Chem.-BioL Interactions, 33 (1981) 229--238
© Elsevier/North-HollandScientific Publishers Ltd.
METABOLISM OF DIMETHYLNITROSAMINE AND SUBSEQUENT REMOVAL OF O6-bIETHYLGUANINE FROM DNA BY ISOLATED RAT I~EPATOCYTES
DIANE R. UMBENHAUERand ANTHONYE. PEGG Department of Physiology and Specialized Cancer Research Center, The Milton S. Hershey Medical Center, The Pennsylvania State University, Hershey, PA 17033 (U.S.A.)
(Received May 5th, 1980) (Revision received August 24th, 1980) (Accepted August 25th, 1980)
SUMMARY Freshly prepared isolated hepatocytes were shown to metabolize dimethylnitrosamine producing an alkylating agent which reacted with cellular DNA giving rise to 7-methylguanine and O6-methylguanine. Alkylation of DNA was prevented by aminoacetonitrile and was proportional to the dimethylnitrosamine concentration over the range of 1--90 ~M. Isolated hepatocytes were able to catalyze the removal of O~-methylguanine from their DNA. After formation of this product by reaction with N-methyl-N-nitrosourea or dimethylnitrosamine to give extents of alkylation in the range of 0.1--15.0 ~mol O6-methylguanine per mole of guanine in DNA, the loss produced in 3 h incubation was comparable to that seen in vivo from liver DNA alkylated to the same extent. The isolated hepatocytes therefore, provide a useful system in which factors influencing O6-methylguanine persistence in DNA can be studied.
INTRODUCTION Liver cells m a y be protected from the carcinogenic potential of alkylating agents by their possession of a repair system which catalyzes the removal of O6-methylguanine from D N A [1--3]. There is widespread exposure to low levels of some of these carcinogens such as dimethylnitrosamine [4,5] and factors affecting the activityof this system may, therefore, play an important role in determining susceptibility to these agents. At present it is difficult to study the removal of 0 6-alkylguanine from D N A in mammalian cells because this activity is present in m u c h larger amounts in liverthan in fibroblasts or lymphocytes convenient for cell culture techniques [2,6]. Therefore, studies have been carried out either by following the persistence of O 6-alkylguanine in D N A in vivo [1,2,7,8] or by using crude cell extracts in vitro [6,9,10].
230 Many informative experiments could be accomplished more conveniently by use of isolated whole cell preparations. For this reason, we have tested the ability of isolated hepatocytes to carry o u t this reaction. It is known that liver extracts contain at least t w o and probably more forms of the enzyme system capable of converting dimethylnitrosamine to an alkylating species [10--13] b u t only the form with the lowest Kin-value is likely to play any physiological role in activation of the carcinogen. Therefore, we also investigated the ability of isolated hepatocytes to activate dimethylnitrosamine when added at low concentrations consistent with in vivo experimentation. The results showed that freshly prepared isolated hepatocytes were able to convert dimethylnitrosamine to an alkylating species which reacted with the cellular DNA and that the 06-methylguanine produced was rapidly removed on continued incubation. MATERIALS AND METHODS
Chemicals. Unlabeled dimethylnitrosamine and aminoacetonitrile were purchased from Aldrich Chemical Company, Milwaukee, WI. Aminoacetonitrile was recrystallized from ethanol before using. Collagenase T y p e II was from Worthington Biochemical Corp., Freehold, NJ. Pronase (B grade) was from Calbiochem-Behring Corp., La Jolla, CA. [~H]Dimethylnitrosamine (3.49 Ci/mmol), N-[3H]methyl-N-nitrosourea (1.0 Ci/mmol) and ['4C]dimethylnitrosamine (46.75 mCi/mmol) were obtained from New England Nuclear, Boston, MA. All other chemicals were from Sigma Chemical Company, St. Louis, MO. Preparation of hepatocytes. Female Sprague--Dawley rats (Charles River Breeding Laboratories, Wilmington, MA) weighing 180--210 g were used. Livers were perfused in situ at 37°C  and hepatocytes were prepared as described by Jefferson . The perfusion medium equilibrated with 95% O: : 5% CO2 consisted of a modified Krebs-Henseleit bicarbonate buffer (pH 7.2), with 10% bovine erythrocytes, glucose at 27.4 mM, and monosodium glutamate and sodium pyruvate at 5 mM each b u t without calcium, bovine serum albumin or amino acids. Livers were perfused in a non-recirculating system for 15 min at a flow of 14 ml/min, after which 35 mg of collagenase was added to the 150 ml of perfusate in the recirculating system. Following a 35-min digestion, each liver was removed and gently forced through a stainless steel sieve (10 mesh) into 75 ml of erythrocyte-free buffer . This suspension was filtered through c o t t o n gauze and the cells were washed four times in 50 ml conical polycarbonate tubes using a sedimentation-resuspension procedure. The cell suspensions were accelerated to 800 rev./min in a Sorval Model GLC-2 centrifuge and allowed to stop without braking. After removing the supernatant by aspiration, the remaining pellet was resuspended in buffer and poured off, leaving the tight clump of cells at the b o t t o m of the tube. For the last two washes and subsequent incubation, the buffer also contained 3% bovine serum albumin, plasma levels of amino acids and 2.4 mM CaC12.
231 Incubation. Polycarbonate flasks (250 ml capacity) were used to incubate cell suspensions in a shaking water bath at 37°C. Hepatocytes were suspended in the erythrocyte-free perfusion buffer containing 3% bovine serum albumin, plasma levels of amino acids and 2.4 mM CaC12. Each flask contained 15--40 ml of cell suspension at concentrations in the range of 106--7.5 × 106 cells/ml and was supplied with 95% 02 : 5% CO2. The high density of cells was used to minimize the amount of radioactive carcinogen needed and maximize the cellular alkylation. Even with these high cell densities, cells exhibited a minimum of clumping and viabilities as determined by trypan blue exclusion were 90--95% initially and greater than 80% after 5 h of incubation. Nmethyl-N-nitrosourea, dimethylnitrosamine and/or aminoacetonitrile were added to the flasks at appropriate times. At the end of the incubation, the cells were pelleted (1000 rev./min for I rain) and frozen. All experiments were carried o u t several times with essentially similar results to the representative values shown in the Tables and Figure. However, differences in the cell preparations and densities used for each experiment prevented statistical analysis of the results. D N A isolation. DNA was isolated by a modification of the technique used by Bodell and Banerjee . Cells were homogenized in 10 ml of buffer (0.1 M NaC1, 5 mM EDTA, 0.1 M Tris--HC1, pH 8.0) and 0.1 volume of 10% sodium dodecyl sulfate was added and the homogenate was shaken for 5 rain. After adding solid NaCIO4 to a concentration of I M, i volume of chloroform/ isoamyl alcohol (10/1, v/v) was added and the mixture was shaken for 10 rain at room temperature, then centrifuged for 10 min at 10 000 × g. The aqueous phase was removed and reextracted with chloroform/isoamyl alcohol. The aqueous phase was again removed, 0.1 volume of 4 M sodium acetate was added followed by 2 volumes of cold ethanol. The precipitated DNA was washed once with ethanol, dissolved in 0.01 X SSC (1 X SSC is 0.15 M NaC1, 0.015 M Na citrate, pH 6.8), and the solution was then made 0.1 X SSC with the addition of 10 X SSC. The DNA was digested with RNAase (100 ~g/ml) (freed from DNAase by heating at 100°C for 2 min) for 90 rain at 37°C. Pronase (100 ~g/ml) (self
232 TABLE I A L K Y L A T I O N OF HEPATOCYTE DNA FOLLOWING EXPOSURE TO DIMETHYLNITROSAMINE Primary hepatocytes were prepared and incubated as described under Materials and Methods at a concentration of 1 ml of packed cells (about 5 × 107 cells) per 15 ml of incubation volume with the amount of [3H]dimethylnitrosamine (spec. act., 5.8--3490 Ci/mol according to the concentration) shown for 3 h. Concentration of dimethylnitrosamine (uM)
0.9 9.9 91 541
Alkylation of DNA a 7-Methylguanine
1.8 22.2 200 251
0.1 1.1 22.4 40.7
aExpressed as umol methylated guanine/mol guanine.
activity in the fractions corresponding to the methylated purines was determined by adding 10 ml of Formula 947 LSC Cocktail (New England Nuclear) and counting in a Beckman LS-3133T liquid scintillation counter. In some experiments methylated base content was determined following separation by HPLC using a Whatman Partisil 10/25 SCX column eluted with 0.02 M ammonium formate (pH 4) (Bennett and Pegg, unpublished data}. R E S ULTS
When freshly prepared hepatocytes were incubated in the presence of labeled dimethylnitrosamine and then DNA was isolated from them and analyzed it was found to be alkylated as shown in Table I. Both 7-methylTABLE II E F F E C T OF A M I N O A C E T O N I T R I L E ON A L K Y L A T I O N OF HEPATOCYTE DNA BY DIMETHYLNITROSAMINE Primary hepatocytes were prepared and incubated as described under Materials and Methods at a concentration of 0.75 ml of packed cells per 15 ml of incubation volume with 36 uM [~4C]dimethylnitrosamine (46.75 mCi/mmol) for 2 h in the presence of the concentration of aminoacetonitrile shown. Aminoacetonitrile (raM)
0 0.02 0.10 1.00
Alkylation of DNA a 7-Methylguanine
203 145 64 11
14.5 8.2 3.0 0.03
aExpressed as ~mol methylated guanine/tool guanine.
233 guanine and O6-methylguanine were found to be present in the DNA. The extent of alkylation as measured by the presence of 7-methylguanine was proportional to the initial co.ncentration of dimethylnitrosamine added over the range 0.9--91 pM when measurements were made after 3 h of exposure. Higher concentrations of the nitrosamine led to less than expected levels of alkylation but may not have been metabolized completely within the 3-h period. It is known that metabolic conversion of dimethylnitrosamine to an alkylating species in vivo is inhibited by treatment with the drug, aminoacetonitrile [17,18]. As shown in Table II, aminoacetonitrile inhibited DNA alkylation in the isolated hepatocytes exposed to dimethylnitrosamine in a dosedependent manner. Significant inhibition pertained at 0.1 mM concentration but complete inhibition required 1 mM aminoacetonitrile. These results indicate that the isolated hepatocyte preparations are capable of metabolizing dimethylnitrosamine to an alkylating agent during incubation in vitro. Previous studies from this and other laboratories have demonstrated that O6-alkylguanine is removed from liver DNA by an enzymatic process [2,6,10]. This process is quite efficient after alkylation to low extent removing more than 90% of the lesions within a few hours. In order to test whether 06methylguanine was removed from DNA in isolated hepatocytes the cells were alkylated by exposure to either labeled N-methyl-N-nitrosourea or to dimethylnitrosamine. N-Methyl-N-nitrosourea is a direct acting alkylating agent which does not require metabolic activation. Its decomposition at the pH of the incubation mixture is very rapid and, as measured by the production of 7-methylguanine in the hepatocyte DNA, alkylation by this compound was complete within 20 min. As shown in Table III, O6-methyl guanine present in the hepatocyte DNA after reaction with N-methyl-NTABLE III REMOVAL OF 06-METHYLGUANINE FROM HEPATOCYTE DNA DURING INCUBATION IN VITRO Hepatocyte DNAwas alkylated by incubation for 20 min with 0.16 mM N - [ m e t h y l - 3 H ] - N nitrosourea (1.0 Ci/mmol) (Expts. A and B ) o r by incubation with 2.7 uM [3H]dimethylnitrosamine(3.49 Ci/mmol) for 90 min (Expt. C) or with 0.7 ~M [SH]dimethylnitrosamine (Expt. D) for 180 min. The O 6-methylguanine present in the DNA at this time was determined in one aliquot of the hepatocyte preparation. The remainder of the hepatocytes were incubated for the time shown as described under Materials and Methods before determination of the amount of O6-methylguanineremaining. N.D., not determined. Incubation time (h)
0 1 2 4
0 6 -Methylguanine
1.9 1.1 0.6 N.D.
1.2 0.8 N.D. 0.5
0.27 N.D. 0.10 N.D.
0.14 N.D. 0.07 N.D.
aExpressed as umol/mol guanine.
234 nitrosourea was lost quite rapidly declining by a b o u t 60% within 2 h. Similar rapid removal o f O6-methylguanine from DNA was seen following alkylation by dimethylnitrosamine. In these experiments (Table III), t he cells were exposed to d i m e t h y l n i t r o s a m i n e f or 90--180 min and then collected by centrifugation, washed and suspended in fresh m e d i u m w i t h o u t t he nitrosamine. In all o f the experiments shown in Table III the e x t e n t o f alkylation leading to O6-methylguanine was in the range o f 0.1--2.0 p m o l / m o l guanine. This level o f alkylation is p r o d u c e d in vivo in the liver following exposure to doses o f d i m e t h y l n i t r o s a m i n e o f 100 gg/kg or less. Table IV shows for comparative purposes the levels o f O6-methylguanine present in hepatic DNA within 0.25--4 h after administration o f dimethylnitrosamine. It can be seen th a t the rate of loss o f O6-methylguanine from h e p a t o c y t e DNA in vitro (Table III) is slightly slower but comparable to t hat in vivo after doses o f 5--100 pg/kg (Table IV). Figure 1 shows t he persistence o f O6-methylguanine in h e p a t o c y t e DNA after alkylation by exposure t o 108 pM dimethylnitrosamine for 2 h. Subsequent incubation for a f u r t h e r 3 h led to a progressive decline a m o u n t i n g to a total o f 43% o f t he starting value. Table IV, Expt. A shows that a similar rate o f decline in O6-methylguanine is seen in vivo after a dose o f 760 pg/kg which produces a comparable a m o u n t o f alkylation. It should be n o t e d t h a t there was only a slight, insignificant decline in 7-methylguanine c o n t e n t in t he isolated h e p a t o c y t e preparations during the 3-h period in b o t h t he e x p e r i m e n t shown in Fig. 1 and in t hat o f Table III (where 7-methylguanine levels are n o t given). This result is in agreement with th e relatively long tin o f this p r o d u c t in DNA in vivo [2,3]. TABLE IV REMOVAL OF O6-METHYLGUANINE FROM HEPATIC DNA IN VIVO FOLLOWING ADMINISTRATION OF DIMETHYLNITROSAMINE Rats were treated with [ 3H ] dimethylnitrosamine (3.49---0.046 Ci/mmol according to the dose) by intraperitoneal injection. At the times shown the rats were killed and hepatic DNA analyzed for the content of methylated bases. For each dose the content of 7methylguanine did not differ significantly over the time period shown indicating that dimethylnitrosamine metabolism was complete and that the loss of O6-methylguanine was due to a specific enzymatic mechanism and not to generalized degradation of alkylated DNA. N.D., not determined. Time after administration (h) 0.25 1 2 4
06-Methylguanine contenta Expt. A (0.76 mg/kg)
Expt. B (0.1 mg/kg)
Expt. C (0.05 mg/kg)
Expt. D (0.005 mg/kg)
N.D. 15.3 12.1 10.2
1.15 0.75 0.48 N.D.
0.59 0.26 0.12 N.D.
0.043 0.022 0.006 N.D.
aExpressed as #tool/tool guanine.
A®250t g 200
Fig. 1.7-Methyl- and 06-methylguanine content in hepatocyte DNA following incubation with dimethylnitrosarnine. Hepatocytes were incubated with 108 pM ['4C]dimethylnitrosamine (46.75 mCi/mmol) at a concentration of 3 ml packed cells per 20 ml buffer for 2 h, then washed and incubated for the time shown as described in Materials and Methods. DNA was then isolated and analyzed for the content of 7-methylguanine (e •) or 0 ~-methylguanine (w • ). DISCUSSION The present experiments show by direct measurement t h a t freshly prepared isolated hepatocytes are able to metabolize dimethylnitrosamine generating an alkylating agent which reacts with DNA. Such interaction has previously been inferred from the ability of the nitrosamine to transform hepatocytes , cause mutations in other cells cultured with the hepatocytes [20,21] or to induce DNA repair synthesis in them [22--24]. However, it should be noted that liver microsome preparations contain at least two 'dimethylnitrosamine demethylase' activities of which only the form having a low Kin-value is likely to be of pathophysiological importance [11--13]. Doses of dimethylnitrosamine below the LDs0 are unlikely to give rise to concentrations above 0.5 mM and the relevance of results obtained in vitro with concentrations in excess of this is questionable. The present data indicate t h a t micromolar concentrations of dimethylnitrosamine are metabolized rapidly by the isolated hepatocytes which therefore provide a suitable system for studying the interaction with DNA and its subsequent repair. The extent of alkylation produced by a given concentration of dimethylnitrosamine is dependent on the dilution of the hepatocyte suspension used for incubation. Comparison of the results in Table I with other values obtained in our laboratory for alkylation of liver DNA in vivo after administration of dimethylnitrosamine  suggests that
236 the extent of reaction with DNA is 7--10 times less in vitro under our incubation conditions (as in Table I) than in vivo. To some extent this may be explained by the greater reaction of the alkylating species with water when the cells are diluted such that the equivalent of about 0.5 g of liver is suspended in 15 ml. Studies of the extent to which the alkylating species generated by hepatocyte metabolism of dimethylnitrosamine can interact with extracellular material would be of considerable interest. The present experiments were carried out with hepatocytes from the rat, a species which is relatively resistant to carcinogenesis by single doses of dimethylnitrosamine [4,25] and is relatively active in removing OO-methyl guanine from DNA [2,3,7]. Syrian golden hamsters are more sensitive to carcinogenesis and have a lesser capacity to remove O6-methylguanine [4,25]. It has been claimed that cells from Chinese hamsters are unable to carry out this reaction at all [26,27] although these results should be regarded with some caution as measurements were made at only one level of alkylation and it is well known that the process is saturated at high doses [8,10,25]. The isolated hepatocyte system provides a convenient means by which these species differences might be investigated. The enzymatic reaction leading to the removal of O6-methylguanine from h e p a t o c y t e DNA is not fully understood but does not involve a glycosylase releasing the free base . Rapid removal of a substantial fraction of the O6-methylguanine occurs only when the degree of alkylation is low suggesting that the process is saturated or inhibited at higher doses [7,8,10]. Even in liver where activity is higher than in any other tissue only a small a m o u n t of O6-methylguanine can be removed in a few hours. But, as shown in Table III and Fig. 1, the loss of O6-methylguanine from hepatocyte DNA over a 3 hour period is comparable to that seen from liver DNA in vivo and more than 40% of the starting level was removed in this time. Excellent viability of the hepatocytes is maintained over such an incubation period. Therefore, the freshly prepared hepatocytes offer a useful system in which the removal process can be studied. In particular, the effects of potential inhibitory and activatory substances on the process may be tested much more conveniently than in the intact animal. ACKNOWLEDGEMENTS
The research was supported by grants CA 18137 and 1P 30 CA18450 from NCI, DHEW. REFERENCES 1 R. Goth and M.F. Rajewsky, Persistence of O~-ethylguanine in rat brain DNA: correlation with nervous system-specific carcinogenesis by ethyl nitrosourea, Proc. Natl. Acad. Sci. U.S.A., 71 (1974) 639. 2 A.E. Pegg, Formation and metabolism of alkylated nucleosides: possible role in carcinogenesis by nitroso compounds and alkylating agents, Adv. Cancer Res., 25
237 3 G.P. Margison and P.J. O'Connor, Nucleic acid modification by N-nitroso compounds, in: P.L. Grover (Ed.), Chemical Carcinogens and DNA, CRC Press, Florida, 1979, pp. 111--159. 4 IARC Monographs on the evaluation of the carcinogenic risk of chemicals to humans, Vol. 18, International Agency for Research on Cancer, Lyon, France, 1978. 5 L. Fishbein, Overview of some aspects of occurrence, formation and analysis of nitrosamines, Sci. Total Environ., 13 (1979) 157. 6 A.E. Pegg, Enzymatic removal of O 4-methylguanine from DNA by mammalian cell extracts, Biochem. Biophys. Res. Commun., 84 (1978) 166. 7 A.E. Pegg, Alkylation of rat liver DNA by dimethylnitrosamine: effect of dosage on O6-methylguanine levels, J. Natl. Cancer Inst., 58 (1977) 681. 8 P. Kleihues and G.P. Margison, Exhaustion and recovery of repair excision of O ' methylguanine from rat liver DNA, Nature, 259 (1976) 153. 9 R. Montesano, H. Brdsil, G. Planche-Martel, G. Margison and A.E. Pegg, Effect of chronic treatment of rats with dimethylnitrosamine on the removal of O 4-methylguanine from DNA, Cancer Res., 40 (1980) 452. 10 A.E. Pegg, Dimethylnitrosamine inhibits enzymatic removal of O6-methylguanine from DNA, Nature, 274 (1978) 182, 11 J.C. Arcos, D.L. Davies, C.E.L. Brown and M.F. Argus, Repressible and inducible enzymic forms of DMN-demethylase, Z. Krebsforsch, 89 (1977) 181. 12 M.B. Kroeger-Koepke and C.J. Michejda, Evidence for several demethylase enzymes in the oxidation of dimethylnitrosamine and phenylmethylnitrosamine by rat liver fractions, Cancer Res., 39 (1979) 1587. 13 A.E. Pegg, Metabolism of N-nitrosodimethylamine, in: Molecular and Cellular Aspects of Carcinogen Screening Tests, International Agency for Cancer Research, Lyon, France, I A R C ScientificPublication No. 27, 1980, pp. 3--22. 14 J.H. Exton, The perfused rat liver,Methods Enzymol., 37 (1975) 25. 15 R.C. Feldhoff, J.M. Taylor and L.S. Jefferson, Synthesis and secretion of rat albumin in vivo, in perfused liver, and in isolated hepatocytes, J. Biol. Chem., 252 (1977) 3611. 16 W.J. Bodell and M.R. Banerjee, Reduced D N A repair in mouse satelliteD N A after treatment with methyl methanesulfonate and N-methyl-N-nitrosourea, Nucleic Acid Res., 3 (1976) 1689. 17 L. Flume, G. Campadelli-Fiume, P.N. Magee and J. Holsman, Cellular injury and carcinogenesis. Inhibition of metabolism of dimethylnitrosamine by aminoacetonitrile,Biochem. J., 120 (1970) 601. 18 D. Hadjiolov and D. Mundt, Effect of aminoacetonitrile on the metabolism of dimethylnitrosamine and methylation of R N A during liver carcinogenesis, J. Natl. Cancer Inst., 52 (1974) 753. 19 G.M. Williams, The use of liver epithelialcultures for the study of chemical carcinogenesis, A m . J. Pathol., 85 (1976) 739. 20 A.D. Tates, I. Neuteboorn, M. Hofker, and L. den Engelse, A micronucleus technique for detecting clastogenic effects of mutagens/carcinogens ( D E N , D M N ) in hepatocytes of rat liverin vivo. Murat. Res., 74 (1980) 11. 21 C.A. Jones and E. Huberman, A sensitive hepatocyte-mediated assay for the metabolism of nitrosamines to mutagens for mammalian cells, Cancer Res., 40 (1980) 406. 22 G.M. Williams and M.F. Laspia, The detection of various nitrosamines in the hepatocyte primary culture/DNA repair test, Cancer Lett., 6 (1979) 199. 23 T. Mendoza-Figueroa, R. L6pez-Revilla and S. Villa-TreviKo, Dose-dependent D N A ruptures induced by the procarcinogen dimethylnitrosamine on primary rat liver cultures, Cancer Res., 39 (1979) 3254. 24 G. Michalopoulos, G.L. Sattler, L. O'Connor and H.C. Pitot, Unscheduled D N A synthesis induced by procarcinogens in suspensions and primary cultures of hepatocytes on collagen membranes, Cancer Res., 38 (1978) 1866.
238 25 R. Stumpf, G.P. Margison, R. Montesano and A.E. Pegg, Formation and loss of alkylated purines from D N A of hamster liver after administration of dimethylnitrosamine, Cancer Res., 39 (1979) 50. 26 G.P. Margison, J.A. SwindeIl, C.H. Ockey and A.W. Craig, The effects of a single dose of dimethylnitrosamine in the Chinese hamster and the persistence of D N A alkylation products in selected tissues, Carcinogenesis, 1 (1980) 91. 27 R. Goth-Goldstein, Inability of Chinese hamster ovary cells to excise O"-alkylguanine, Cancer Res., 40 (1980) 2623.