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DNA adduct formation in rat, human and hamster pancreas treated with methylnitrosourea
Cancer Letters, 26 (1985) 269-276 Elsevier Scientific Publishers Ireland Ltd.
269
DNA ADDUCT FORMATION IN RAT, HUMAN AND HAMSTER PANCREAS TREATED WITH METHYLNITROSOUREA
S. FRIEDMAN
and I. PARSA
State University of New York Downstate Medical Center, Brooklyn, NY 11203 (U.S.A.) (Received 20 December 1984) (Accepted 16 January 1984)
SUMMARY
The methylation of cellular macromolecules with dimethylnitrosamine (DMN) and methylnitrosourea (MNU) was studied in organ cultured rat, hamster and human pancreatic explants. At concentrations of DMN and MNU that caused similar methylation of protein in human explants DMN caused only 2.6% and 0.3% of the methylation of DNA and RNA that was produced by MNU. The DNA of explants treated with MNU was analyzed. The 06-methylguanine (06-MeG)/7-methylguanine (7-MeG) ratio was greater in the hamster DNA than in DNA isolated from either rat or human. The time course of removal of methyl adducts from DNA was followed for 6 h after treatment with MNU. No decline in 06-MeG occurred during this period in hamster explants, although there was a decline in the content of 7-MeA and 3-MeA, whereas there was removal of 06-MeG in the DNA from human pancreas explants. INTRODUCTION
Existing evidence suggests that environmental chemicals and in particular nitroso compounds contribute to the development of human pancreatic cancer. The induction of malignancy in organcultured pancreas by nitroso compounds was reported from this laboratory [l-3] . Nitroso compounds, directly or after becoming metabolically active, form adducts with nucleophilic sites of macromolecules. The occurrence of both 7-MeG and 06-MeG in pancreatic DNA of hamster treated with N-nitrosobis (2-oxopropyl)amine (BOP) has been studied by Lawson et al. [4]. The persistence of 06alkylguanine in DNA extracted from organs of carcinogen-treated rodents has been thought to correlate with tumor formation in these tissues [ 51 . Recent reports, however, have described the persistence of 06-MeG in DNA of several tissues of MNU-treated hamsters [ 61 and Mongolian gerbils [ 71 without the appearance of tumor in these organs. Immunohistochemical 0304-3835/85/$03.30 o 1985 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
270
studies of 06-ethylguanine half-life in rat brain cells has not revealed any obvious correlation between the presence of this adduct in specific cell types and the type of brain tumors induced by ethylnitrosourea [ 81. The present report compares the formation and removal of carcinogen-DNA adducts in organcultured adult rat, human and hamster pancreas. MATERIALS AND METHODS
Human pancreas Pancreases including tail and body were obtained in the operating room from cadaveric donors with no pancreatic disease. The main pancreatic duct and a sleeve of the parenchyma 0.5 cm in diameter were dissected out from each pancreas. Explants were prepared by parallel incisions 1 mm apart and perpendicular to the main pancreatic duct. Rat pancreas Pancreases from Luis rats (200-300 g) were removed under ether anesthesia. Explants of l-2 mm in thickness were prepared by parallel incisions perpendicular to the longitudinal axis of the pancreas. Hamster pancreas Pancreases from Syrian golden hamster (150-200 ether anesthesia and explants l-2 mm in thickness dissection of the adipose tissue.
g) were obtained under were prepared after
Organ culture Explants from each of the 3 species were rinsed with prewarmed (37°C) medium and placed on 5 X 50 mm Millipore filter strips floated on 5 ml of a chemically defined [9] medium in a roller tube. Up to 20 explants were placed on a filter strip and each roller tube contained up to 4 strips of filter. The roller tubes were incubated at 37°C in an atmosphere of 10% CO, in air saturated with water vapor, and turned at 0.5 rev./min. Two hours after the initiation of culture the explants were rinsed and placed in medium containing [methyl-‘%] DMN or MNU and incubated in this medium for 15 min to 24 h. For evaluation of the removal of adducts, the explants were then rinsed in medium without carcinogen and re-incubated for up to 24 h. At various time intervals (0,1,2,4,6,8,12 and 24 h) after the 2 h of incubation with N-[methyl-14C]MNU or DMN, explants were removed, rinsed in medium without isotope, and were stored at -80°C until processed for DNA extraction. Treatment of explant with radioactive alkylating agent N-[methyl-14C]DMN (spec. act. 15 Ci/mol) was directly added to the medium to obtain a final concentration of 20 pg/ml and used immediately. N-[methyl-14C]MNU (spec. act. 17 Ci/mol) was obtained as pellets (Amers-
271
ham Corp., 2636 South Clearbrook Dr., Arlington Heights, IL 60005) to which medium was added before use to a concentration of 3.6 yCi/ml. DNA methylation was studied using 0.04-0.20 pmol/ml of MNU. Radioactivity
of RNA, DNA and protein
Pancreas explants treated with either 3.6 ctCi/ml of MNU or DMN for 2 h were homogenized in a Potter-Elvehjem homogenizer in 4 ~01s. cold 4% HC104. The homogenate was centrifuged at 5000 X g for 5 min. After washing the precipitate with cold 4% HClO* , RNA, DNA and protein were extracted as described by Munro and Fleck [lo]. RNA and DNA were determined spectrophotometrically and protein was measured by the method of Lowry et al. [ 111. Aliquots of DNA, RNA and protein were counted in a liquid scintillation counter and corrected for quenching with an internal standard. DNA adducts
DNA was extracted from pancreatic explants by the method of Kirby [ 121 as modified by Lawley and Thatcher [ 131. The DNA, 0.5-l mg, was hydrolyzed in 0.5 ml of 0.1 M HCl for 45 min at 70°C. The hydrolyzate was clarified by centrifugation. Appropriate marker bases were added to the supernatant which was adjusted to pH 2.9 and applied to a 110 X 1 cm Sephadex G-10 column. The column was eluted with 0.05 M ammonium formate buffer containing 0.02% NaN3 (pH 6.75) [ 141. Fractions of 4 ml were collected and the amount of adenine and guanine present determined by their absorbance at 260 nm. The fractions were then dried at 6O”C, and dissolved in 0.2 ml of 0.2 M NH3 and 5 ml of a triton based scintillation fluid added. They were counted in a liquid scintillation counter. RESULTS
Human
explants
Alkylation
of DNA, RNA and protein
by MNU and DMN
Total radioactivity present in DNA, RNA and protein fractions of MNUand DMN-treated human explants are shown in Table 1. The amount of radioactivity incorporated into the protein fraction of MNU-treated explants was about 1.5 times the radioactivity incorporated into the protein fraction from DMN-treated explants. Radioactivities of DNA and RNA in MNUtreated explants were 40- and 260-fold, respectively, higher than radioactivities of DNA and RNA in DMN-treated explants. Dose effects
There was an increase in methylation of DNA with increasing dose of MNU. This increase was proportional to the dose over the range used in these experiments (Fig. 1). The same relationship was observed for the amount
212 TABLE 1 ALKYLATION
OF RNA, DNA AND PROTEIN BY DMN AND MNU
DNA RNA Protein
DMN (cpm/mg)
MNU (cpm/mg)
29 51 331
1126 13,483 497
of 3-MeA and 7-MeG present in the DNA as a result of increasing dose. Over the dose range 0.04-0.2 pmol/ml the 3-MeA/7-MeG ratio remained constant. Such a relationship was not observed for 06-MeG. The amount of 06-MeG present in the DNA increased faster with dose than did 7-MeG or 3-MeA as demonstrated by the increase in the 06-MeG/7-MeG ratio with dose, from 0.029 to 0.052 at the lowest (0.04 pmol/ml) and highest doses (0.2 I.tmol/ml) used, respectively. Adduct formation in rat, hamster and human pancreas explants The peak of DNA-labeling in explants treated with N-[methyl-14Cl MNU 60-
O.b4
CONCENTRATION
(mM)
Fig. 1. Dose-response curve of purine adducts formed in DNA after treatment of pancreatic explants with [ “C]MNU for 2 h. 0, 7-methylguanine; A, 3-methyladenine; l , OS-methylguanine.
273
for 15 min to 24 h occurred between 1 and 2 h after exposure to MNU. The explants therefore were incubated with MNU for 2 h and analyzed. The amount of label incorporated in pancreas explants under these conditions varied with species. The highest was observed in rat explants and the lowest in those of human. The ratio of methylated base formed to amount of parent base measured was highest for 7-MeG and lowest for l-MeA. While the ratios of l-MeA/A and 3-MeA/A were of the same range in all 3 species, the ratio of 7-MeA/A in rat was twice that of hamster and 20 times that of human. The ratio of 06-MeGjG in hamster was on the other hand, significantly greater than those of rat and human. Interspecies differences in base methylation were also noted when the percentage of methylated bases per amount of 7-MeG present in each DNA (rat, hamster and human) were compared (Table 2). Removal
of adducts
in hamster
and human pancreas
Due to lack of preservation, removal of adducts in rat pancreas was not studied. In hamster explants removal of 7-MeA and 3-MeA was observed over the’6-h period (Fig. 2A). However there was no removal of 06-MeG from the DNA. In contrast in human explants (Fig. 2B) removal of 3-methyladenine occurred, but the content of 7-MeA, which was low compared to that present in hamster DNA at the first time point, does not decline further. 06-Methylguanine appeared to be removed only early in the course of recovery from MNU treatment in the human explants. TABLE
2
ALKYLATED pmol/mi)
FOR
BASE
IN DNA
FROM
EXPLANTS
TREATED
WITH
[14C]MNU
(0.21
2 h pmol
methylated
baae/mol
Rat 1-Methyladenine 3-Methyladenine 7-Methyladenine 7-Methylguanine 06-Methylguanine
3-Methyladenine 7-Methyladenine 06-Methylguanine % S.E. bNo. of determinations. ‘Not done. dSignificance at P 0.05.
base in DNA
Hamster 1.1
7.6 8.6 176.0 8.1
+ 0.5a (3)b f3.2 f1.5 f 10.4 + 1.8
Methylated l-Methyladenine
parent
0.88 6.08 6.71 4.58
1.0 8.0 3.5 152.0 11.9
base/7-methylguanine
Human + 0.6 (3) +1.3 f 2.4 + 23.7 f 2.2
_c 5.6 0.4 98.9 4.2
kO.5 zk 0.2d f 26.9 kO.9
(%)
+ 0.45
0.75
zk 2.62 f l.lgd f 0.35
6.64 + 0.17 2.58 f 1.62 7.20 f 0.23d
f 0.42
_c 8.12 f 0.42 0.46 + 0.27 3.90 f 0.59
214
z
0.1 12
HOURS
’
I
2
4
6
1
6
IO
HOURS
Fig. 2. Time course of removal of alkylated purines from DNA of hamster and human explants after treatment with [ ‘%]MNU. Explants were treated for 2 h with 0.2 pmol/ ml [ lYJ]MNU. The medium was changed and DNA was extracted from the explants at the indicated times. A, hamster explants; B, human explants. ‘7, 7-methylguanine; 0, 06. methylguanine; A, 3-methyladenine; 0, 7-methyladenine.
DISCUSSION
The amount of 7-MeG and 3-MeA formation in human pancreas explants was shown to be proportional to the concentration of MNU in culture medium. The dose response curve for 06-MeG was different suggesting presence of a saturable process for removal of 06-MeG from DNA in human pancreatic explants. This is in accord with findings reported for human fibroblasts [ 151. The data indicates that other met&slated bases were removed from the DNA faster than 7-methylguanine (Fig. 2). When the species are compared the 06-methylguanine/7-methylguanine ratio is significantly higher in the DNA extracted from hamster explants than either the human or rat explants. The time course of disappearance of this base from human and hamster DNA also indicates that hamster explants are deficient in their ability to remove the alkylation product from DNA. The kinetics of removal of
I2
275
alkylated bases is complex, even over the short time interval studied. There is rapid removal of several of the alkylation products within 2 h of treatment with little removal thereafter. This could be due to either limitation of the excision process within cells, the fact that some cells in these explants do not have the capacity to remove alkylated products from DNA, or due to toxicity of the drug. There are marked differences in the distribution of radioactivity and extent of alkylation of nucleic acids due to treatment with MNU or DMN. Both these agents are potent carcinogens in this in vitro system and the fact that macromolecules are alkylated by DMN indicates the presence of an enzymatic system capable of converting the drug into an active alkylating agent [ 161. However, at concentrations that cause the incorporation of similar amounts of radioactivity into protein, much less radioactivity is incorporated into nucleic acids with DMN as alkylating agent than with MNU, such that we could not determine the products formed in DNA of pancreatic explants with DMN at concentrations that would allow the explants to survive. The finding that much less radioactivity from DMN is incorporated into RNA than into protein in rat pancreas was first reported bs Magee and Farber [ 171 and the pancreas was the only tissue analyzed in which less radioactivity was incorporated into RNA than into protein. We observe the same phenomena when the pancreas is treated with this drug in vitro. ACKNOWLEDGEMENT
This work was supported in part by grant no. 08RlCA30354 National Cancer institute.
from
REFERENCES 1 Parsa, I., Marsh, W.H. and Sutton, A.L. (1981) An in vitro model of human pancreas carcinogenesis: effects of nitroso compounds. Cancer, 47, 1543-1551. 2 Parsa, I., Marsh, W.H., Sutton, A.L. and Butt, K.M.H. (1981) Effects of dimethylnitrosamine on organ-cultured adult human pancreas. Am. J. Pathol., 102, 403-411. 3 Parsa, I. (1984) Methylnitrosourea-induced carcinoma in organ-cultured fetal human pancreas, Cancer Res., 44, 3530-3538. 4 Lawson, T.A., Gingel, R., Nagel, D., Hines, L.A. and Ross, A. (1981) Methylation of hamster DNA by the carcinogen N-nitrosobis( 2-oxopropyl)amine. Cancer Letters, 11,251-256. 5 Pegg, A.E. (1983) Alkylation and subsequent repair of DNA after exposure of dimethylnitrosamine and related carcinogens. In: Reviews in Biochemical Toxicology, Vol. 5, pp. 83-133. Editors: E. Hodgson, J.R. and Bend and R.M. Philpot. Elsevier/ North-Holland Biomedical Press, Amsterdam. 6 Likhachev,A.J., Inanov, M.N., Bresil, N., Planche-Martel, G., Montesono, R. and Margison, G. (1983) Carcinogenicity of single doses of N-nitroso-N-methylurea and N-nitroso-N-ethylurea in Syrian golden hamsters and persistence of alkylated purines in the DNA of various tissues. Cancer Res., 43,829-833. 7 Kleihues, P., Bamborschke, S. and Doerjer, G. (1980) Persistence of alkylated DNA bases in the Mongolian gerbil follo.wing a single dose of methylnitrosourea. Carcinogenesis, l, lll-113.
276
8 Heyting,C.,VanDer Laken,C.J.,Van Raamsdonk, W. and Pool, C.W. (1983) Immunohistochemical detection of O6 -ethyldeoxyguanosine in the rat brain after in ZGO applications of N-ethyl-N-nitrosourea. Cancer Res., 44, 2935-2941. 9 Parsa, I., Marsh, W.H. and Fitzgerald, P.J. (1970) A chemically defined medium for organ cultured differentiation of rat pancreas anlage. Exp. Cell Res., 59, 171-175. 10 Munro, H.N. and Fleck, A. (1966) Determination of nucleic acids. Methods Biochem. Anal., 14, 114-176. 11 Lowry, O.H., Rosenbrough, N.J., Farr, A.L. and Randall, R.J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193, 265-275. 12 Kirby, K.S. (1952) A new method for the isolation of deoxyribonucleic acids. Biochem. J., 66, 495-504. 13 Lawley, P.D. and Thatcher, C.J. (1970) Methylation of deoxyribonucleic acid in cultured mammalian cells by N-methyl-N’nitro-N-nitrosoguanidine. Biochem. J., 116,693-707. 14 Lawley, P.D. and Shah, S.A. (1972) Methylation of RNA by the carcinogens dimethyl sulphate, N-methyl-N-nitrosourea and N-methyl-N’nitro-N-nitroso-guanidine. Biothem. J., 128,117-132. 15 Medcalf, A.S.C. and Lawley, P.D. (1981) Time course of 06-methylguanine removal from DNA of N-methyl-N-nitrosourea treated human fibroblasts. Nature, 289, 796798. 16 Magee, P.N. and Hultin, T. (1962) Toxic liver injury and carcinogenesis methylation of proteins of rat-liver slices by dimethylnitrosamine in vitro. Biochem. J., 83,106114. I.7 Magee, P.N. and Farber, E. (1962) Toxic liver injury and carcinogenesis methylation of rat-liver nucleic acids by dimethylnitrosamine in vivo. Biochem. J., 83, 114-124.
269
DNA ADDUCT FORMATION IN RAT, HUMAN AND HAMSTER PANCREAS TREATED WITH METHYLNITROSOUREA
S. FRIEDMAN
and I. PARSA
State University of New York Downstate Medical Center, Brooklyn, NY 11203 (U.S.A.) (Received 20 December 1984) (Accepted 16 January 1984)
SUMMARY
The methylation of cellular macromolecules with dimethylnitrosamine (DMN) and methylnitrosourea (MNU) was studied in organ cultured rat, hamster and human pancreatic explants. At concentrations of DMN and MNU that caused similar methylation of protein in human explants DMN caused only 2.6% and 0.3% of the methylation of DNA and RNA that was produced by MNU. The DNA of explants treated with MNU was analyzed. The 06-methylguanine (06-MeG)/7-methylguanine (7-MeG) ratio was greater in the hamster DNA than in DNA isolated from either rat or human. The time course of removal of methyl adducts from DNA was followed for 6 h after treatment with MNU. No decline in 06-MeG occurred during this period in hamster explants, although there was a decline in the content of 7-MeA and 3-MeA, whereas there was removal of 06-MeG in the DNA from human pancreas explants. INTRODUCTION
Existing evidence suggests that environmental chemicals and in particular nitroso compounds contribute to the development of human pancreatic cancer. The induction of malignancy in organcultured pancreas by nitroso compounds was reported from this laboratory [l-3] . Nitroso compounds, directly or after becoming metabolically active, form adducts with nucleophilic sites of macromolecules. The occurrence of both 7-MeG and 06-MeG in pancreatic DNA of hamster treated with N-nitrosobis (2-oxopropyl)amine (BOP) has been studied by Lawson et al. [4]. The persistence of 06alkylguanine in DNA extracted from organs of carcinogen-treated rodents has been thought to correlate with tumor formation in these tissues [ 51 . Recent reports, however, have described the persistence of 06-MeG in DNA of several tissues of MNU-treated hamsters [ 61 and Mongolian gerbils [ 71 without the appearance of tumor in these organs. Immunohistochemical 0304-3835/85/$03.30 o 1985 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
270
studies of 06-ethylguanine half-life in rat brain cells has not revealed any obvious correlation between the presence of this adduct in specific cell types and the type of brain tumors induced by ethylnitrosourea [ 81. The present report compares the formation and removal of carcinogen-DNA adducts in organcultured adult rat, human and hamster pancreas. MATERIALS AND METHODS
Human pancreas Pancreases including tail and body were obtained in the operating room from cadaveric donors with no pancreatic disease. The main pancreatic duct and a sleeve of the parenchyma 0.5 cm in diameter were dissected out from each pancreas. Explants were prepared by parallel incisions 1 mm apart and perpendicular to the main pancreatic duct. Rat pancreas Pancreases from Luis rats (200-300 g) were removed under ether anesthesia. Explants of l-2 mm in thickness were prepared by parallel incisions perpendicular to the longitudinal axis of the pancreas. Hamster pancreas Pancreases from Syrian golden hamster (150-200 ether anesthesia and explants l-2 mm in thickness dissection of the adipose tissue.
g) were obtained under were prepared after
Organ culture Explants from each of the 3 species were rinsed with prewarmed (37°C) medium and placed on 5 X 50 mm Millipore filter strips floated on 5 ml of a chemically defined [9] medium in a roller tube. Up to 20 explants were placed on a filter strip and each roller tube contained up to 4 strips of filter. The roller tubes were incubated at 37°C in an atmosphere of 10% CO, in air saturated with water vapor, and turned at 0.5 rev./min. Two hours after the initiation of culture the explants were rinsed and placed in medium containing [methyl-‘%] DMN or MNU and incubated in this medium for 15 min to 24 h. For evaluation of the removal of adducts, the explants were then rinsed in medium without carcinogen and re-incubated for up to 24 h. At various time intervals (0,1,2,4,6,8,12 and 24 h) after the 2 h of incubation with N-[methyl-14C]MNU or DMN, explants were removed, rinsed in medium without isotope, and were stored at -80°C until processed for DNA extraction. Treatment of explant with radioactive alkylating agent N-[methyl-14C]DMN (spec. act. 15 Ci/mol) was directly added to the medium to obtain a final concentration of 20 pg/ml and used immediately. N-[methyl-14C]MNU (spec. act. 17 Ci/mol) was obtained as pellets (Amers-
271
ham Corp., 2636 South Clearbrook Dr., Arlington Heights, IL 60005) to which medium was added before use to a concentration of 3.6 yCi/ml. DNA methylation was studied using 0.04-0.20 pmol/ml of MNU. Radioactivity
of RNA, DNA and protein
Pancreas explants treated with either 3.6 ctCi/ml of MNU or DMN for 2 h were homogenized in a Potter-Elvehjem homogenizer in 4 ~01s. cold 4% HC104. The homogenate was centrifuged at 5000 X g for 5 min. After washing the precipitate with cold 4% HClO* , RNA, DNA and protein were extracted as described by Munro and Fleck [lo]. RNA and DNA were determined spectrophotometrically and protein was measured by the method of Lowry et al. [ 111. Aliquots of DNA, RNA and protein were counted in a liquid scintillation counter and corrected for quenching with an internal standard. DNA adducts
DNA was extracted from pancreatic explants by the method of Kirby [ 121 as modified by Lawley and Thatcher [ 131. The DNA, 0.5-l mg, was hydrolyzed in 0.5 ml of 0.1 M HCl for 45 min at 70°C. The hydrolyzate was clarified by centrifugation. Appropriate marker bases were added to the supernatant which was adjusted to pH 2.9 and applied to a 110 X 1 cm Sephadex G-10 column. The column was eluted with 0.05 M ammonium formate buffer containing 0.02% NaN3 (pH 6.75) [ 141. Fractions of 4 ml were collected and the amount of adenine and guanine present determined by their absorbance at 260 nm. The fractions were then dried at 6O”C, and dissolved in 0.2 ml of 0.2 M NH3 and 5 ml of a triton based scintillation fluid added. They were counted in a liquid scintillation counter. RESULTS
Human
explants
Alkylation
of DNA, RNA and protein
by MNU and DMN
Total radioactivity present in DNA, RNA and protein fractions of MNUand DMN-treated human explants are shown in Table 1. The amount of radioactivity incorporated into the protein fraction of MNU-treated explants was about 1.5 times the radioactivity incorporated into the protein fraction from DMN-treated explants. Radioactivities of DNA and RNA in MNUtreated explants were 40- and 260-fold, respectively, higher than radioactivities of DNA and RNA in DMN-treated explants. Dose effects
There was an increase in methylation of DNA with increasing dose of MNU. This increase was proportional to the dose over the range used in these experiments (Fig. 1). The same relationship was observed for the amount
212 TABLE 1 ALKYLATION
OF RNA, DNA AND PROTEIN BY DMN AND MNU
DNA RNA Protein
DMN (cpm/mg)
MNU (cpm/mg)
29 51 331
1126 13,483 497
of 3-MeA and 7-MeG present in the DNA as a result of increasing dose. Over the dose range 0.04-0.2 pmol/ml the 3-MeA/7-MeG ratio remained constant. Such a relationship was not observed for 06-MeG. The amount of 06-MeG present in the DNA increased faster with dose than did 7-MeG or 3-MeA as demonstrated by the increase in the 06-MeG/7-MeG ratio with dose, from 0.029 to 0.052 at the lowest (0.04 pmol/ml) and highest doses (0.2 I.tmol/ml) used, respectively. Adduct formation in rat, hamster and human pancreas explants The peak of DNA-labeling in explants treated with N-[methyl-14Cl MNU 60-
O.b4
CONCENTRATION
(mM)
Fig. 1. Dose-response curve of purine adducts formed in DNA after treatment of pancreatic explants with [ “C]MNU for 2 h. 0, 7-methylguanine; A, 3-methyladenine; l , OS-methylguanine.
273
for 15 min to 24 h occurred between 1 and 2 h after exposure to MNU. The explants therefore were incubated with MNU for 2 h and analyzed. The amount of label incorporated in pancreas explants under these conditions varied with species. The highest was observed in rat explants and the lowest in those of human. The ratio of methylated base formed to amount of parent base measured was highest for 7-MeG and lowest for l-MeA. While the ratios of l-MeA/A and 3-MeA/A were of the same range in all 3 species, the ratio of 7-MeA/A in rat was twice that of hamster and 20 times that of human. The ratio of 06-MeGjG in hamster was on the other hand, significantly greater than those of rat and human. Interspecies differences in base methylation were also noted when the percentage of methylated bases per amount of 7-MeG present in each DNA (rat, hamster and human) were compared (Table 2). Removal
of adducts
in hamster
and human pancreas
Due to lack of preservation, removal of adducts in rat pancreas was not studied. In hamster explants removal of 7-MeA and 3-MeA was observed over the’6-h period (Fig. 2A). However there was no removal of 06-MeG from the DNA. In contrast in human explants (Fig. 2B) removal of 3-methyladenine occurred, but the content of 7-MeA, which was low compared to that present in hamster DNA at the first time point, does not decline further. 06-Methylguanine appeared to be removed only early in the course of recovery from MNU treatment in the human explants. TABLE
2
ALKYLATED pmol/mi)
FOR
BASE
IN DNA
FROM
EXPLANTS
TREATED
WITH
[14C]MNU
(0.21
2 h pmol
methylated
baae/mol
Rat 1-Methyladenine 3-Methyladenine 7-Methyladenine 7-Methylguanine 06-Methylguanine
3-Methyladenine 7-Methyladenine 06-Methylguanine % S.E. bNo. of determinations. ‘Not done. dSignificance at P 0.05.
base in DNA
Hamster 1.1
7.6 8.6 176.0 8.1
+ 0.5a (3)b f3.2 f1.5 f 10.4 + 1.8
Methylated l-Methyladenine
parent
0.88 6.08 6.71 4.58
1.0 8.0 3.5 152.0 11.9
base/7-methylguanine
Human + 0.6 (3) +1.3 f 2.4 + 23.7 f 2.2
_c 5.6 0.4 98.9 4.2
kO.5 zk 0.2d f 26.9 kO.9
(%)
+ 0.45
0.75
zk 2.62 f l.lgd f 0.35
6.64 + 0.17 2.58 f 1.62 7.20 f 0.23d
f 0.42
_c 8.12 f 0.42 0.46 + 0.27 3.90 f 0.59
214
z
0.1 12
HOURS
’
I
2
4
6
1
6
IO
HOURS
Fig. 2. Time course of removal of alkylated purines from DNA of hamster and human explants after treatment with [ ‘%]MNU. Explants were treated for 2 h with 0.2 pmol/ ml [ lYJ]MNU. The medium was changed and DNA was extracted from the explants at the indicated times. A, hamster explants; B, human explants. ‘7, 7-methylguanine; 0, 06. methylguanine; A, 3-methyladenine; 0, 7-methyladenine.
DISCUSSION
The amount of 7-MeG and 3-MeA formation in human pancreas explants was shown to be proportional to the concentration of MNU in culture medium. The dose response curve for 06-MeG was different suggesting presence of a saturable process for removal of 06-MeG from DNA in human pancreatic explants. This is in accord with findings reported for human fibroblasts [ 151. The data indicates that other met&slated bases were removed from the DNA faster than 7-methylguanine (Fig. 2). When the species are compared the 06-methylguanine/7-methylguanine ratio is significantly higher in the DNA extracted from hamster explants than either the human or rat explants. The time course of disappearance of this base from human and hamster DNA also indicates that hamster explants are deficient in their ability to remove the alkylation product from DNA. The kinetics of removal of
I2
275
alkylated bases is complex, even over the short time interval studied. There is rapid removal of several of the alkylation products within 2 h of treatment with little removal thereafter. This could be due to either limitation of the excision process within cells, the fact that some cells in these explants do not have the capacity to remove alkylated products from DNA, or due to toxicity of the drug. There are marked differences in the distribution of radioactivity and extent of alkylation of nucleic acids due to treatment with MNU or DMN. Both these agents are potent carcinogens in this in vitro system and the fact that macromolecules are alkylated by DMN indicates the presence of an enzymatic system capable of converting the drug into an active alkylating agent [ 161. However, at concentrations that cause the incorporation of similar amounts of radioactivity into protein, much less radioactivity is incorporated into nucleic acids with DMN as alkylating agent than with MNU, such that we could not determine the products formed in DNA of pancreatic explants with DMN at concentrations that would allow the explants to survive. The finding that much less radioactivity from DMN is incorporated into RNA than into protein in rat pancreas was first reported bs Magee and Farber [ 171 and the pancreas was the only tissue analyzed in which less radioactivity was incorporated into RNA than into protein. We observe the same phenomena when the pancreas is treated with this drug in vitro. ACKNOWLEDGEMENT
This work was supported in part by grant no. 08RlCA30354 National Cancer institute.
from
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