvent A with detection at 260 nm. The sample (-) and the incubation mixture of HMBD alone (- - -) are shown. The yield of 8-HMP-dGuo was determined by
comparing the peak area with that of the standard 30 pM 8-HMP-dGuo solution. (B) The reaction mixture was applied to HPLC using solvent B with the fluorometric detection a t 300/ 400 nm. The incubation mixture of HMBD alone did not reveal any fluorescent peaks.
Chart 2
R
I
OH
8-HMP-dGuo
OH
8-HMP-dAdo
2). 8-HMP-dGuo showed an absorption maximum a t 278 nm, and 8-HMP-dAdo a maximum a t 274 nm. 8-HMPdGuo showed fluorescence maxima a t 297 (excitation)/ 402 nm (emission)with a relative molar intensity of 1.45, and 8-HMP-dAdo showed maxima a t 2941386 nm with a relative molar intensity of 1.56 (Figure 2). Products of the reaction of each deoxyribonucleoside with 10 mM HMBD a t pH 7.4 and 37 "C for 2 h were analyzed by HPLC. Figure 3 shows the HPLC profiles of the reaction mixture of dGuo and HMBD. The Wabsorbing peak corresponding to 8-HMP-dGuo was de-
tected, but no other reaction products were detected owing to the presence of many degradation products of HMBD (Figure 3A). The yield of 8-HMP-dGuo was estimated to be 4.3% of dGuo. In addition to the peak due to 8-HMP-dGu0,a minor peak due to G1 was detected by the fluorometric detection (Figure 3B). The fluorescence spectrum of the peak fraction of GI showed maxima a t 2921398 nm, similar to that of 8-HMP-dGuo. Figure 4 shows the HPLC profiles of the reaction mixture of &do and HMBD. The UV-absorbing peak due to 8-HMPdAdo was detected, but no other reaction products were detected owing to the presence of many peaks due to the degradation products of HMBD (Figure 4A). The yield of 8-HMP-dAdo was estimated to be 4.0% a d o . In addition to the peak due to 8-HMP-dAd0, two peaks due to A1 and Az were detected by the fluorometric detection (Figure 4B). The fluorescence spectra of the peak fractions of A1 and A2 showed maxima a t 2971410 nm and 3091381 nm, respectively. No UV-absorbing or fluorescent peaks were detected in the reaction mixtures of dThd and dCyd. Calf thymus DNA was incubated with 0.1 M HMBD a t pH 7.4 and 37 "C for 2 h. The modified DNA was recovered and digested successively with nuclease PI, phosphodiesterase I, and alkaline phosphatase to be converted into nucleosides. By this digestion procedure the control DNA was converted into dGuo, a d o , dThd, and dCyd almost quantitatively when estimated by HPLC with detection at 260 nm (Figure 5A1). The modified DNA was similarly converted into four deoxyribonucleosides (Figure 5B1). The modified DNA gave two major and several minor fluorescent peaks (Figure ~ B z )whereas , the control DNA did not give these peaks (Figure 5Az). The retention times of two major fluorescent peaks were identical with those of 8-HMP-dGuo and 8-HMP-dAdo. Fluorescence spectra of two major peak fractions showed maxima at 304394 nm and 2941377 nm, which were similar to those of the standard 8-HMP-dGuo and 8-HMP-dAd0,respectively. Cochromatography of the digest with 8-HMP-dGuo and 8-HMP-dAdo revealed that these two peaks were indistinguishable from those of the standard nucleosides. Three of the minor fluorescent peaks may have been derived from dGuo and dAdo moieties because the retention times of these peaks were identical with those of AI, GI, and Az, and the fluorescence spectra of these peak fractions showed maxima a t 302/397 nm (similar to that of GI), a t 3011408 nm (similar to that of AI), and a t 3121383 nm (similar to that of Ad.
Chem. Res. Toxicol., Vol. 8, No. 3, 1995
Hiramoto et al. 1 mT
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Retention time (min ) Figure 5. HPLC of the enzymatic digests of the control (A) and the HMBD-modified calf thymus DNA (B). The control and the calf thymus DNA modified with 0.1 M HMBD at pH 7.4 and 37 "C for 2 h were enzymatically digested (see Experimental Procedures section). (A1 and B1) A 10-pL aliquot of the digest (43 pg of DNNmL = 128pM nucleoside) was subjected to HPLC using solvent A with detection at 260 nm. The concentration of dCyd, dGuo, dThd, and dAdo in the digest was determined by comparing the peak area with that of the standard 20 pM nucleoside solution. The sum of the concentrations of the nucleosides was 127 pM for the control (AI)and 134 pM for the modified DNA (B1). (A2 and Bz) A 10-pL aliquot of the digest was analyzed by HPLC using solvent B with the fluorometric detection a t 300/400 nm. The concentrations of 8-HMP-dGuo and 8-HMP-dAdo were determined by comparing the peak area with those of the standard 20 nM solutions. Table 2. 8-HMP-dGuoand 8-HMP-dAd0Levels in Calf Thymus DNA Treated with HMBDa modified nucleoside level/105 nucleosides modified nucleoside +none +lo% ethanol +O. 1 M 2-mercaptoethanol 8-HMP-dGuo 12.2 6.1 6.9 16.8 21.1 8-HMP-dAdo 16.7 33.0 9.5 10.4 18.8 a Calf thymus DNA was incubated with 0.1 M HMBD a t pH 7.4 and 37 "C for 2 h in the absence and presence of ethanol or 2-mercaptoethanol. The modified DNA was digested into nucleosides, and the levels of 8-HMP-dGuo and 8-HMP-dAdo were determined by HPLC (Figure 5Bz).
The levels of 8-HMP-dGuo and 8-HMP-dAdo in the modified DNA were determined by HPLC with the fluorometric determination. Three determinations indicated that the level of 8-HMP-dGuo was 12-21/105 nucleosides and that of 8-HMP-dAdo was 16-33/105 nucleosides (Table 2). The levels of these modified nucleosides were markedly decreased when DNA was treated with HMBD in the presence of ethanol and 2-mercaptoethanol. Release of malonaldehyde from calf thymus DNA treated with HMBD was examined (Table 1,lower part). It was found that DNA treated with 0.5 or 1mM HMBD released malonaldehyde, as well a s DNA treated with hydroxyl radical generated from Fenton reagent. Hence, the deoxyribose moiety of DNA was damaged by HMBD. Mechanisms of the inhibition of the HMBD-induced modification of DNA base and sugar moieties by ethanol, Cys, and 2-mercaptoethanol were investigated. The ESR spin-trapping technique indicated that the intensity of 6 line signals of the DMPO carbon-centered radical with hyperfine splitting constants of aN = 1.59 mT and aH =
-.
C 4.
D -.
Figure 6. ESR spectra of the incubation mixture of 10 mM HMBD and 0.1 M DMPO (A) in the presence of 10% ethanol (B), 0.1 M Cys (C), and 0.1 M 2-mercaptoethanol (D).Receiver gain was set at 250. I
8 m
e
4
9
d
200
300 400 Wavelength ( nm )
0 10 20 Retention time ( min )
Figure 7. Absorption spectra (A) and HPLC (B) of the mixture of HMBD and Cys. 0.1 mM HMBD was mixed with 0 (a),0.025 (b),0.05 (c), 0.075 (d), 0.1 (e), or 0.2 mM (0Cys in 10 mM sodium phosphate buffer (pH 7.0). (A) Absorption spectra were recorded within 5 min after mixing. (B) HPLC was performed immediately after mixing using solvent F with detection at 320 nm.
2.43 mT appearing in the incubation mixture of HMBD and DMPO (Figure 6A) (15)was extensively decreased by addition of ethanol (Figure 6B). This decrease may be caused by donating a hydrogen atom to 44hydroxymethy1)phenyl radical and thus scavenging the radical (15). When Cys (Figure 6C) or 2-mercaptoethanol (Figure 6D) was added to the incubation mixture, the signals of the adduct did not appear. When HMBD was mixed with increasing amounts of Cys a t pH 7.0, the W spectrum of the compound showing an absorption maximum at 275 nm was spontaneously changed (Figure 7A). The adduct with an absorption maximum at 320 nm was produced. HPLC of the mixture revealed two peaks a t retention times of 5 and 22 min, the former corresponding to HMBD and the latter to the adduct (Figure 7B). The adduct was too unstable to be isolated. When pH of the neutral mixture of HMBD and an excess amount of Cys was made a t 1.0, the W spectrum of the mixture showed an absorption maximum a t 275 nm and HPLC of the mixture showed a single peak with a retention time a t 5 min. When pH of the acidic mixture was readjusted a t 7.0, the mixture showed a n absorption maximum a t 320 nm and a single peak with a retention time at 22 min (data not shown).
Chem. Res. Toxicol., Vol. 8, No. 3, 1995 361
DNA Modification by a Carcinogen in Mushroom
Scheme 1 Purine base damage
NiN
DNA
Y
CHZOH
CHZOH I
if
Pyrimidine base damage CH3CH20H
2-Mercaptoethanol
1
Deoxyribose damage I
t
Adduct
9 CHZOH
Hence, HMBD formed certain reversible adduct with Cys at the neutral pH. The reaction of HMBD with 2-mercaptoethanol gave similar results. Hence, Cys and 2-mercaptoethanol may mask HMBD by formation of reversible adducts a t the neutral pH so as not to generate 4-(hydroxymethyl)phenyl radical.
Discussion HMBD is a potent carcinogen or mutagen in mushroom A. bisporus (3, 8, 11-14). The mechanism of action of HMBD has not yet been elucidated. Our previous study (15)has shown that the compound induces DNA single strand breaks at the nonspecific sites of the phosphodiester bonds of DNA. It enters cells to break the intracellular double stranded DNA into smaller fragments. The active species for the DNA breaking has been suggested to be carbon-centered 4-(hydroxymethyl)phenyl radical generated by removal of nitrogen molecule according to the Gomberg-Bachman reaction (18). Generation of 4-(hydroxymethyl)phenyl radical has been elucidated by its conversion into benzyl alcohol in the presence of a hydrogen donor ethanol, and by the formation of spin adducts of the carbon-centered radical. In addition to the previous finding, it was clearly demonstrated in the present study that DNA base and sugar moieties were modified by the carbon-centered radical. It has been known that the reaction of purine nucleosides with arenediazonium salts affords 8-arylpurine nucleosides or 8-arylpurine bases by removal of nitrogen molecule under relatively rigorous conditions (19-21 1. Stock and his co-workers (20, 21) have shown that carbon-centered radicals generated in the reactions participate in the 8-aryl substitution. In these reactions, however, 8-arylguanine nucleosides were obtained (201, but 8-aryladenine nucleosides were not obtained because the glycosyl bonds were cleaved (211. In the reactions of dGuo and dAdo with HMBD under milder conditions, 8-HMP-dGuo and 8-HMP-dAdo were produced without accompanying cleavage of the glycosyl bonds. The same adducts were also detected in DNA modified with HMBD. The results clearly demonstrate that carbon-centered 4-(hydroxymethyl)phenyl radical can directly attack a t the 8-positions of the purine base moieties of DNA
t Strand breaking
without generating oxygen-derived radicals. Hydroxyphenyl radicals generated from diazoquinones, structural analogs of arenediazonium salts, can add to the 8-position of purine nucleosides via the similar carbon-centered radical mechanisms (22). Because the extents of the loss of dGuo and dAdo by HMBD were greater than the yields of 8-HMP-dGuo and 8-HMP-dAdoin the nucleoside level reactions, other unidentified compounds may be produced. The loss of dThd and dCyd induced by HMBD was extensive in the nucleoside level reactions, but no products could be identified. The levels of 8-HMP-dGuo and 8-HMP-dAdo in calf thymus DNA modified with HMBD were estimated to be 10-40/105nucleosides. The modification of DNA was conducted at a high concentration of HMBD (0.1 MI in order to determine the modified nucleosides by fluorometry, whose sensitivity is generally lower than that obtained using the radiometric method. This concentration was much higher than the reasonable intracellular concentrations a t below 1 mM. Therefore, the levels of the modified purine nucleosides in DNA treated with the meaningful concentrations of HMBD may be considered to be much lower. In contrast, because HMBD a t above 1mM concentration effectively cleaves the DNA strands a t the nonspecific sites (15),it is likely that the larger DNA fragments were recovered but the smaller DNA fragments were not recovered in the workup process of the present study. Considering the possibility that base modification accelerates the cleavage of the phosphodiester bonds, the modified purine bases may be rich in the unrecovered smaller DNA fragments. Hence, a considerable extent of purine base modification could be expected in the modification of DNA with HMBD a t reasonable intracellular concentrations. As well as hydroxyl radical generated by Fenton reaction, HMBD induced the release of malonaldehyde from deoxyribose and DNA. It is suggested that hydroxyl radical reacts with deoxyribose by abstracting hydrogen atom, leading to the sugar radicals which in turn release malonaldehyde (16). Two explanations for the mechanism of the release of malonaldehyde by HMBD could be made. One is the direct abstraction of hydrogen atom from deoxyribose by the carbon-centered radical, and another is the abstraction of hydrogen atom by the
362 Chem. Res. Toxicol., Vol. 8, No. 3, 1995 oxygen radicals, i.e., hydroxyl radical, derived from the carbon-centered radical. The sugar radicals thus generated swiftly reacted with oxygen molecules to form peroxy sugar radicals which were in turn broken down into malonaldehyde. It is likley that molecular oxygen is required for the release of malonaldehyde. The base modification and the damage of deoxyribose in DNA by HMBD were effectively inhibited by ethanol, Cys, or 2-mercaptoethanol. Inhibition by ethanol has been found due to its scavenging activity of the carboncentered radical (151,but the mechanism of the inhibition of Cys and 2-mercaptoethanol was different. Cys and 2-mercaptoethanol reacted with HMBD to form unstable adducts which may prevent the generation of the carboncentered radical. In conclusion, HMBD generated carbon-centered 44hydroxymethy1)phenyl radical which reacted with the purine bases to produce 8-(4-hydroxymethyl)phenyl-substituted purine bases, which damage all the bases and deoxyribose in DNA molecules (Scheme 1). As well as the DNA strand breaking activity of the radical (151, these modifications may be relevant to the carcinogenicity and mutagenicity of HMBD (11,12).
Acknowledgment. This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Japan. References Levenberg, B. (1960) Isolation and enzymatic reactions of agaritine, a new amino acid derivative from Agaricaceae. Fed. Proc., Fed. Am. Sac. Exp. Biol. 19, 6. Levenberg, B. (1961) Structure and enzymatic cleavage of agaritine. A phenylhydrazide of L-glutamic acid isolated from Agari83, 503-505. caceae. J . Am. Chem. SOC. Levenberg, B. (1962) An aromatic diazonium compound in the mushroom Agaricus bisporus. Biochem. Biophys. Acta 63,212214.
Levenberg, B. (1964) Isolation and structure of agaritine, a y-glutamyl-substituted arylhydrazine derivative from Agaricaceae. J. Biol. Chem. 239, 2267-2273. Daniels, E. G., Kelly, R. B., and Hinman, J. W. (1961) Agaritine: An improved isolation procedure and confirmation of structure by synthesis. J . Am. Chem. SOC. 83, 3333-3334. Kelly, R., Daniels, E. G., and Hinman, J. W. (1962) Agaritine: Isolation, degradation, and synthesis. J . Org. Chem. 27, 32293231.
Gigliotti, H. J., and Levenberg, B. (1964)Studies on the y-glutamyl transferase ofAgaricus bisporus. J . Biol. Chem. 239,2274-2284.
Hiramoto et al. ( 8 ) Ross, A. E., Nagel, D. L., and Toth, B. (1982) Evidence for the
occurrence and formation of diazonium ions in the Agaricus bisporus mushroom and its extracts. J . Agric. Food Chem. 30, 521-525. (9) Toth, B., Raha, C. R., Wallcave, L., and Nagel, D. (1981)
Attempted tumor induction with agaritine in mice. Anticancer Research and Clinical Oncology 93, 109-121. (10) Toth, B., and Sornson, H. (1984) Lack of carcinogenicity of agaritine by subcutaneous administration in mice. Mycopathologia 86,75-79. (11) Toth, B., Patil, K., and Jae, H. (1981) Carcinogenesis of 4-(hydroxymethy1)benzenediazonium ion (tetrafluoroborate)of Agaricus bisporus. Cancer Res. 41, 2444-2449. (12) Toth, B., Nagel, D., and Ross, A. (1982)Gastric tumorigenesis by a single dose of 4-(hydroxymethyl)benenediazoniumion of Agaricus bisporus. Br. J . Cancer 46, 417-422. (13) Friederich, U., Fischer, B., Luthy, J., Hann, D., Schlatter, C., and Wurgler, F. E. (1986) The mutagenic activity of agaritine-a constituent of the cultivated mushroom Agaricus bisporus-and its derivatives detected with the Salmonelldmammalian microsome assay (Ames Test). 2.Lebensm.-Unters. Forsch. 183,8589. (14) Rogan, E. G., Walker, B. A,, Gingell, R., Nagel, D. L., and Toth, B. (1982) Microbial mutagenicity of related hydrazines. Mutat. Res. 102, 413-424. (15) Hiramoto, K., Kaku, M., Kato, T., and Kikugawa, K. (1995) DNA
strand breaking by the carbon-centered radical generated from 4-(hydroxymethyl)benzenediazoniumsalt, a carcinogen in mushroom Agaricus bisporus. Chem.-Biol. Interact. 94, 21-36. (16) Greenwald, R. A., Rush, S. W., Moak, S. A,, and Weitz, Z. (1989)
Conversion of superoxide generated by polymorphonuclear leukocytes to hydroxyl radical: A direct spectrophotometric detection system based on degradation of deoxyribose. Free Radical BioE. Med. 6, 385-392. (17) Kosugi, H., Kojima, T., and Kikugawa, K. (1993) Characteristics of the thiobarbituric acid reactivity of human urine as a possible consequence of lipid peroxidation. Lzpids 28, 337-343. (18)Bachmann, W. E., and Hoffman, R. A. (1944) The preparation of unsymmetrical biaryls by the diazo reaction and the nitrosoacetylamine reaction. Org. React. (N.Y.) 2, 224-241. (19) Hoffmann, H.-D., and Muller, W. (1965) Simple arylation of guanosine in the 8-position by diazonium salt. Biochim. Biophys. Acta 123, 421-424. (20) Hung, M.-H., and Stock, L. M. (1982) Reaction of benzenediazonium ions with guanine and its derivatives. J . Org. Chem. 47, 448-453. (21) Chin, A,, Hung, M.-H., and Stock, L. M. (1981) Reactions of benzenediazonium ions with adenine and its derivatives. J . Org. Chem. 46, 2203-2207. (22) Kikugawa, K., Kato, T., and Kojima, K. (1992) Substitution ofpand o-hydroxyphenyl radicals at the 8 position of purine nucleo-
sides by reaction with mutagenic p- and o-diazoquinones. Mutat. Res. 268, 65-75.
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