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Formation of DNA adducts and oxidative DNA damage in rats treated with 1,6-dinitropyrene
Cancer Letters. 71 (1993) 51-56 Elsevier Scientific Publishers Ireland
51 Ltd.
Formation of DNA adducts and oxidative treated with 1,6-dinitropyrene Z. DjuriC”, D.W. Potterb,
S.J. Gulp”, D.A. Luongoa
DNA damage in rats
and F.A. Beland’
“Department of Internal Medicine. Wayne State Universiiy, Detroit. MI 48201. hTo.uicology Division. Rohme and Haas Co., Spring House, PA 19477 and ‘DiGion of Biochemical Toxicology. National Cenrer for To.Gological Research. Je/ferson. AR 72079 /USA) (Received (Accepted
29 January 1993) 29 March 1993)
Summary In vitro metabolism studies have indicated that the tumorigenic environmental pollutant 1,6dinitropyrene has the potential to bind covalently to DNA and to induce oxidative DNA damage. We have determined if 1,6-dinitropyrene treatment will cause both types of DNA damage in vivo. Female Sprague-Dawley rats were given a single intraperitoneal injection of 1,6_dinitropyrene, and covalent DNA adduct formation, as indicated by the presence of N-(deoxyguanosin-8-yl)-I -amino6-nitropyrene, and oxidative DNA damage, as indicated by increases in 5-hydroxymethyl-2’deoxyuridine and 8-hydroxy-2’-deoxyguanosine, were assessed at 3, 12, 24 and 48 h after dosing. 32P-postlabeling analyses of DNA isolated from liver, mammary gland, bladder and nucleated blood cells indicated the formation of N-(deoxyguanosin-8-yl)-1-amino-6-nitropyrene, with the levels being highest in the bladder. 5-hydroxymethyl-2’-deoxyuridine was detected in DNA from each of these tissues, and the levels of this oxidized nucleoside were higher in the mammary glands and livers of 1,6-dinitropyrene-treated rats. 1,6-Dinitropyrene dosing did not affect the levels of 8-hydroxy-2’-deoxyguanosine in these two tissues. These results indicate that exposure to 1,6Correspondence IO: Dr. Zora DjuriC. Hematology/Oncology. P.O. Box 02143. Detroit. MI 48201, USA. 0304-3835/93/$06.00 0 1993 Elsevier Scientific Printed and Published in Ireland
Publishers
Ireland
dinitropyrene can result in increased levels of 5hydroxymethyl-2’-deoxyuridine in addition to covalent DNA adduct formation.
Keywords: 3zP-postiabeling; mass spectrometry; N(deoxyguanosin-8-yl)-l-amino-6-nitropyrene; 5-hydroxymethyl-2’-deoxyuridine; S-hydroxy-2’-deoxyguanosine Introduction 1,6-Dinitropyrene is an environmental pollutant that induces tumors in a variety of tissues in experimental animals. In newborn CD-l mice, for instance, intraperitoneal injection of this nitropolycyclic aromatic hydrocarbon elicits the formation of liver tumors [25], while a similar treatment in newborn CD rats causes a very rapid induction of malignant fibrous histocytomas [ 151. The mammary gland also appears to be a target tissue in CD rats; however, the aggressive nature of the histocytomas precludes a significant tumorigenic response in this tissue. Malignant fibrous histocytomas have also been detected in mice [23] and rats [20] injected subcutaneously with 1,6dinitropyrene, while intrapulmonary administration causes lung tumors in both rats [ 16,181 and hamsters [22]. The mechanism of 1,6-dinitropyrene tumorigenesis is thought to involve metabolic reduction to a Ltd
52
DNA-binding species [2,9]. Thus. the in vitro nitroreduction of 1,6-dinitropyrene to N-hydroxyI-amino-6-nitropyrene and its O-acetylated derivative has been shown to result in the formation of N-(deoxyguanosin-8-yl)-1-amino-6-nitropyrene [6,8], and the same DNA adduct has been detected in target and non-target tissues in vivo [3,8,21]. In addition to DNA adduct formation, the in vitro nitroreduction of 1,6-dinitropyrene produces metabolites that can react with molecular oxygen to give reactive oxygen species [7,10,12]. This suggests that 1,6-dinitropyrene could cause both DNA adduct formation and oxidative DNA damage in vivo. To investigate this possibility, we treated Sprague-Dawley rats with a single dose of 1,6-dinitropyrene and assayed for the formation of N-(deoxyguanosin-8-yl)-l-amino-6-nitropyrene as well as for the production of oxidative DNA damage, as indicated by increases in 5-hydroxymethyl-2’-deoxyuridine and 8-hydroxy-2’-deoxyguanosine. Materials and Methods Sixteen female Sprague-Dawley rats (150- 170 g; 45 days old; obtained from the breeding colony at the National Center for Toxicological Research) were treated by intraperitoneal injection with 32 pg of 1,6-dinitropyrene (Sigma Chemical Co., St. Louis, MO) in 160 ,J trioctanion (tricaprylin, C8:0, Pfaltz and Bauer, Waterbury, Conn.). A similar number of control rats received trioctanion only. Three, 12, 24, and 48 h following treatment, four control and four experimental rats were exposed to carbon dioxide and killed by decapitation. After collecting blood in heparinized tubes, the liver, bladder and mammary glands (the fourth and fifth mammae) were excised. Nuclei were isolated from frozen blood by the method of Ciulla et al. [5], except that 50 mM mannitol was added to the sucrose buffer used to lyse the cells. Mammary epithelial cells were prepared from fresh tissue by the method of Moon et al. [ 191. Hepatic nuclei were isolated from frozen tissue as described by Basler et al. [ 11. Bladders were stored frozen. DNA was isolated from thawed nuclei, cells, or tissues, as previously described [8]. The DNA was then dissolved in 5 mM Bis-Tris, 0.1 mM EDTA. pH
7.1, quantified by UV spectrometry, and divided into aliquots for analyses of DNA adducts and oxidative DNA damage. DNA adducts were assayed by 32P-postlabeling as described by Smith et al. [21]. Adducts were identified through comparison with a DNA standard containing N-(deoxyguanosin-8-yl)-l-amino6-nitropyrene, which was prepared by reacting Nhydroxy- 1-amino-6-nitropyrene with DNA at pH 5 [21]. Adduct levels were quantified with a Molecular Dynamics 400E phosphorimager through reference to a DNA standard obtained from the lungs of rats administered [4,5,9,10-‘H] 1,6dinitropyrene. This standard has been shown to contain N-(deoxyguanosin-8-yl)1-amino-6-nitropyrene at a level of 3.8 adducts/107 nucleotides [21]. DNA standards were included in all “Ppostlabeling assays. Oxidative DNA damage was assessed after enzymatically hydrolyzing the DNA to nucleosides. The levels of 5-hydroxymethyl-2’-deoxyuridine were determined by gas chromatography with mass spectral detection as reported by Djurid et al. [l 11. 8-Hydroxy-2’-deoxyguanosine was quantified by high-pressure liquid chromatography (HPLC) with electrochemical detection [ 131. Results
32P-postlabeling analyses of DNA from the mammary glands, liver, bladder and nucleated blood cells of rats treated with 1,6-dinitropyrene indicated the presence of a single major adduct that was not detected in DNA from rats treated with the solvent alone. Representative chromatograms from mammary gland DNA are shown in Fig. 1; similar chromatograms were obtained with DNA from the other tissues. As shown in Fig. 1, the adduct detected in 1,6_dinitropyrenetreated animals had elution characteristics identical to the DNA adduct standard, N-(deoxyguanosin-8-yl)-1-amino-6-nitropyrene. With the exception of nucleated blood cells, the levels of this adduct within a particular tissue were relatively constant at each of the time points examined (Table I). The highest adduct levels were detected in the bladder, which had an average value of
Fig. 1. Autoradiograms of 32P-postlabeled DNA adducts from (A) DNA modified to give N-(deoxyguanosin-X-yl)-1-amino-6-nitropyrene (dG-CS-ANP), (B) mammary mary gland DNA from a rat treated with 1,6-dinitropyrene.
34.4 f 7.9 adducts/lO’ nucleotides. In the mammary gland the average concentration was fivefold lower, with a value of 6.7 + 2.3 adducts/lO* nucleotides, which was approximately twice the limit of detection (3.8 adducts/lO’ nucleotides) for this tissue under the assay conditions. With the liver and nucleated blood cells, the adduct was detected visually in 50% of the samples analyzed from the 1,6-dinitropyrene-treated rats, and the average concentration was less than twice the limit of detection for these tissues. Table I.
Levels of N-(deoxyguanosin-8-y])-1-amino-6-nitropyrene
Tissue
Group
Bladder Liver Nucleated cells
blood
Treated Control Treated Control Treated Control Treated
_%Hydroxymethyl-2’-deoxyuridine Mass spectral analyses indicated the presence of 5-hydroxymethyl-2’-deoxyuridine in both control and experimental rats. In control rats the levels of Shydroxymethyl-2’-deoxyuridine were about twofold higher in liver than in mammary gland, bladder or nucleated blood cells (Table II). Following treatment with 1,6-dinitropyrene, the levels of this oxidized base were elevated in mammary gland DNA at each time point, with the increase being statistically significant at 12, 24 and in tissues from female rats treated
with 1.6-dinitropyrene”.
Time after treatment 3h
Adducrs/108nucleotides Mammary gland Control
in vitro with N-hydroxy-I-amino-6-nitropyrene gland DNA from a control rat. and (C) mam-
3.8 4.8 2.7 41.5 2.9 2.4 1.5 5.1
12 h
* f f f + f
0.7 0.3b 1.4 10.5c 0.9 1.5 l 0.7 f l.lC
3.8 9.8 2.7 30.1 2.9 4.6 1.5 2.3
24 h
f 0.7 f 5.5c l 1.4 f 25.5b,C + 0.9 f 1.4’ f 0.7 l 1.3
3.8 6.8 2.7 25.4 2.9 4.6 1.5 1.6
48 h
* f f +
0.7 3.0’ 1.4 16.1’ l 0.9 f 1.9 l 0.7 i 0.4
3.8 5.2 2.7 40.5 2.9 2.9 1.5 1.2
+ 0.7 f 4.7 f 1.4 + 20.7’ f 0.9 ZIE1.8 f 0.7 f 0.7
“Groups of four rats were treated by intraperitoneal injection with either trioctanion or I ,6-dinitropyrene in trioctanion and sacriticed at the indicated times after dosing. DNA was isolated from the tissues and DNA adducts were assayed by 3”P-postlabeling. The data for the treated rats is presented as the mean f the standard deviation for three to four animals at each time point except for the nucleated blood cell DNA, for which only two samples were analyzed. At least one control rat was analyzed at each time point and the data are presented as the mean f S.D. for all control samples within a tissue group (n 2 4). bOnly two rats were analyzed. ‘Significantly different from control DNA at the same time point (one-tailed !-test; p < 0.05).
54
Table II. Levels of 5-hydroxymethyl-2’-deoxyuridine dinitropyrene”. Tissue
Group
and 8-hydroxy-2’-deoxyguanosine
Time after treatment 3h
5-Hydroxymethvl-2’deoxyuridine/104 Mammary
gland
Control Treated Control Treated Control Treated Control Treated
Liver Bladder Nucleated cells
blood
in tissues from female rats treated with 1,6-
12 h
24 h
48 h
thymidine residues 1.75 1.96 3.77 6.95 1.93 1.69 1.22 1.27
f zt f f f + f f
0.11 1.1X 1.27 2.32b 0.65 0.58 0.85 0.67
1.32 2.38 3.38 3.32 1.72 2.79 1.96 1.76
f f f f f f f f
0 36 0.28’ 0.34 1.28 0.40 1.08 0.90 0.67
I .47 2.83 2.78 4.71 2.05 2.10 1.29 1.84
0. I3 + 0.81 h l 1.04 f 0.41” f 0.19 f 0.23 f 0.34 f 0.43
1.35 f I.85 f 3.46 + 4.89 f 1.65 f 2.28 f 1.37 f 1.47c
0.19 0.24b 0.59 2.64 0.22 1.40 0.51
2.06 1.68 1.83 2.03
+ f + f
0.68 0.32 0.44 0.36
I.44 1.52 1.65 I.51
f f + +
I.38 I.49 1.34 1.18
0.15 0.35 0.04 0.11
l
8-Hydro.~y-2’-deox~~~gguanosine/lO5deo.~yguanosine residues Mammary
gland
Control Treated Control Treated
Liver
2.13 2.17 2.18 I.34
zt + zt f
0.24 0.60 I.17 0.05
0.24 0.04 0.26 0.17
f f f +
“Groups of four rats were treated by intraperitoneal injection with either trioctanion or I,6-dinitropyrene in trioctanion and killed at the times indicated. DNA was isolated from the tissues and 5_hydroxymethyl- 2’.deoxyuridine and 8-hydroxy-2’-deoxyguanosine were assayed by mass spectrometry and HPLC with electrochemical detection. respectively. The data for the treated rats is presented as the mean f S.D. for three to four animals at each time ooint. bSignificantly different from control DNA at the same time point (two-tailed t-test; p < 0.05). COnly one rat was assayed. .
48 h (Table II). l,&Dinitropyrene administration also caused the levels of S-hydroxymethyl-2’deoxyuridine to increase in liver DNA, the increases being significant at 3 and 24 h following dosing. Slightly elevated levels of 5-hydroxymethyl-2’-deoxyuridine were detected in the bladder, but 1,Gdinitropyrene treatment appeared to have no effect on the levels of this hydroxylated nucleoside in nucleated blood cells. 8-Hydroxy-y-2’-deoxyguanosine
Due to the amount of DNA necessary for the HPLC analyses using electrochemical detection ( - 100 pg), 8-hydroxy-2’-deoxyguanosine was assessed only in liver and mammary gland DNA. As shown in Table II, the levels of this oxidized base were similar in both these tissues from control rats, and treatment with 1,6-dinitropyrene had no effect on the concentration of this hydroxylated purine in either tissue. Discussion Previous
in
vitro
metabolism
studies
have
indicated that the nitroreduction of I .&dinitropyrene could result in the generation of redoxactive species in addition to electrophilic dinitropyrene metabolites [7,10,12]. This suggests that 1,6_dinitropyrene has the potential to damage DNA in vivo by a dual mechanism, through DNA adduct formation as well as by the induction of oxidized DNA bases. 32P-postlabeling analyses indicated that 1,6_dinitropyrene administration did lead to DNA adduction, with N-(deoxyguanosin-S-y&l -amino-6-nitropyrene being the major adduct detected. In accord with earlier work using [4.5,9, 10-3H] 1,6-dinitropyrene combined with HPLC analyses [8], the highest levels of N-(deoxyguanosin-8-yl)-I-amino-6-nitropyrene occurred in the bladder, with lower values being found in the mammary gland and liver. The same adduct was also detected by 3’P-postlabeling analyses in nucleated blood cell DNA. Two forms of oxidative DNA damage, 5hydroxy-2 ‘-deoxyuridine and 8-hydroxy-2 ‘-deoxyguanosine, were quantified, and these hydroxylated nucleosides were found in both control and experimental rats. As compared with other
55
tissues, the levels of 5-hydroxymethyl-2’-deoxyuridine were twofold higher in the liver. This may be a reflection of lower levels of 5-hydroxymethyl2 ‘-deoxyuridine glycosylase in the liver as compared with other tissues [4]. 1,6_Dinitropyrene treatment increased the levels of 5-hydroxymethyl2 ‘-deoxyuridine in liver and mammary gland DNA, but had no effect on the levels of 8-hydroxy2’-deoxyguanosine in the same tissues. If reactive oxygen species are produced during the in vivo metabolism of 1,6-dinitropyrene, then presumably the levels of both hydroxylated nucleosides should have been increased. As this was not the case, 1,6-dinitropyrene exposure may increase the levels of 5-hydroxymethyl-2’-deoxyuridine via another mechanism. For instance, increased levels of this nucleoside may be a biological response to 1,6-dinitropyrene exposure, since 5-hydroxymethyl-2’-deoxyuridine appears to be involved in normal cellular functions such as gene regulation [4]. Alternatively, the levels of both hydroxylated nucleosides may be increased upon 1,6-dinitropyrene treatment, but S-hydroxy2’-deoxyguanosine may be repaired more rapidly than 5-hydroxymethyl-2’-deoxyuridine. In mice treated with y-rays, for example, 50% of the 8-hydroxy-2’-deoxyguanosine residues were excised in about one hour [17]. In another experiment, SENCAR mice had much lower levels of 8hydroxy-2’-deoxyguanosine as compared with 5hydroxymethyl-2 ‘-deoxyuridine in their skin DNA following treatment with the tumor promoter 12-0-tetradecanoylphorbol-13-acetate [24]. In addition, when using low doses of 12-O-tetradecanoylphorbol13-acetate increases of the 8-hydroxy-2 ‘-deoxyguanosine over control values were much less than those of 5-hydroxymethyl-2 ‘-deoxyuridine. which suggests that increases in 8-hydroxy-2 ‘-deoxyguanosine may be more difficult to detect. In summary, we have found increased levels of 5-hydroxy-2’-deoxyuridine and DNA adduct formation in liver and mammary gland DNA of rats treated with 1,6_dinitropyrene. Other investigators have suggested that for carcinogenic polycyclic aromatic hydrocarbons. such as benzo[a]pyrene. DNA adducts and oxidative DNA damage are involved in tumor initiation and promotion, respec-
tively [14]. A similar scenario may be occurring with nitropolycyclic aromatic hydrocarbons, such as 1,6_dinitropyrene. Acknowledgement This work was supported in part by the Wayne State University Ben Kasle Trust Fund for Cancer Research. References 1
2
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8
Basler, J., Hastie, N.D.. Pietras, D.. Matsui, S., Sandberg. A.A. and Berezney, R. (1981) Hybridization of nuclear attached deoxyribonucleic acid fragments. matrix Biochemistry, 20, 6921-6929. Beland, F.A. and Kadlubar. F.F. (1990) Metabolic activation and DNA adducts of aromatic amines and nitroaromatic hydrocarbons. In: Handbook of Experimental Pharmacology, Vol. 94/I, pp. 267-325. Editors: C.S. Cooper and P.L. Grover. Springer-Verlag. Berlin/ Heidelberg. Beland, F.A., Fullerton. N.F.. Smith, B.A. and Heflich, R.H. (1993) Formation of DNA adducts and induction of mutations in rats treated with tumorigenic doses of 1.6dinitropyrene. Environ. Health Perspect., in press. Boorstein. R.J.. Chiu. L. and Teebor, G.W. (1989) Phylogenetic evidence of a role for 5-hydroxymethyluracilDNA glycosylase in the maintenance of 5-methylcytosine in DNA. Nucleic Acids Res.. 17. 7653-7661. Ciulla. T.A.. Sklar. R.M. and Hauser, S.L. ( 1988) A simple method for DNA purification from peripheral blood. Anal. Biochem.. 174. 485-488. Djurid, Z., Fifer. E.K. and Beland. F.A. (1985) Acetyl coenzyme A-dependent binding of carcinogenic and mutagenic dinitropyrenes to DNA. Carcinogenesis, 6. 941-944. Djurid. Z., Potter, D. W., Heflich, R.H. and Beland, F.A. (1986) Aerobic and anaerobic reduction of nitrated pyrenes in vitro. Chem.-Biol. Interact., 59. 309-324. Djuric. Z., Fifer. E.K., Yamazoe, Y. and Beland. F.A. (1988) DNA binding by I-nitropyrene and 1.6dinitropyrene in vitro and in viva: effects of nitroreductase induction. Carcinogenesis. 9. 357-364. Djurid, Z. (1989) Metabolism and DNA-binding of nitrated pyrenes. In: Reviews in Biochemical Toxicology, Vol. 10, pp. I-40. Editors: E. Hodgson. J.R. Bend and R.M. Philpot. Elsevier. New York. Djuric. Z. and McGunagle. D.L. (1989) Differences in reduction of 1.6-dinitropyrene and I-nitro-6-nitrosopyrene by rat liver cytosolic enzymes and formation of oxygen-reactive metabolites from nitrosoreduction. Cancer Lett.. 48. I3- 18. Djuric, Z.. Luongo. D.A. and Harper. D.A. (1991) Quantitation of 5-(hydroxymethylnrracil in DNA by gas chro-
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12
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matography with mass spectral detection. Chem. Res. Toxicol., 4. 687-691. Djurid, Z. (1992) Comparative reduction of I-nitro-3nitrosopyrene and I-nitro-6-nitrosopyrene: implications for the tumorigenicity of dinitropyrenes. Cancer Lett., 65, 73-78. Floyd, R.A., Watson. J.J., Wong. P.K., Altmiller. D.H. and Rickard, R.C. (1986) Hydroxyl free radical adduct of deoxyguanosine: sensitive detection and mechanisms of formation. Free Rad. Res. Comms., I. 163-172. Frenkel, K. (1989) Oxidation of DNA bases by tumor promoter-activated processes. Environ. Health Perspect., 81. 45-54. Imaida, K.. Lee. M., Wang. C.Y. and King, C.M. (1991) Carcinogenicity of dinitropyrenes in the weanhng female CD rat. Carcinogenesis, 12. 1187-l 191. Iwagawa. M., Maeda, T.. Izumi. K.. Otsuka. H.. Nishifuji, K., Ohnishi. Y. and Aoki. S. (1989) Comparative doseresponse study on the pulmonary carcinogenicity of 1,6dinitropyrene and benzo[a]pyrene in F344 rats. Carcinogenesis, 10, 1285-1290. Kasai. H.. Crain. P.F.. Kuchino, Y., Nishimura, S.. Ootsuyama, A. and Tanooka, H. (1986) Formation of 8hydroxyguanine moiety in cellular DNA by agents producing oxygen radicals and evidence for its repair. Carcinogenesis, 7, 1849-1851. Maeda, T., Izumi, K., Otsuka. H., Manabe. Y., Kinouchi. T. and Ohnishi. Y. (1986) Induction of squamous cell carcinoma in the rat lung by 1.6-dinitropyrene. J. Natl. Cancer Inst.. 76, 693-701.
19
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Moon. R.C., Janss, D.H. and Young, S. (1969) Preparation of fat cell-‘free’ rat mammary gland. J. Histochem. Cytochem.. 17, 182-186. Ohgaki. H.. Hasegawa. H., Kato, T.. Negishi, C.. Sato. S. and Sugimura, T. (1985) Absence of carcinogenicity of Initropyrene, correction of previous results, and new demonstration of carcinogenicity of 1.6-dinitropyrene in rats. Cancer Lett., 25, 239-245. Smith. B.A.. Fullerton, N.F., Aidoo, A., Heflich, R.H. and Beland. F.A. (1992) DNA adduct formation in relation to lymphocyte mutations and lung tumor induction in F344 rats treated with the environmental pollutant 1.6dinitropyrene. Environ. Health Perspect.. in press. Takayama, S.. Ishikawa. T., Nakajima. H. and Sato. S. (1985) Lung carcinoma induction in Syrian golden hamsters by intratracheal instillation of 1.6-dinitropyrene. Jpn. J. Cancer Res. (Gann), 76, 457-461. Tokiwa, H., Otofuji, T.. Horikawa, K., Kitamori, S.. Otsuka. H.. Manabe, Y., Kinouchi, T. and Ohnishi, Y. (1984) 1,6_dinitropyrene: mutagenicity in Salmonella and carcinogenicity in BALBic mice. J. Natl. Cancer Inst., 73. 1359- 1363. Wei, H. and Frenkel, K. (1991) In vivo formation of oxidized DNA bases in tumor promoter-treated mouse skin. Cancer Res.. 51. 4443-4449. Wislocki, P.G.. Bagan. ES.. Lu. A.Y.H.. Dooley, K.L.. Fu, P.P., Han-Hsu, H., Beland. F.A. and Kadlubar. F.F. (1986) Tumorigenicity of nitrated derivatives of pyrene. benz[a]anthracene, chrysene and benzo[a]pyrene in the newborn mouse assay. Carcinogenesis. 7, 13 17- 1322.
51 Ltd.
Formation of DNA adducts and oxidative treated with 1,6-dinitropyrene Z. DjuriC”, D.W. Potterb,
S.J. Gulp”, D.A. Luongoa
DNA damage in rats
and F.A. Beland’
“Department of Internal Medicine. Wayne State Universiiy, Detroit. MI 48201. hTo.uicology Division. Rohme and Haas Co., Spring House, PA 19477 and ‘DiGion of Biochemical Toxicology. National Cenrer for To.Gological Research. Je/ferson. AR 72079 /USA) (Received (Accepted
29 January 1993) 29 March 1993)
Summary In vitro metabolism studies have indicated that the tumorigenic environmental pollutant 1,6dinitropyrene has the potential to bind covalently to DNA and to induce oxidative DNA damage. We have determined if 1,6-dinitropyrene treatment will cause both types of DNA damage in vivo. Female Sprague-Dawley rats were given a single intraperitoneal injection of 1,6_dinitropyrene, and covalent DNA adduct formation, as indicated by the presence of N-(deoxyguanosin-8-yl)-I -amino6-nitropyrene, and oxidative DNA damage, as indicated by increases in 5-hydroxymethyl-2’deoxyuridine and 8-hydroxy-2’-deoxyguanosine, were assessed at 3, 12, 24 and 48 h after dosing. 32P-postlabeling analyses of DNA isolated from liver, mammary gland, bladder and nucleated blood cells indicated the formation of N-(deoxyguanosin-8-yl)-1-amino-6-nitropyrene, with the levels being highest in the bladder. 5-hydroxymethyl-2’-deoxyuridine was detected in DNA from each of these tissues, and the levels of this oxidized nucleoside were higher in the mammary glands and livers of 1,6-dinitropyrene-treated rats. 1,6-Dinitropyrene dosing did not affect the levels of 8-hydroxy-2’-deoxyguanosine in these two tissues. These results indicate that exposure to 1,6Correspondence IO: Dr. Zora DjuriC. Hematology/Oncology. P.O. Box 02143. Detroit. MI 48201, USA. 0304-3835/93/$06.00 0 1993 Elsevier Scientific Printed and Published in Ireland
Publishers
Ireland
dinitropyrene can result in increased levels of 5hydroxymethyl-2’-deoxyuridine in addition to covalent DNA adduct formation.
Keywords: 3zP-postiabeling; mass spectrometry; N(deoxyguanosin-8-yl)-l-amino-6-nitropyrene; 5-hydroxymethyl-2’-deoxyuridine; S-hydroxy-2’-deoxyguanosine Introduction 1,6-Dinitropyrene is an environmental pollutant that induces tumors in a variety of tissues in experimental animals. In newborn CD-l mice, for instance, intraperitoneal injection of this nitropolycyclic aromatic hydrocarbon elicits the formation of liver tumors [25], while a similar treatment in newborn CD rats causes a very rapid induction of malignant fibrous histocytomas [ 151. The mammary gland also appears to be a target tissue in CD rats; however, the aggressive nature of the histocytomas precludes a significant tumorigenic response in this tissue. Malignant fibrous histocytomas have also been detected in mice [23] and rats [20] injected subcutaneously with 1,6dinitropyrene, while intrapulmonary administration causes lung tumors in both rats [ 16,181 and hamsters [22]. The mechanism of 1,6-dinitropyrene tumorigenesis is thought to involve metabolic reduction to a Ltd
52
DNA-binding species [2,9]. Thus. the in vitro nitroreduction of 1,6-dinitropyrene to N-hydroxyI-amino-6-nitropyrene and its O-acetylated derivative has been shown to result in the formation of N-(deoxyguanosin-8-yl)-1-amino-6-nitropyrene [6,8], and the same DNA adduct has been detected in target and non-target tissues in vivo [3,8,21]. In addition to DNA adduct formation, the in vitro nitroreduction of 1,6-dinitropyrene produces metabolites that can react with molecular oxygen to give reactive oxygen species [7,10,12]. This suggests that 1,6-dinitropyrene could cause both DNA adduct formation and oxidative DNA damage in vivo. To investigate this possibility, we treated Sprague-Dawley rats with a single dose of 1,6-dinitropyrene and assayed for the formation of N-(deoxyguanosin-8-yl)-l-amino-6-nitropyrene as well as for the production of oxidative DNA damage, as indicated by increases in 5-hydroxymethyl-2’-deoxyuridine and 8-hydroxy-2’-deoxyguanosine. Materials and Methods Sixteen female Sprague-Dawley rats (150- 170 g; 45 days old; obtained from the breeding colony at the National Center for Toxicological Research) were treated by intraperitoneal injection with 32 pg of 1,6-dinitropyrene (Sigma Chemical Co., St. Louis, MO) in 160 ,J trioctanion (tricaprylin, C8:0, Pfaltz and Bauer, Waterbury, Conn.). A similar number of control rats received trioctanion only. Three, 12, 24, and 48 h following treatment, four control and four experimental rats were exposed to carbon dioxide and killed by decapitation. After collecting blood in heparinized tubes, the liver, bladder and mammary glands (the fourth and fifth mammae) were excised. Nuclei were isolated from frozen blood by the method of Ciulla et al. [5], except that 50 mM mannitol was added to the sucrose buffer used to lyse the cells. Mammary epithelial cells were prepared from fresh tissue by the method of Moon et al. [ 191. Hepatic nuclei were isolated from frozen tissue as described by Basler et al. [ 11. Bladders were stored frozen. DNA was isolated from thawed nuclei, cells, or tissues, as previously described [8]. The DNA was then dissolved in 5 mM Bis-Tris, 0.1 mM EDTA. pH
7.1, quantified by UV spectrometry, and divided into aliquots for analyses of DNA adducts and oxidative DNA damage. DNA adducts were assayed by 32P-postlabeling as described by Smith et al. [21]. Adducts were identified through comparison with a DNA standard containing N-(deoxyguanosin-8-yl)-l-amino6-nitropyrene, which was prepared by reacting Nhydroxy- 1-amino-6-nitropyrene with DNA at pH 5 [21]. Adduct levels were quantified with a Molecular Dynamics 400E phosphorimager through reference to a DNA standard obtained from the lungs of rats administered [4,5,9,10-‘H] 1,6dinitropyrene. This standard has been shown to contain N-(deoxyguanosin-8-yl)1-amino-6-nitropyrene at a level of 3.8 adducts/107 nucleotides [21]. DNA standards were included in all “Ppostlabeling assays. Oxidative DNA damage was assessed after enzymatically hydrolyzing the DNA to nucleosides. The levels of 5-hydroxymethyl-2’-deoxyuridine were determined by gas chromatography with mass spectral detection as reported by Djurid et al. [l 11. 8-Hydroxy-2’-deoxyguanosine was quantified by high-pressure liquid chromatography (HPLC) with electrochemical detection [ 131. Results
32P-postlabeling analyses of DNA from the mammary glands, liver, bladder and nucleated blood cells of rats treated with 1,6-dinitropyrene indicated the presence of a single major adduct that was not detected in DNA from rats treated with the solvent alone. Representative chromatograms from mammary gland DNA are shown in Fig. 1; similar chromatograms were obtained with DNA from the other tissues. As shown in Fig. 1, the adduct detected in 1,6_dinitropyrenetreated animals had elution characteristics identical to the DNA adduct standard, N-(deoxyguanosin-8-yl)-1-amino-6-nitropyrene. With the exception of nucleated blood cells, the levels of this adduct within a particular tissue were relatively constant at each of the time points examined (Table I). The highest adduct levels were detected in the bladder, which had an average value of
Fig. 1. Autoradiograms of 32P-postlabeled DNA adducts from (A) DNA modified to give N-(deoxyguanosin-X-yl)-1-amino-6-nitropyrene (dG-CS-ANP), (B) mammary mary gland DNA from a rat treated with 1,6-dinitropyrene.
34.4 f 7.9 adducts/lO’ nucleotides. In the mammary gland the average concentration was fivefold lower, with a value of 6.7 + 2.3 adducts/lO* nucleotides, which was approximately twice the limit of detection (3.8 adducts/lO’ nucleotides) for this tissue under the assay conditions. With the liver and nucleated blood cells, the adduct was detected visually in 50% of the samples analyzed from the 1,6-dinitropyrene-treated rats, and the average concentration was less than twice the limit of detection for these tissues. Table I.
Levels of N-(deoxyguanosin-8-y])-1-amino-6-nitropyrene
Tissue
Group
Bladder Liver Nucleated cells
blood
Treated Control Treated Control Treated Control Treated
_%Hydroxymethyl-2’-deoxyuridine Mass spectral analyses indicated the presence of 5-hydroxymethyl-2’-deoxyuridine in both control and experimental rats. In control rats the levels of Shydroxymethyl-2’-deoxyuridine were about twofold higher in liver than in mammary gland, bladder or nucleated blood cells (Table II). Following treatment with 1,6-dinitropyrene, the levels of this oxidized base were elevated in mammary gland DNA at each time point, with the increase being statistically significant at 12, 24 and in tissues from female rats treated
with 1.6-dinitropyrene”.
Time after treatment 3h
Adducrs/108nucleotides Mammary gland Control
in vitro with N-hydroxy-I-amino-6-nitropyrene gland DNA from a control rat. and (C) mam-
3.8 4.8 2.7 41.5 2.9 2.4 1.5 5.1
12 h
* f f f + f
0.7 0.3b 1.4 10.5c 0.9 1.5 l 0.7 f l.lC
3.8 9.8 2.7 30.1 2.9 4.6 1.5 2.3
24 h
f 0.7 f 5.5c l 1.4 f 25.5b,C + 0.9 f 1.4’ f 0.7 l 1.3
3.8 6.8 2.7 25.4 2.9 4.6 1.5 1.6
48 h
* f f +
0.7 3.0’ 1.4 16.1’ l 0.9 f 1.9 l 0.7 i 0.4
3.8 5.2 2.7 40.5 2.9 2.9 1.5 1.2
+ 0.7 f 4.7 f 1.4 + 20.7’ f 0.9 ZIE1.8 f 0.7 f 0.7
“Groups of four rats were treated by intraperitoneal injection with either trioctanion or I ,6-dinitropyrene in trioctanion and sacriticed at the indicated times after dosing. DNA was isolated from the tissues and DNA adducts were assayed by 3”P-postlabeling. The data for the treated rats is presented as the mean f the standard deviation for three to four animals at each time point except for the nucleated blood cell DNA, for which only two samples were analyzed. At least one control rat was analyzed at each time point and the data are presented as the mean f S.D. for all control samples within a tissue group (n 2 4). bOnly two rats were analyzed. ‘Significantly different from control DNA at the same time point (one-tailed !-test; p < 0.05).
54
Table II. Levels of 5-hydroxymethyl-2’-deoxyuridine dinitropyrene”. Tissue
Group
and 8-hydroxy-2’-deoxyguanosine
Time after treatment 3h
5-Hydroxymethvl-2’deoxyuridine/104 Mammary
gland
Control Treated Control Treated Control Treated Control Treated
Liver Bladder Nucleated cells
blood
in tissues from female rats treated with 1,6-
12 h
24 h
48 h
thymidine residues 1.75 1.96 3.77 6.95 1.93 1.69 1.22 1.27
f zt f f f + f f
0.11 1.1X 1.27 2.32b 0.65 0.58 0.85 0.67
1.32 2.38 3.38 3.32 1.72 2.79 1.96 1.76
f f f f f f f f
0 36 0.28’ 0.34 1.28 0.40 1.08 0.90 0.67
I .47 2.83 2.78 4.71 2.05 2.10 1.29 1.84
0. I3 + 0.81 h l 1.04 f 0.41” f 0.19 f 0.23 f 0.34 f 0.43
1.35 f I.85 f 3.46 + 4.89 f 1.65 f 2.28 f 1.37 f 1.47c
0.19 0.24b 0.59 2.64 0.22 1.40 0.51
2.06 1.68 1.83 2.03
+ f + f
0.68 0.32 0.44 0.36
I.44 1.52 1.65 I.51
f f + +
I.38 I.49 1.34 1.18
0.15 0.35 0.04 0.11
l
8-Hydro.~y-2’-deox~~~gguanosine/lO5deo.~yguanosine residues Mammary
gland
Control Treated Control Treated
Liver
2.13 2.17 2.18 I.34
zt + zt f
0.24 0.60 I.17 0.05
0.24 0.04 0.26 0.17
f f f +
“Groups of four rats were treated by intraperitoneal injection with either trioctanion or I,6-dinitropyrene in trioctanion and killed at the times indicated. DNA was isolated from the tissues and 5_hydroxymethyl- 2’.deoxyuridine and 8-hydroxy-2’-deoxyguanosine were assayed by mass spectrometry and HPLC with electrochemical detection. respectively. The data for the treated rats is presented as the mean f S.D. for three to four animals at each time ooint. bSignificantly different from control DNA at the same time point (two-tailed t-test; p < 0.05). COnly one rat was assayed. .
48 h (Table II). l,&Dinitropyrene administration also caused the levels of S-hydroxymethyl-2’deoxyuridine to increase in liver DNA, the increases being significant at 3 and 24 h following dosing. Slightly elevated levels of 5-hydroxymethyl-2’-deoxyuridine were detected in the bladder, but 1,Gdinitropyrene treatment appeared to have no effect on the levels of this hydroxylated nucleoside in nucleated blood cells. 8-Hydroxy-y-2’-deoxyguanosine
Due to the amount of DNA necessary for the HPLC analyses using electrochemical detection ( - 100 pg), 8-hydroxy-2’-deoxyguanosine was assessed only in liver and mammary gland DNA. As shown in Table II, the levels of this oxidized base were similar in both these tissues from control rats, and treatment with 1,6-dinitropyrene had no effect on the concentration of this hydroxylated purine in either tissue. Discussion Previous
in
vitro
metabolism
studies
have
indicated that the nitroreduction of I .&dinitropyrene could result in the generation of redoxactive species in addition to electrophilic dinitropyrene metabolites [7,10,12]. This suggests that 1,6_dinitropyrene has the potential to damage DNA in vivo by a dual mechanism, through DNA adduct formation as well as by the induction of oxidized DNA bases. 32P-postlabeling analyses indicated that 1,6_dinitropyrene administration did lead to DNA adduction, with N-(deoxyguanosin-S-y&l -amino-6-nitropyrene being the major adduct detected. In accord with earlier work using [4.5,9, 10-3H] 1,6-dinitropyrene combined with HPLC analyses [8], the highest levels of N-(deoxyguanosin-8-yl)-I-amino-6-nitropyrene occurred in the bladder, with lower values being found in the mammary gland and liver. The same adduct was also detected by 3’P-postlabeling analyses in nucleated blood cell DNA. Two forms of oxidative DNA damage, 5hydroxy-2 ‘-deoxyuridine and 8-hydroxy-2 ‘-deoxyguanosine, were quantified, and these hydroxylated nucleosides were found in both control and experimental rats. As compared with other
55
tissues, the levels of 5-hydroxymethyl-2’-deoxyuridine were twofold higher in the liver. This may be a reflection of lower levels of 5-hydroxymethyl2 ‘-deoxyuridine glycosylase in the liver as compared with other tissues [4]. 1,6_Dinitropyrene treatment increased the levels of 5-hydroxymethyl2 ‘-deoxyuridine in liver and mammary gland DNA, but had no effect on the levels of 8-hydroxy2’-deoxyguanosine in the same tissues. If reactive oxygen species are produced during the in vivo metabolism of 1,6-dinitropyrene, then presumably the levels of both hydroxylated nucleosides should have been increased. As this was not the case, 1,6-dinitropyrene exposure may increase the levels of 5-hydroxymethyl-2’-deoxyuridine via another mechanism. For instance, increased levels of this nucleoside may be a biological response to 1,6-dinitropyrene exposure, since 5-hydroxymethyl-2’-deoxyuridine appears to be involved in normal cellular functions such as gene regulation [4]. Alternatively, the levels of both hydroxylated nucleosides may be increased upon 1,6-dinitropyrene treatment, but S-hydroxy2’-deoxyguanosine may be repaired more rapidly than 5-hydroxymethyl-2’-deoxyuridine. In mice treated with y-rays, for example, 50% of the 8-hydroxy-2’-deoxyguanosine residues were excised in about one hour [17]. In another experiment, SENCAR mice had much lower levels of 8hydroxy-2’-deoxyguanosine as compared with 5hydroxymethyl-2 ‘-deoxyuridine in their skin DNA following treatment with the tumor promoter 12-0-tetradecanoylphorbol-13-acetate [24]. In addition, when using low doses of 12-O-tetradecanoylphorbol13-acetate increases of the 8-hydroxy-2 ‘-deoxyguanosine over control values were much less than those of 5-hydroxymethyl-2 ‘-deoxyuridine. which suggests that increases in 8-hydroxy-2 ‘-deoxyguanosine may be more difficult to detect. In summary, we have found increased levels of 5-hydroxy-2’-deoxyuridine and DNA adduct formation in liver and mammary gland DNA of rats treated with 1,6_dinitropyrene. Other investigators have suggested that for carcinogenic polycyclic aromatic hydrocarbons. such as benzo[a]pyrene. DNA adducts and oxidative DNA damage are involved in tumor initiation and promotion, respec-
tively [14]. A similar scenario may be occurring with nitropolycyclic aromatic hydrocarbons, such as 1,6_dinitropyrene. Acknowledgement This work was supported in part by the Wayne State University Ben Kasle Trust Fund for Cancer Research. References 1
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