- Email: [email protected]
High binding of formic acid to biomacromolecules in mice
Nuclear Instruments and Methods in Physics Research B 223–224 (2004) 745–749 www.elsevier.com/locate/nimb
High binding of formic acid to biomacromolecules in mice H.F. Wang a, L.H. Xu b, H.F. Sun a, B. Xue a, Y.F. Liu K.X. Liu c, H.J. Ma c, Z.Y. Guo c a
a,*
, S.X. Peng c,
Department of Applied Chemistry and Department of Chemical Biology, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, PR China b Department of Environmental Science and Engineering, Fudan University, Shanghai 200433, PR China c Institute of Heavy Ion Physics, School of Physics, Peking University, Beijing 100871, PR China
Abstract As an air and water pollutant and a common metabolite of methanol, formaldehyde and acetone, formic acid has won the interest of toxicologists. Using accelerator mass spectrometry (AMS) associated with liquid scintillation counting (LSC), we investigated the formation and persistence of the formate induced liver/kidney DNA adducts and hemoglobin (Hb) adducts in mice to obtain more toxicological information of formic acid. Both the kidney/liver DNA adducts levels and the Hb adducts levels increase with increasing dose (10.0 lg/kg b.w. to 100.0 mg/kg b.w.) in a good log/log linear correlation. Even at the lowest dose, the number of formate-DNA adducts was so high compared with the published data of other chemicals. Adduction mechanisms are proposed. 2004 Elsevier B.V. All rights reserved. PACS: 87.15.R Keywords: Formic acid; Adduction; Accelerator mass spectrometry (AMS)
1. Introduction Formic acid has replaced the mineral acids in many cases of modern chemical industry. It has attracted great attention of toxicologists because it is a common metabolite of methanol, formaldehyde and acetone [1]. Also, formic acid is a widely distributed air and water pollutant [2]. The toxicology of formic acid has often been investigated in connection with methanol [3]. The majority of
*
Corresponding author. Tel.: +86-10-6275-7196; fax: +8610-6275-5409. E-mail address: [email protected] (Y.F. Liu).
formic acid is oxidized to CO2 and H2 O in tissues, such as liver, erythrocytes, kidney etc., via tetrahydrofolic acid-dependent pathway [4]. According to the limited published papers, formic acid was concluded to be neither carcinogenic nor mutagenic nor a tumor promoter [2]. It was not mutagenic in the tests of Salmonella typhimurium strains TA100, TA1535, TA97, or TA98 and the sister-chromatid exchange (SCE). Mutagenicity was reported in the test for induction of the sex-linked recessive lethal mutations and the test of chromosomal aberrations [5,6]. Information about macromolecular adduction with formic acid is unavailable. The study of the formation of adducts is an increasingly important
0168-583X/$ - see front matter 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.nimb.2004.04.138
746
H.F. Wang et al. / Nucl. Instr. and Meth. in Phys. Res. B 223–224 (2004) 745–749
research area in the field of contemporary toxicology. The presence and amount of the specific adducts correlated with a chemical exposure are most significant for the hazard identification and the cancer risk assessment [7,8]. In this study, using the ultra-sensitive accelerator mass spectrometry (AMS) and liquid scintillation counting (LSC), we investigated the formation and persistence of the formate-DNA adducts and formateHb adducts in mice to obtain more toxicological information of formic acid.
2. Experimental 2.1. Animal treatment Male Kunming mice (25–30 g), obtained from the Institute of Zoology of the Chinese Academy of Sciences (China) were used for the experiments. Mice were randomized into treatment groups (8– 10 mice/group). Formate administrations were carried out by stomach intubation (gavage) with 0.9% NaCl as the vehicle. Control animals were given 0.9% NaCl only. The formate solutions were prepared in different concentrations, spiked with 14 C-labeled sodium formate (purity 98%, specific activity 50.0 mCi/mmol, American Radiolabeled Chemical Inc.). The specific activities of these solutions were measured using the LSC (Packard Tri-cab 2750 TR/LL, Packard Co, USA). In the dose-response study, mice were sacrificed at 6 h after being administrated with 0.01, 0.10, 1.0, 10.0 or 100.0 mg/kg b.w.. In the time course study, mice were treated with a single dose of 100.0 mg/kg b.w. formate and then sacrificed at 2, 6, 24, 72, 120 h post administration, respectively. Livers and kidneys were collected for DNA isolation (livers from every two mice or kidneys from four mice were merged into one sample). Blood samples from every two mice were collected for hemoglobin (Hb) isolation. 2.2. DNA and Hb isolation DNA was extracted from liver or kidney samples using the method of Gupta [9]. The purity of the extracted DNA was monitored by UV A260 nm /
A280 nm ratio. Only DNA with an absorbance ratio of 1.82 ± 0.05 was used. Hb was isolated from the blood samples followed the used method of Wang et al. [10]. The isolated DNA/Hb was dried in vacuum. Through these long biochemical isolation procedures, the contamination of unbound 14 C onto the final DNA/Hb extract was cleaned. In the separation procedure we always did careful operation, such as multiple rinses and using new disposable tubes. In order to prove that there is no unbound 14 C in DNA except the 14 C-formate bound with DNA and the natural abundance of 14 C in DNA, we designed a checking experiment. A tracer of 14 C-formate was perfused into the liver tissues of mice without being injected 14 C-formate just before the liver tissues were homogenized. Then, the liver tissues were subjected to the separation of DNA by the regular protocol. The AMS measurement results indicated that no unbound 14 C was found in DNA. The same negative results were obtained in another checking experiment using a different 14 C-labeled compound, sodium 14 C-benzoate. Therefore, we may conclude that the protocol we use to separate DNA does exclude the existence of unbound 14 C. 2.3. Sample preparation and measurement Dry DNA/Hb sample was converted to graphite following the protocol of Vogel [11]. The graphite (around 1 mg) mixed with Co powder was used as an ion source in AMS measurement. We took great care in keeping the vacuum system in high vacuum and without being contaminated. The 14 C/ 12 C ratios of the samples were measured by the 2 · 6 MV EN Tandem AMS facility at the Institute of Heavy Ion Physics, Peking University with a sensitivity of 7 · 1015 and a counting precision of around 2% [12]. The ratio could be converted to the number of adducts based on some fundamental data (1 MC ¼ 5.9 · 1010 14 C/g C; 1 lg DNA ¼ 3240 pmol of nucleotides; 1 mol Hb ¼ 574 mol amino-acid residues; C content in DNA ¼ 30%; C content in Hb ¼ 50%; T1=2 of 14 C ¼ 5730 a) [13]. In our previous biomolecular measurements the detection limit achieved was 1 adduct per 1011 nucleotides [13] or 3 adducts per 1012 amino-acid residues [10].
10
5
10
4
10
3
10
2
10
1
747
10
When the 14 C/12 C ratio of a sample was too high to be measured by AMS, LSC was employed to measure the 14 C content of the sample. The LSC samples were prepared following a modified method of Mahin [14]. Briefly, dried DNA or Hb samples were placed in individual counting vials. After adding distilled water and perchloric acid, the vials were warmed to 64C for 0.5 h. For Hb samples, 30% hydrogen peroxide was added to each vial and then warmed to 64C for another 0.5 h. The DNA/Hb sample preparation was completed by addition of liquid scintillation cocktail. The prepared samples were measured by the LSC (Packard Tri-cab 2750 TR/LL). The number of adducts could be calculated from the radioactivity measured by the LSC.
Adducts/10 amino-acid residues
H.F. Wang et al. / Nucl. Instr. and Meth. in Phys. Res. B 223–224 (2004) 745–749
0
10 -3 10
10
-2
10
-1
10
0
10
1
10
2
10
3
Dose, mg/kg b.w. Fig. 2. Dependence of Hb adduct yields on formate concentration. Each point represents the mean ± SD of 2–4 samples (from 4 to 8 mice). The point at the highest dose was measured by LSC, and the others by AMS.
3. Results
10
4
Liver Kidney
4
5
Adducts/10 amino acid residues or 10 nucleotides
10
10
3
10
2
10
1
10
0
6
8
Adducts/10 nucleotides
The dose responses of binding of formate to hepatic/kidney DNA and Hb are shown in Figs. 1 and 2, respectively. Both of the DNA adducts and the Hb adducts increase with increasing dose in a good log/log linear mode (r P 0:998) over the entire range of doses. These data show that the dose responses of DNA and Hb adducts are very simi-
lar, presenting a positive correlation (relevant figure neglected here). At the same dose, the level of liver DNA adducts is slightly higher than that of kidney, which is consistent with the liver being the main metabolic site for formate. The DNA adduct level is almost one hundred-fold of the Hb adduct level at the same dose. The number of DNA adducts of formate is much higher than that of other
10
-3
10
-2
10
-1
10
0
10
1
10
2
10
3
Dose, mg/kg b.w.
Fig. 1. Dependence of hepatic/kidney DNA adduct yields on formate dose. Each point represents the mean ± SD of 2–4 samples (from 4 to 8 mice), except the point of hepatic DNA at 10.0 mg/kg b.w. (1 sample from 2 mice). The point of kidney DNA at 10.0 mg/kg b.w. was measured by both AMS and LSC, the points at lower doses by AMS, and the others by LSC.
18
Hb
16
H e p a tic D N A
14 12 10 8 6 4 2 0 -2
0
20
40
60
80
100
120
Time, hr
Fig. 3. Time course of the hepatic DNA/Hb adducts induced by formate. Each point represents the mean ± SD of 2–4 samples (from 4 to 8 mice), except the point of the DNA adducts at 120 h (1 sample from 2 mice). The points of Hb samples at 24, 72 and 120 h were measured by both AMS and LSC, and the others by LSC.
748
H.F. Wang et al. / Nucl. Instr. and Meth. in Phys. Res. B 223–224 (2004) 745–749
chemicals, including some well-known carcinogens, such as 2-amino-3, 8-dimethylimidazo (4,5-f) quinoxaline (MeIQx), 4-(methylnitrosamino)-1-(3pyridyl-1-butanone) (NNK) and benzene. The time curves of the formate induced hepatic DNA adducts levels and the Hb adducts levels after a single exposure of large dose (100.0 mg/kg b.w.) are shown in Fig. 3. The hepatic DNA adducts level reaches a peak at 24 h and then drops down. Formate induced Hb adducts level peaks within 2 h. From 2 to 6 h, the Hb adducts level decreases quickly. Then the Hb adducts level increases and reaches nearly a plateau 24 h later.
clue that one possible pathway of DNA adduction with formate is through the formylation of 50 -Opentose of nucleotides, presumably leading to the DNA strand cleavage. We are interested in the mechanism of the high DNA binding of formate. To clarify the formylation mechanism we are conducting the analysis of the formate-nucleosides adducts, which are formed from the metabolites of formate-DNA adducts in vivo, by means of the combination of AMS and HPLC. In addition, in this study we should not rule out the another pathway of binding, the tetrahydrofolic acid involving incorporation of formate into purines and pyrimidines, and then DNA [4].
4. Discussion Acknowledgements Compared with the published data of the adducts of other chemicals, the number of hepatic DNA adducts is very high. Under similar experimental conditions, the formate-DNA adducts numbers are about 100· that of MeIQx [15] and nicotine [13] and >25-fold larger than NNK [13]. In our experiments, the possibility of unbound 14 C-formic acid contamination onto the separated pure DNA adducts has been excluded by a checking experiment described in the preceding paragraph of DNA and Hb isolation. After a single exposure of formate, the DNA adducts in liver reach a maximum and then decrease to about 3.0 adducts/104 nucleotides after 72 h. This level appears to be stably bound to DNA. Although the role of DNA adducts in carcinogenesis process is still not fully established, it was reported that the adduct levels of carcinogens generally correlated positively with the risk of developing tumors and, the persistent DNA adduct levels after short-term (days) administration of carcinogens also influenced the tumor incidence [16]. But formic acid is not a mutagen, carcinogen and cancer promoter. A possible mechanism of Hb adduction with formate is that formate is coordinated to the heme iron through O atom. This was already verified by the NMR analysis [17]. In 1997 Kryukov reported the formylation of nucleosides via an esterification reaction between 50 -OH group of pentose and formic acid [18]. Their findings have afforded us a
This work was supported by the National Natural Science Foundation of China (Grant No. 19935020), and partly supported by the Institute of Heavy Ion Physics of PKU.
References [1] M.F. Bogeiger, Am. Ind. Hyg. Assoc. J. 48 (1987) 900. [2] National Toxicology Program (NTP), NTP toxicity report series 19, NIH Publication 92-3342. Research Triangle Park, NC: National Toxicology Program, 1992. [3] O. Roe, CRC Crit. Rev. Toxicol. 10 (1982) 275. [4] M.F. Boeniger, Am. Ind. Hyg. Assoc. J. 48 (1987) 900. [5] B.F.A. Stumm-Gegethoff, Theor. Appl. Genet. 39 (1969) 330. [6] T. Morita, K. Takeda, K. Okumura, Mutat. Res. 240 (1990) 195. [7] P.B. Farmer, Clin. Chem. 40 (1994) 1438. [8] E.R. Nestmann, D.W. Bryant, C.J. Carr, Regul. Toxicol. Pharmacol. 24 (1996) 9. [9] R.C. Gupta, Proc. Nat. Acad. Sci. USA 81 (1984) 6943. [10] Y. Wang, H.F. Wang, H.F. Sun, X.Y. Wang, Y.F. Liu, K.X. Liu, X.Y. Lu, H.J. Ma, K. Li, Chem. J. Chin. Univ. 23 (2002) 809 (in Chinese). [11] J.S. Vogel, Radiocarbon 34 (1992) 344. [12] C.E. Chen, Z.Y. Guo, S.Q. Yan, Nucl. Instr. and Meth. B 52 (1990) 306. [13] X.S. Li, H.F. Wang, X.Y. Wang, Y.F. Liu, K. Li, Z.Y. Guo, K.X. Liu, X.Y. Lu, J.J. Wang, Radiocarbon 38 (1996) 347. [14] D.T. Mahin, R.T. Lofberg, Anal. Biochem. 16 (1966) 500.
H.F. Wang et al. / Nucl. Instr. and Meth. in Phys. Res. B 223–224 (2004) 745–749 [15] K.W. Turteltaub, J.S. Felton, B.L. Gledhill, J.S. Vogel, J.R. Southon, M.W. Caffee, R.C. Finkel, D.E. Nelson, I.D. Proctor, J.C. Davis, Proc. Nat. Acad. Sci. USA 87 (1990) 5288. [16] M. Otteneder, W.K. Lutz, Mutat. Res. 42 (1999) 237.
749
[17] S. Aime, M. Fasano, S. Paoletti, F. Cutruzzola, A. Desideri, M. Bolognesi, M. Rizzi, P. Ascenzi, Biophys. J. 70 (1996) 482. [18] V.I. Kryukov, K.V. Antonov, A.I. Miroshnikov, Bioorg. Chem. 23 (1997) 903 (in Russian).
High binding of formic acid to biomacromolecules in mice H.F. Wang a, L.H. Xu b, H.F. Sun a, B. Xue a, Y.F. Liu K.X. Liu c, H.J. Ma c, Z.Y. Guo c a
a,*
, S.X. Peng c,
Department of Applied Chemistry and Department of Chemical Biology, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, PR China b Department of Environmental Science and Engineering, Fudan University, Shanghai 200433, PR China c Institute of Heavy Ion Physics, School of Physics, Peking University, Beijing 100871, PR China
Abstract As an air and water pollutant and a common metabolite of methanol, formaldehyde and acetone, formic acid has won the interest of toxicologists. Using accelerator mass spectrometry (AMS) associated with liquid scintillation counting (LSC), we investigated the formation and persistence of the formate induced liver/kidney DNA adducts and hemoglobin (Hb) adducts in mice to obtain more toxicological information of formic acid. Both the kidney/liver DNA adducts levels and the Hb adducts levels increase with increasing dose (10.0 lg/kg b.w. to 100.0 mg/kg b.w.) in a good log/log linear correlation. Even at the lowest dose, the number of formate-DNA adducts was so high compared with the published data of other chemicals. Adduction mechanisms are proposed. 2004 Elsevier B.V. All rights reserved. PACS: 87.15.R Keywords: Formic acid; Adduction; Accelerator mass spectrometry (AMS)
1. Introduction Formic acid has replaced the mineral acids in many cases of modern chemical industry. It has attracted great attention of toxicologists because it is a common metabolite of methanol, formaldehyde and acetone [1]. Also, formic acid is a widely distributed air and water pollutant [2]. The toxicology of formic acid has often been investigated in connection with methanol [3]. The majority of
*
Corresponding author. Tel.: +86-10-6275-7196; fax: +8610-6275-5409. E-mail address: [email protected] (Y.F. Liu).
formic acid is oxidized to CO2 and H2 O in tissues, such as liver, erythrocytes, kidney etc., via tetrahydrofolic acid-dependent pathway [4]. According to the limited published papers, formic acid was concluded to be neither carcinogenic nor mutagenic nor a tumor promoter [2]. It was not mutagenic in the tests of Salmonella typhimurium strains TA100, TA1535, TA97, or TA98 and the sister-chromatid exchange (SCE). Mutagenicity was reported in the test for induction of the sex-linked recessive lethal mutations and the test of chromosomal aberrations [5,6]. Information about macromolecular adduction with formic acid is unavailable. The study of the formation of adducts is an increasingly important
0168-583X/$ - see front matter 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.nimb.2004.04.138
746
H.F. Wang et al. / Nucl. Instr. and Meth. in Phys. Res. B 223–224 (2004) 745–749
research area in the field of contemporary toxicology. The presence and amount of the specific adducts correlated with a chemical exposure are most significant for the hazard identification and the cancer risk assessment [7,8]. In this study, using the ultra-sensitive accelerator mass spectrometry (AMS) and liquid scintillation counting (LSC), we investigated the formation and persistence of the formate-DNA adducts and formateHb adducts in mice to obtain more toxicological information of formic acid.
2. Experimental 2.1. Animal treatment Male Kunming mice (25–30 g), obtained from the Institute of Zoology of the Chinese Academy of Sciences (China) were used for the experiments. Mice were randomized into treatment groups (8– 10 mice/group). Formate administrations were carried out by stomach intubation (gavage) with 0.9% NaCl as the vehicle. Control animals were given 0.9% NaCl only. The formate solutions were prepared in different concentrations, spiked with 14 C-labeled sodium formate (purity 98%, specific activity 50.0 mCi/mmol, American Radiolabeled Chemical Inc.). The specific activities of these solutions were measured using the LSC (Packard Tri-cab 2750 TR/LL, Packard Co, USA). In the dose-response study, mice were sacrificed at 6 h after being administrated with 0.01, 0.10, 1.0, 10.0 or 100.0 mg/kg b.w.. In the time course study, mice were treated with a single dose of 100.0 mg/kg b.w. formate and then sacrificed at 2, 6, 24, 72, 120 h post administration, respectively. Livers and kidneys were collected for DNA isolation (livers from every two mice or kidneys from four mice were merged into one sample). Blood samples from every two mice were collected for hemoglobin (Hb) isolation. 2.2. DNA and Hb isolation DNA was extracted from liver or kidney samples using the method of Gupta [9]. The purity of the extracted DNA was monitored by UV A260 nm /
A280 nm ratio. Only DNA with an absorbance ratio of 1.82 ± 0.05 was used. Hb was isolated from the blood samples followed the used method of Wang et al. [10]. The isolated DNA/Hb was dried in vacuum. Through these long biochemical isolation procedures, the contamination of unbound 14 C onto the final DNA/Hb extract was cleaned. In the separation procedure we always did careful operation, such as multiple rinses and using new disposable tubes. In order to prove that there is no unbound 14 C in DNA except the 14 C-formate bound with DNA and the natural abundance of 14 C in DNA, we designed a checking experiment. A tracer of 14 C-formate was perfused into the liver tissues of mice without being injected 14 C-formate just before the liver tissues were homogenized. Then, the liver tissues were subjected to the separation of DNA by the regular protocol. The AMS measurement results indicated that no unbound 14 C was found in DNA. The same negative results were obtained in another checking experiment using a different 14 C-labeled compound, sodium 14 C-benzoate. Therefore, we may conclude that the protocol we use to separate DNA does exclude the existence of unbound 14 C. 2.3. Sample preparation and measurement Dry DNA/Hb sample was converted to graphite following the protocol of Vogel [11]. The graphite (around 1 mg) mixed with Co powder was used as an ion source in AMS measurement. We took great care in keeping the vacuum system in high vacuum and without being contaminated. The 14 C/ 12 C ratios of the samples were measured by the 2 · 6 MV EN Tandem AMS facility at the Institute of Heavy Ion Physics, Peking University with a sensitivity of 7 · 1015 and a counting precision of around 2% [12]. The ratio could be converted to the number of adducts based on some fundamental data (1 MC ¼ 5.9 · 1010 14 C/g C; 1 lg DNA ¼ 3240 pmol of nucleotides; 1 mol Hb ¼ 574 mol amino-acid residues; C content in DNA ¼ 30%; C content in Hb ¼ 50%; T1=2 of 14 C ¼ 5730 a) [13]. In our previous biomolecular measurements the detection limit achieved was 1 adduct per 1011 nucleotides [13] or 3 adducts per 1012 amino-acid residues [10].
10
5
10
4
10
3
10
2
10
1
747
10
When the 14 C/12 C ratio of a sample was too high to be measured by AMS, LSC was employed to measure the 14 C content of the sample. The LSC samples were prepared following a modified method of Mahin [14]. Briefly, dried DNA or Hb samples were placed in individual counting vials. After adding distilled water and perchloric acid, the vials were warmed to 64C for 0.5 h. For Hb samples, 30% hydrogen peroxide was added to each vial and then warmed to 64C for another 0.5 h. The DNA/Hb sample preparation was completed by addition of liquid scintillation cocktail. The prepared samples were measured by the LSC (Packard Tri-cab 2750 TR/LL). The number of adducts could be calculated from the radioactivity measured by the LSC.
Adducts/10 amino-acid residues
H.F. Wang et al. / Nucl. Instr. and Meth. in Phys. Res. B 223–224 (2004) 745–749
0
10 -3 10
10
-2
10
-1
10
0
10
1
10
2
10
3
Dose, mg/kg b.w. Fig. 2. Dependence of Hb adduct yields on formate concentration. Each point represents the mean ± SD of 2–4 samples (from 4 to 8 mice). The point at the highest dose was measured by LSC, and the others by AMS.
3. Results
10
4
Liver Kidney
4
5
Adducts/10 amino acid residues or 10 nucleotides
10
10
3
10
2
10
1
10
0
6
8
Adducts/10 nucleotides
The dose responses of binding of formate to hepatic/kidney DNA and Hb are shown in Figs. 1 and 2, respectively. Both of the DNA adducts and the Hb adducts increase with increasing dose in a good log/log linear mode (r P 0:998) over the entire range of doses. These data show that the dose responses of DNA and Hb adducts are very simi-
lar, presenting a positive correlation (relevant figure neglected here). At the same dose, the level of liver DNA adducts is slightly higher than that of kidney, which is consistent with the liver being the main metabolic site for formate. The DNA adduct level is almost one hundred-fold of the Hb adduct level at the same dose. The number of DNA adducts of formate is much higher than that of other
10
-3
10
-2
10
-1
10
0
10
1
10
2
10
3
Dose, mg/kg b.w.
Fig. 1. Dependence of hepatic/kidney DNA adduct yields on formate dose. Each point represents the mean ± SD of 2–4 samples (from 4 to 8 mice), except the point of hepatic DNA at 10.0 mg/kg b.w. (1 sample from 2 mice). The point of kidney DNA at 10.0 mg/kg b.w. was measured by both AMS and LSC, the points at lower doses by AMS, and the others by LSC.
18
Hb
16
H e p a tic D N A
14 12 10 8 6 4 2 0 -2
0
20
40
60
80
100
120
Time, hr
Fig. 3. Time course of the hepatic DNA/Hb adducts induced by formate. Each point represents the mean ± SD of 2–4 samples (from 4 to 8 mice), except the point of the DNA adducts at 120 h (1 sample from 2 mice). The points of Hb samples at 24, 72 and 120 h were measured by both AMS and LSC, and the others by LSC.
748
H.F. Wang et al. / Nucl. Instr. and Meth. in Phys. Res. B 223–224 (2004) 745–749
chemicals, including some well-known carcinogens, such as 2-amino-3, 8-dimethylimidazo (4,5-f) quinoxaline (MeIQx), 4-(methylnitrosamino)-1-(3pyridyl-1-butanone) (NNK) and benzene. The time curves of the formate induced hepatic DNA adducts levels and the Hb adducts levels after a single exposure of large dose (100.0 mg/kg b.w.) are shown in Fig. 3. The hepatic DNA adducts level reaches a peak at 24 h and then drops down. Formate induced Hb adducts level peaks within 2 h. From 2 to 6 h, the Hb adducts level decreases quickly. Then the Hb adducts level increases and reaches nearly a plateau 24 h later.
clue that one possible pathway of DNA adduction with formate is through the formylation of 50 -Opentose of nucleotides, presumably leading to the DNA strand cleavage. We are interested in the mechanism of the high DNA binding of formate. To clarify the formylation mechanism we are conducting the analysis of the formate-nucleosides adducts, which are formed from the metabolites of formate-DNA adducts in vivo, by means of the combination of AMS and HPLC. In addition, in this study we should not rule out the another pathway of binding, the tetrahydrofolic acid involving incorporation of formate into purines and pyrimidines, and then DNA [4].
4. Discussion Acknowledgements Compared with the published data of the adducts of other chemicals, the number of hepatic DNA adducts is very high. Under similar experimental conditions, the formate-DNA adducts numbers are about 100· that of MeIQx [15] and nicotine [13] and >25-fold larger than NNK [13]. In our experiments, the possibility of unbound 14 C-formic acid contamination onto the separated pure DNA adducts has been excluded by a checking experiment described in the preceding paragraph of DNA and Hb isolation. After a single exposure of formate, the DNA adducts in liver reach a maximum and then decrease to about 3.0 adducts/104 nucleotides after 72 h. This level appears to be stably bound to DNA. Although the role of DNA adducts in carcinogenesis process is still not fully established, it was reported that the adduct levels of carcinogens generally correlated positively with the risk of developing tumors and, the persistent DNA adduct levels after short-term (days) administration of carcinogens also influenced the tumor incidence [16]. But formic acid is not a mutagen, carcinogen and cancer promoter. A possible mechanism of Hb adduction with formate is that formate is coordinated to the heme iron through O atom. This was already verified by the NMR analysis [17]. In 1997 Kryukov reported the formylation of nucleosides via an esterification reaction between 50 -OH group of pentose and formic acid [18]. Their findings have afforded us a
This work was supported by the National Natural Science Foundation of China (Grant No. 19935020), and partly supported by the Institute of Heavy Ion Physics of PKU.
References [1] M.F. Bogeiger, Am. Ind. Hyg. Assoc. J. 48 (1987) 900. [2] National Toxicology Program (NTP), NTP toxicity report series 19, NIH Publication 92-3342. Research Triangle Park, NC: National Toxicology Program, 1992. [3] O. Roe, CRC Crit. Rev. Toxicol. 10 (1982) 275. [4] M.F. Boeniger, Am. Ind. Hyg. Assoc. J. 48 (1987) 900. [5] B.F.A. Stumm-Gegethoff, Theor. Appl. Genet. 39 (1969) 330. [6] T. Morita, K. Takeda, K. Okumura, Mutat. Res. 240 (1990) 195. [7] P.B. Farmer, Clin. Chem. 40 (1994) 1438. [8] E.R. Nestmann, D.W. Bryant, C.J. Carr, Regul. Toxicol. Pharmacol. 24 (1996) 9. [9] R.C. Gupta, Proc. Nat. Acad. Sci. USA 81 (1984) 6943. [10] Y. Wang, H.F. Wang, H.F. Sun, X.Y. Wang, Y.F. Liu, K.X. Liu, X.Y. Lu, H.J. Ma, K. Li, Chem. J. Chin. Univ. 23 (2002) 809 (in Chinese). [11] J.S. Vogel, Radiocarbon 34 (1992) 344. [12] C.E. Chen, Z.Y. Guo, S.Q. Yan, Nucl. Instr. and Meth. B 52 (1990) 306. [13] X.S. Li, H.F. Wang, X.Y. Wang, Y.F. Liu, K. Li, Z.Y. Guo, K.X. Liu, X.Y. Lu, J.J. Wang, Radiocarbon 38 (1996) 347. [14] D.T. Mahin, R.T. Lofberg, Anal. Biochem. 16 (1966) 500.
H.F. Wang et al. / Nucl. Instr. and Meth. in Phys. Res. B 223–224 (2004) 745–749 [15] K.W. Turteltaub, J.S. Felton, B.L. Gledhill, J.S. Vogel, J.R. Southon, M.W. Caffee, R.C. Finkel, D.E. Nelson, I.D. Proctor, J.C. Davis, Proc. Nat. Acad. Sci. USA 87 (1990) 5288. [16] M. Otteneder, W.K. Lutz, Mutat. Res. 42 (1999) 237.
749
[17] S. Aime, M. Fasano, S. Paoletti, F. Cutruzzola, A. Desideri, M. Bolognesi, M. Rizzi, P. Ascenzi, Biophys. J. 70 (1996) 482. [18] V.I. Kryukov, K.V. Antonov, A.I. Miroshnikov, Bioorg. Chem. 23 (1997) 903 (in Russian).