- Email: [email protected]
Effect of various chemicals on macromolecular binding during oxidative dealkylation of dimethylnitrosamine by hamster liver microsomes
Toxicology Letters,5 (1980) 345-351 o Elsevier/North-Holland Biomedical Press
345
EFFECT OF VARIOUS CHEMICALS ON MACROMOLECULAR BINDING DURING OXIDATIVE DEALKYLATION OF DIMETHYLNITROSAMINE BY HAMSTER LIVER MICROSOMES
YOUNG SOOK HONG*, SANGDUK KIM and PRABHAKAR Fels Research Institute and the Department Medicine, Philadelphia, PA 19140, U.S.A.
of Biochemistry,
D. LOTLIKAR** Temple University School of
(Received October 2&h, 1979) (Accepted December 12th, 1979)
SUMMARY
Differential inhibitory effects of various chemicals on both formaldehyde formation and total macromolecular binding have been examined during oxidative demethylation of [ 14C] dimethylnitrosamine (DMN) by hamster liver microsomes. One group of chemicals such as diethyldithiocarbamate (DEDTC), aminoacetonitrile (AAN), 2-[ (2,4-dichloro-6-phenyl) phenoxy ] ethylamine (DPEA), azide and ethanol inhibits both HCHO formation and total macromolecular binding. A second group of chemicals such as reduced glutathione, semicarbazide and N-(l-naphthyl)thiourea (NTU) inhibits macromolecular binding without appreciably affecting HCHO formation. Possible mechanisms of these differential inhibitory effects by these chemicals are discussed.
INTRODUCTION
Oxidative demethylation of DMN is considered to be an activation pathway in its carcinogenic process [6, 19- -211. Inhibition of formaldehyde formation by CO [3, 51 and reconstitution studies [lo, 171 have shown involvement of cytochrome P-450 enzyme system in the microsomal oxidation of this carcinogen. During oxidative demethylation of DMN, in addition to formaldehyde formation, a reactive alkylating species is generated which interacts covalently with various cellular macromolecules [6,19-211. In *Present address: Department of Biochemistry, College of Medicine, Ewha Woman’s University, Seoul, Korea 33-0151(702). **To whom requests for reprints should be addressed. Abrreviations: AAN, aminoacetonitrile; DEDTC, diethyldithiocarbamate; DEN, diethylnitrosamine; DMA, N,N-dimethylaniline; DMN, dimethylnitrosamine; DPEA, 2-[( 2,4dichloro-6-phenyl)phenoxy ]ethylamine; GSH, reduced glutathione; NTU, N-( l-naphthyl)thiourea; SKF525-A, 2-(diethylamino)ethyl-2,2-diphenylvalerate hydrochloride; TCA, trichloroacetic acid.
346
earlier studies, either the formaldehyde formation or macromolecular binding, but not both, were examined during microsomal oxidation of this carcinogen [ 3--5, 171. However, in recent studies, we have shown that during NADPH dependent microsomal oxidation of DMN, in addition to formaldehyde formation, methylation of proteins occurred [ 131. The present report now demonstrates the differential effects of various chemicals on both formaldehyde formation and total macromolecular binding during oxidative demethylation of DMN by hamster liver microsomes. MATERIALS
AND METHODS
Chemicals used in the present study were from the following sources in the U.S.A.: Calf thymus DNA, yeast sRNA, bovine pancreatic ribonuclease A, calf thymus histone II-A, egg white lysozyme, DEDTC, GSH and sodium azide, Sigma Chemical Co., St. Louis, MO: NADPH, Boehringer Mannheim Biochemicals, Indianapolis, IN: DMN, DEN, NTU, and 5,5-dimethyl-1,3cyclohexanedione, Eastman Kodak Co., Rochester, NY; [ 14C] DMN (spec. radioact. 5.2 mCi/mmol), New England Nuclear Corp., Boston, MA; methanol and potassium cyanide, J.T. Baker Chemical Co., Phillipsburg, NJ: reagent grade aniline, DMA and semicarbazide hydrochloride, Fisher Scientific Co., Fairlawn, NJ; AAN, Aldrich Chemical Co., Milwaukee, WI; absolute ethanol, Publicker Industries Co., Linfield, PA; SKF-525A, a gift of Smith Kline & French Laboratories, Philadelphia, PA; DPEA was kindly provided by Dr. Robert McMahon, Eli Lilly Laboratories, Indianapolis, IN; all other chemicals were of reagent grade. Liver microsomes were made from adult male Syrian golden hamsters (100-150 g) as described previously [ 181. Cytochrome P-450 content of such microsomal preparations was assayed by the method of Omura and Sat0 [22]. The incubation medium for oxidation studies contained 100 mM potassium phosphate buffer, pH 6.5,2 mM NADPH, 2 mM DMN containing [ 14C] 0MN (final spec. act. 1.0 mCi/mmol), liver microsomal protein containing 1.0 nmol of cytochrome P-450 and various chemicals as indicated to a total volume of 0.5 ml. Duplicate samples were incubated in air for 30 min at 37°C. After incubation, 1 pmol of semicarbazide was added to all samples. When histone or other exogenous macromolecules were not present in samples during incubation, 5 mg histone, as a carrier protein, was also added to these samples after incubation. The reaction was terminated by the addition of 1.5 ml of 50% TCA. Blank samples were prepared by adding NADPH after the addition of TCA at zero times. After centrifugation of all samples in a clinical centrifuge for 15 min, the supernatant and the precipitate were used for the determinations of HCHO and macromolecular binding respectively. I-II4 CHO was precipitated as formaldemethone by the procedure of Frisell and MacKenzie [8] as modified by Palk and Kim [23] using 8 mg of carrier formaldehyde. Radioactivity in the precipitated formaldemethone (theoret-
341
ical yield, 78 mg) was measured by scintillation spectrometry as described previously [ 171. Macromolecular binding in the TCA precipitate was determined by washing the precipitate three times with 5 ml of ice-cold 15% TCA and once with 5 ml of ethanol. Finally, the radioactivity present in the washed precipitate was measured by scintillation spectrometry after dissolving the precipitate in 10 ml of scintillation liquid (New England Nuclear formula, 963). Radioactivity obtained with blank samples was deducted from radioactivity obtained with incubated samples. RESULTS AND DISCUSSION
We have shown previously that during DMN oxidation by hamster liver microsomes, both formaldehyde formation and protein methylation were NADPH and enzyme dependent [13]. Optimum pH for these product formations was about 6.5 [13, 171. In the present study therefore, both of these phenomena, HCHO formation and macromolecular binding have been examined at their optimum pH (Tables I and II). Under these conditions, in the absence of any exogenous macromolecules, the amount of total binding was about 3% of HCHO formation (Table I). In the presence of histone during incubation, HCHO formation was decreased whereas total binding was increased, thus, in the presence of histone, the amount of binding was about 12% of HCHO formation. Ribonuclease A did not have much effect on HCHO formation, but it increased the total binding by 60%. Presence of either sRNA or DNA inhibited both reactions to some extent. Since presence of histone gave the maximum amount of total binding in these studies, TABLE I EFFECT OF EXOGENOUS MACROMOLECULES ON HCHO FORMATION AND TOTAL MACROMOLECULAR BINDING DURING DIMETHYLNITROSAMINE OXIDATION BY HAMSTER LIVER MICROSOMES All details are described in MATERIALS AND METHODS. Where indicated, 5 mg of variour macromolecules, except DNA, was added to the incubation medium. Due to solubility problems, only 1.5 mg of calf thymus DNA was added to the incubation medium. Results are given as averages of three analyses. Experimental variations were less than 10% of the average of three analyses. Macromolecules
___ Histone, II-A Lysozyme Ribonuclease A sRNA DNA
added
nmol formed/nmol
P-450/30
HCHO
Total binding
40 19 35 36 24 32
1.2 2.2 1.5 1.9 0.6 0.8
min
348 TABLE II EFFECT OF VARIOUS CHEMICALS ON HCHO FORMATION AND MACROMOLECULAR BINDING DURING OXIDATIVE DEMETHYLATION OF DIMETHYLNITROSAMINE BY HAMSTER LIVER MICROSOMAL PREPARATIONS All details are described in MATERIALS AND METHODS. Where indicated, 5 mg bistone was added to the medium during incubation. Control hamster liver microsmomes, in the absence and presence of histone during the incubation, formed 45 f 4 and 27 f 2 nmol of H14CH0 respectively per nmol cytochrome P-450/30 min during oxidative metabolism of DMN. Under these conditions, 1.3 ?: 0.15 and 2.3 + 0.20 nmol of W!H, were incorporated into total macromolecules respectively per nmol of cytochrome P-450/30 min in the absence and presence of histone during the incubation. These control results are given as means + S.E.M. of three analyses. Other results are averages of three analyses and are expressed as percentages of the control; experimental variations were less than 10% of the averages of three analyses. Compound added
H14CH0 formation
Macromolecular binding % of control
___ SKF525A, 1 mM DPEA, 0.1 mM 0.5 mM AAN, 1 mM DEDTC, 0.1 mM Ethanol, 200 mM KCN, 1 mM NaN,, 1 mM Aniline, 5 mM DMA, 5 mM DEN, 5 mM Methanol, 200 mM NTU, 1 mM semicarbazide, 2 mM GSH
Histone +
Hktone +
100 62 72 23 57 11 10 51 8 0 1 11 102 102 90 115
100 74 75 38 53 4 18 136 5 0 7 19 79 33 13 48
100 88 71 22 54 7 8 20 13 8 1 33 88 75 116 107
100 89 68 39 78 17 21 368 22 0 0 23 95 64 13 53
it was used routinely in comparative studies where various inhibitors were tested. Similar to our previous studies [13], we have also observed in the present study that about 45-50% of total binding is due to methylation of carboxyl residues in either endogenous protein or histone (data not shown). Radioactivity present as carboxylmethylated protein is labile and is released as methanol under alkaline conditions [ 131. Even though presence of histone increased the total amount of binding (Table I), radioactivity incorporation in both the stable and labile forms was about equal. When the stable fraction is further fractionated by the standard procedure described previously [ 131,
349
it is found that about 40%, 5% and 10% of total binding are found in the protein, lipid and nucleic acid fractions respectively. The effects of various chemicals on both formaldehyde formation and macromolecular binding are summarized in Table II. SKF525-A and DPEA are both known to be potent inhibitors of cytochrome P-450 mediated microsomal oxidation of several xenobiotics [2, 241. In the present study during DMN oxidation, SKF525A at 1 mM level inhibited both processes to some extent especially in the absence of histone during incubation. Lake et al. [ 161 have also observed similar degree of inhibition of formaldehyde formation with rat liver preparations with SKF525A during DMN oxidation. However, our results with SKF525A are not in agreement with recent data of other investigators obtained with rat liver microsomes [9]. They have shown 90% inhibition of macromolecular binding with 1 mM SKF525A without appreciably affecting HCHO formation. There is a possibility that in their studies, a large amount of binding was lost during isolation procedure due to the labile nature of the binding as previously shown by us [ 131. Compared to SKF525A, DPEA at 0.5 mM level, is a much more potent inhibitor of both processes (Table II). AAN [ 7,11, 151, DEDTC [ 1, 161, and ethanol [25] have been shown to be potent inhibitors of DMN oxidation in vivo and in vitro studies. In the present study, DEDTC compared to AAN was found to be a much more potent inhibitor of both processes. In these studies, methanol did not have any appreciable inhibitory effects whereas the same concentration of ethanol inhibited both processes to a large extent. Cyanide and azide have been shown to be potent inhibitors of DMN oxidation by rat liver preparations [14]. Inhibition by these two compounds and by DEDTC suggests the possible involvement of a non-cytochrome P-450 enzyme system in the metabolic oxidation of DMN. In the present study (Table II), it was striking to observe that in contrast to azide, which inhibited both processes completely, cyanide inhibited only formaldehyde formation with several fold increase in binding especially in the presence of histone during incubation. This increase in macromolecular binding in the presence of cyanide was found predominantly in the labile fraction. Activity in the labile fraction, however, was not due to the presence of carboxyl methyl derivatives of proteins (data not shown). Aniline [ 121, DMA [ 261 and DEN [ 201 are also oxidized by various microsomal enzymes. These compounds (5 mM) in the present study (Table II) inhibited both processes to a large extent probably by competing for either NADPH or enzymes involved in the oxidative metabolism of DMN. Semicarbazide, a trapping agent for aldehydes, NTU, a potent inhibitor of mixed function amine oxidase [27] and reduced glutathione are found to inhibit macromolecular binding without appreciably affecting the metabolic oxidation of DMN (Table II). It is known that metabolic oxidation of DMN is required for generation of a reactive carbonium ion which is involved in macromolecular binding [ 6, 19--211. Our present results suggest that there are two groups of inhibitors
350
of macromolecular binding. One group of chemicals such as DEDTC, AAN, DPEA, azide and ethanol inhibit macromolecular binding during DMN oxidation by inhibiting at the oxidative level. A second group of chemicals, such as reduced glutathione, semicarbazide, and NTU, inhibit the binding by acting after the oxidative stage at the carbonium ion level. ACKNOWLEDGEMENTS
The research was supported by grants CA-10604 and CA-12227 National Cancer Institute, United States Public Health Service.
from the
REFERENCES 1 S.E. Abanobi, J.A. Popp, S.K. Chang, G.W. Harrington, P.D. Lotlikar, D. Hadjiolov, M. Levitt, S. Rajalakshmi and D.S.R. Sarma, Inhibition of dimethylnitrosamine-induced strand breaks in liver DNA and liver cell necrosis by diethyldithiocarbamate, J. Natl. Cancer Inst., 58 (1977) 263-271. 2 M.W. Anders, Enhancement and inhibition of drug metabolism, Annu. Rev. Pharmacol. 11 (1971) 37-56. 3 M.F. Argus, J.C. Amos, K.M. Pastor, B.C. Wu and N. Venkatesan, Dimethylnitrosaminedemethylase: absence of increased enzyme catabolism and multiplicity of effector sites in repression. Hemoprotein involvement, Chem.-Biol. Interact., 13 (1976) 127-140. 4 A.E. Chin and H.B. Bosmann, Microsome-mediated methylation of DNA by N,Ndimethylnitrosamine in vitro, Biochem. Pharmacol., 25 (1976) 1921-1926. 5 P. Czygan, H. Greim, A.J. Garro, F. Hutterer, F. Schaffner, H. Popper, 0. Rosenthal and D. Cooper, Microsomal metabolism of dimethylnitrosamine and cytochrome P-450 dependency of its activation to a mutagen, Cancer Res., 33 (1973) 2983-2986. 6 H. Druckrey, R. Preusmann, S. Ivankovic and D. Schmal, Organotrope carcinogene wirkungen bei 65 verschiedenen N-nitroso-verbindungen an BD-ratten, Z. Krebsforsch., 69 (1967) 103-201. 7 L. Fiume, G. Campadelli-Fiume, P.N. Magee and J. Holsman, Cellular injury and carcinogenesis: Inhibition of metabolism of dimethylnitrosamine by aminoacetonitrile, Biochem. J. 120 (1970) 601-605. 8 W.R. Frisell and C.G. MacKenzie, The determination of formaldehyde and serine in biological systems, in: D. Glick (Ed.) Methods of Biochemical Analysis, vol 6, Interscience, New York, 1958, pp. 63-77. 9 H.M. Godoy, MI. Diaz Gomez and J.A. Castro, Mechanism of dimethylnitrosamine metabolism and activation in rats, J. Natl. Cancer Inst., 61 (1978) 1285-1289. 10 F.P. Guengerich, Separation and purification of multiple forms of cytochrome P-450. Activities of different forms of cytochrome P-450 toward several compounds of environmental interest, J. Biol. Chem., 252 (1977) 3970-3979. 11 D. Hadjiolov and D. Mundt, Effect of aminoacetonitrile on the metabolism of dimethylnitrosamine and methylation of RNA during liver carcinogenesis, J. Natl. Cancer Inst., 52 (1974) 753-756. 12 M. Kiese, The biochemical production of ferrihemoglobin forming derivatives from aromatic amines, and mechanisms of ferrihemoglobin formation, Pharmacol. Rev., 18 (1966) 1091-1161. 13 S. Kim, P.D. Lotlikar, W. Chin and P.N. Magee, Protein bound carboxyl-methyl ester as a precursor of methanol formation during oxidation of dimethylnitrosamine in vitro, Cancer Lett., 2 (1977) 279-284.
351 14
15
16 17
18
19 20 21 22 23 24
25
26
27
B.G. Lake, M.J. Minski, J.C. Phillips, S.D. Gangolli and A.G. Lloyd, Investigations into the hepatic metabolism of dimethylnitrosamine in the rat, Life Sci., 17 (1976) 1599-1606. B.G. Lake, J.C. Phillips, S.D. Gangolli and A.G. Lloyd, Further studies on the inhibition of rat hepatic dimethylnitrosamine metabolism in vitro, Biochem. SOC. Trans., 4 (1976) 684-685. B.G. Lake, J.C. Phillips, C.E. Heading and SD. Gangolli, Studies on the in vitro metabolism of dimethylnitrosamine by rat liver, Toxicology, 5 (1976) 297-309. P.D. Lotlikar, W.J. Baldy Jr. and E.N. Dwyer, Dimethylnitrosamine demethylation by reconstituted liver microsomal cytochrome P-450, Biochem. J., 152 (1975) 705-708. P.D. Lotlikar, L. Luha and K. Zaleski, Reconstituted hamster liver microsomal enzyme system for N-hydroxylation of the carcinogen 2-acetylaminofluorene, Biochem. Biophys. Res. Commun., 59 (1974) 1349-1355. P.N. Magee and J.M. Barnes, Carcinogenic nitroso compounds, Adv. Cancer Res., 10 (1967) 163-246. P.N. Magee, A.E. Pegg and P.F. Swann, Molecular mechanisms of chemical carcinogenesis. Handb. Alleg. Pathol., 6 (1975) 329-420. J.A. Miller, Carcinogenesis by chemicals: An overview - G.H.A. Clowes memorial lecture, Cancer Res., 30 (1970) 559-576. T. Omura and R. Sato, The carbon monoxide-binding pigment of liver microsomes. I. Evidence for its hemoprotein nature, J. Biol. Chem., 239 (1964) 2370-2378. W.K. Paik and S. Kim, e-Alkyllysinase: New assay method, purification and biological significance, Arch. Biochem. Biophys., 165 (1974) 369-378. C.J. Parli, N.W. Lee and R.E. McMahon, The relationship between the metabolism of 2,4-dichloro-6-phenylphenoxyethylamine (DPEA) and related compounds and their activities as microsomal mono-oxygenase inhibitors, Drug Metab. Disp., 1 (1973) 628-633. J.C. Phillips, B.G. Lake, SD, Gangolli, P. Grass0 and A.G. Lloyd, Effects of pyrazole and 3-amino-1,2,4-triazole on the metabolism and toxicity of dimethylnitrosamine in the rat, J. Natl. Cancer Inst., 58 (1977) 629-633. D.M. Ziegler and F.H. Pettit, Formation of an intermediate N-oxide in the oxidative demethylation of N,N-dimethylaniline catalyzed by liver microsomes, Biochem. Biophys. Res. Commun., 15 (1964) 188-193. D.M. Ziegler and C.H. Mitchell, Microsomal oxidase IV: Properties of a mixed-function amine oxidase isolated from pig liver microsomes, Arch. Biochem. Biophys., 150 (1972) 116-125.
345
EFFECT OF VARIOUS CHEMICALS ON MACROMOLECULAR BINDING DURING OXIDATIVE DEALKYLATION OF DIMETHYLNITROSAMINE BY HAMSTER LIVER MICROSOMES
YOUNG SOOK HONG*, SANGDUK KIM and PRABHAKAR Fels Research Institute and the Department Medicine, Philadelphia, PA 19140, U.S.A.
of Biochemistry,
D. LOTLIKAR** Temple University School of
(Received October 2&h, 1979) (Accepted December 12th, 1979)
SUMMARY
Differential inhibitory effects of various chemicals on both formaldehyde formation and total macromolecular binding have been examined during oxidative demethylation of [ 14C] dimethylnitrosamine (DMN) by hamster liver microsomes. One group of chemicals such as diethyldithiocarbamate (DEDTC), aminoacetonitrile (AAN), 2-[ (2,4-dichloro-6-phenyl) phenoxy ] ethylamine (DPEA), azide and ethanol inhibits both HCHO formation and total macromolecular binding. A second group of chemicals such as reduced glutathione, semicarbazide and N-(l-naphthyl)thiourea (NTU) inhibits macromolecular binding without appreciably affecting HCHO formation. Possible mechanisms of these differential inhibitory effects by these chemicals are discussed.
INTRODUCTION
Oxidative demethylation of DMN is considered to be an activation pathway in its carcinogenic process [6, 19- -211. Inhibition of formaldehyde formation by CO [3, 51 and reconstitution studies [lo, 171 have shown involvement of cytochrome P-450 enzyme system in the microsomal oxidation of this carcinogen. During oxidative demethylation of DMN, in addition to formaldehyde formation, a reactive alkylating species is generated which interacts covalently with various cellular macromolecules [6,19-211. In *Present address: Department of Biochemistry, College of Medicine, Ewha Woman’s University, Seoul, Korea 33-0151(702). **To whom requests for reprints should be addressed. Abrreviations: AAN, aminoacetonitrile; DEDTC, diethyldithiocarbamate; DEN, diethylnitrosamine; DMA, N,N-dimethylaniline; DMN, dimethylnitrosamine; DPEA, 2-[( 2,4dichloro-6-phenyl)phenoxy ]ethylamine; GSH, reduced glutathione; NTU, N-( l-naphthyl)thiourea; SKF525-A, 2-(diethylamino)ethyl-2,2-diphenylvalerate hydrochloride; TCA, trichloroacetic acid.
346
earlier studies, either the formaldehyde formation or macromolecular binding, but not both, were examined during microsomal oxidation of this carcinogen [ 3--5, 171. However, in recent studies, we have shown that during NADPH dependent microsomal oxidation of DMN, in addition to formaldehyde formation, methylation of proteins occurred [ 131. The present report now demonstrates the differential effects of various chemicals on both formaldehyde formation and total macromolecular binding during oxidative demethylation of DMN by hamster liver microsomes. MATERIALS
AND METHODS
Chemicals used in the present study were from the following sources in the U.S.A.: Calf thymus DNA, yeast sRNA, bovine pancreatic ribonuclease A, calf thymus histone II-A, egg white lysozyme, DEDTC, GSH and sodium azide, Sigma Chemical Co., St. Louis, MO: NADPH, Boehringer Mannheim Biochemicals, Indianapolis, IN: DMN, DEN, NTU, and 5,5-dimethyl-1,3cyclohexanedione, Eastman Kodak Co., Rochester, NY; [ 14C] DMN (spec. radioact. 5.2 mCi/mmol), New England Nuclear Corp., Boston, MA; methanol and potassium cyanide, J.T. Baker Chemical Co., Phillipsburg, NJ: reagent grade aniline, DMA and semicarbazide hydrochloride, Fisher Scientific Co., Fairlawn, NJ; AAN, Aldrich Chemical Co., Milwaukee, WI; absolute ethanol, Publicker Industries Co., Linfield, PA; SKF-525A, a gift of Smith Kline & French Laboratories, Philadelphia, PA; DPEA was kindly provided by Dr. Robert McMahon, Eli Lilly Laboratories, Indianapolis, IN; all other chemicals were of reagent grade. Liver microsomes were made from adult male Syrian golden hamsters (100-150 g) as described previously [ 181. Cytochrome P-450 content of such microsomal preparations was assayed by the method of Omura and Sat0 [22]. The incubation medium for oxidation studies contained 100 mM potassium phosphate buffer, pH 6.5,2 mM NADPH, 2 mM DMN containing [ 14C] 0MN (final spec. act. 1.0 mCi/mmol), liver microsomal protein containing 1.0 nmol of cytochrome P-450 and various chemicals as indicated to a total volume of 0.5 ml. Duplicate samples were incubated in air for 30 min at 37°C. After incubation, 1 pmol of semicarbazide was added to all samples. When histone or other exogenous macromolecules were not present in samples during incubation, 5 mg histone, as a carrier protein, was also added to these samples after incubation. The reaction was terminated by the addition of 1.5 ml of 50% TCA. Blank samples were prepared by adding NADPH after the addition of TCA at zero times. After centrifugation of all samples in a clinical centrifuge for 15 min, the supernatant and the precipitate were used for the determinations of HCHO and macromolecular binding respectively. I-II4 CHO was precipitated as formaldemethone by the procedure of Frisell and MacKenzie [8] as modified by Palk and Kim [23] using 8 mg of carrier formaldehyde. Radioactivity in the precipitated formaldemethone (theoret-
341
ical yield, 78 mg) was measured by scintillation spectrometry as described previously [ 171. Macromolecular binding in the TCA precipitate was determined by washing the precipitate three times with 5 ml of ice-cold 15% TCA and once with 5 ml of ethanol. Finally, the radioactivity present in the washed precipitate was measured by scintillation spectrometry after dissolving the precipitate in 10 ml of scintillation liquid (New England Nuclear formula, 963). Radioactivity obtained with blank samples was deducted from radioactivity obtained with incubated samples. RESULTS AND DISCUSSION
We have shown previously that during DMN oxidation by hamster liver microsomes, both formaldehyde formation and protein methylation were NADPH and enzyme dependent [13]. Optimum pH for these product formations was about 6.5 [13, 171. In the present study therefore, both of these phenomena, HCHO formation and macromolecular binding have been examined at their optimum pH (Tables I and II). Under these conditions, in the absence of any exogenous macromolecules, the amount of total binding was about 3% of HCHO formation (Table I). In the presence of histone during incubation, HCHO formation was decreased whereas total binding was increased, thus, in the presence of histone, the amount of binding was about 12% of HCHO formation. Ribonuclease A did not have much effect on HCHO formation, but it increased the total binding by 60%. Presence of either sRNA or DNA inhibited both reactions to some extent. Since presence of histone gave the maximum amount of total binding in these studies, TABLE I EFFECT OF EXOGENOUS MACROMOLECULES ON HCHO FORMATION AND TOTAL MACROMOLECULAR BINDING DURING DIMETHYLNITROSAMINE OXIDATION BY HAMSTER LIVER MICROSOMES All details are described in MATERIALS AND METHODS. Where indicated, 5 mg of variour macromolecules, except DNA, was added to the incubation medium. Due to solubility problems, only 1.5 mg of calf thymus DNA was added to the incubation medium. Results are given as averages of three analyses. Experimental variations were less than 10% of the average of three analyses. Macromolecules
___ Histone, II-A Lysozyme Ribonuclease A sRNA DNA
added
nmol formed/nmol
P-450/30
HCHO
Total binding
40 19 35 36 24 32
1.2 2.2 1.5 1.9 0.6 0.8
min
348 TABLE II EFFECT OF VARIOUS CHEMICALS ON HCHO FORMATION AND MACROMOLECULAR BINDING DURING OXIDATIVE DEMETHYLATION OF DIMETHYLNITROSAMINE BY HAMSTER LIVER MICROSOMAL PREPARATIONS All details are described in MATERIALS AND METHODS. Where indicated, 5 mg bistone was added to the medium during incubation. Control hamster liver microsmomes, in the absence and presence of histone during the incubation, formed 45 f 4 and 27 f 2 nmol of H14CH0 respectively per nmol cytochrome P-450/30 min during oxidative metabolism of DMN. Under these conditions, 1.3 ?: 0.15 and 2.3 + 0.20 nmol of W!H, were incorporated into total macromolecules respectively per nmol of cytochrome P-450/30 min in the absence and presence of histone during the incubation. These control results are given as means + S.E.M. of three analyses. Other results are averages of three analyses and are expressed as percentages of the control; experimental variations were less than 10% of the averages of three analyses. Compound added
H14CH0 formation
Macromolecular binding % of control
___ SKF525A, 1 mM DPEA, 0.1 mM 0.5 mM AAN, 1 mM DEDTC, 0.1 mM Ethanol, 200 mM KCN, 1 mM NaN,, 1 mM Aniline, 5 mM DMA, 5 mM DEN, 5 mM Methanol, 200 mM NTU, 1 mM semicarbazide, 2 mM GSH
Histone +
Hktone +
100 62 72 23 57 11 10 51 8 0 1 11 102 102 90 115
100 74 75 38 53 4 18 136 5 0 7 19 79 33 13 48
100 88 71 22 54 7 8 20 13 8 1 33 88 75 116 107
100 89 68 39 78 17 21 368 22 0 0 23 95 64 13 53
it was used routinely in comparative studies where various inhibitors were tested. Similar to our previous studies [13], we have also observed in the present study that about 45-50% of total binding is due to methylation of carboxyl residues in either endogenous protein or histone (data not shown). Radioactivity present as carboxylmethylated protein is labile and is released as methanol under alkaline conditions [ 131. Even though presence of histone increased the total amount of binding (Table I), radioactivity incorporation in both the stable and labile forms was about equal. When the stable fraction is further fractionated by the standard procedure described previously [ 131,
349
it is found that about 40%, 5% and 10% of total binding are found in the protein, lipid and nucleic acid fractions respectively. The effects of various chemicals on both formaldehyde formation and macromolecular binding are summarized in Table II. SKF525-A and DPEA are both known to be potent inhibitors of cytochrome P-450 mediated microsomal oxidation of several xenobiotics [2, 241. In the present study during DMN oxidation, SKF525A at 1 mM level inhibited both processes to some extent especially in the absence of histone during incubation. Lake et al. [ 161 have also observed similar degree of inhibition of formaldehyde formation with rat liver preparations with SKF525A during DMN oxidation. However, our results with SKF525A are not in agreement with recent data of other investigators obtained with rat liver microsomes [9]. They have shown 90% inhibition of macromolecular binding with 1 mM SKF525A without appreciably affecting HCHO formation. There is a possibility that in their studies, a large amount of binding was lost during isolation procedure due to the labile nature of the binding as previously shown by us [ 131. Compared to SKF525A, DPEA at 0.5 mM level, is a much more potent inhibitor of both processes (Table II). AAN [ 7,11, 151, DEDTC [ 1, 161, and ethanol [25] have been shown to be potent inhibitors of DMN oxidation in vivo and in vitro studies. In the present study, DEDTC compared to AAN was found to be a much more potent inhibitor of both processes. In these studies, methanol did not have any appreciable inhibitory effects whereas the same concentration of ethanol inhibited both processes to a large extent. Cyanide and azide have been shown to be potent inhibitors of DMN oxidation by rat liver preparations [14]. Inhibition by these two compounds and by DEDTC suggests the possible involvement of a non-cytochrome P-450 enzyme system in the metabolic oxidation of DMN. In the present study (Table II), it was striking to observe that in contrast to azide, which inhibited both processes completely, cyanide inhibited only formaldehyde formation with several fold increase in binding especially in the presence of histone during incubation. This increase in macromolecular binding in the presence of cyanide was found predominantly in the labile fraction. Activity in the labile fraction, however, was not due to the presence of carboxyl methyl derivatives of proteins (data not shown). Aniline [ 121, DMA [ 261 and DEN [ 201 are also oxidized by various microsomal enzymes. These compounds (5 mM) in the present study (Table II) inhibited both processes to a large extent probably by competing for either NADPH or enzymes involved in the oxidative metabolism of DMN. Semicarbazide, a trapping agent for aldehydes, NTU, a potent inhibitor of mixed function amine oxidase [27] and reduced glutathione are found to inhibit macromolecular binding without appreciably affecting the metabolic oxidation of DMN (Table II). It is known that metabolic oxidation of DMN is required for generation of a reactive carbonium ion which is involved in macromolecular binding [ 6, 19--211. Our present results suggest that there are two groups of inhibitors
350
of macromolecular binding. One group of chemicals such as DEDTC, AAN, DPEA, azide and ethanol inhibit macromolecular binding during DMN oxidation by inhibiting at the oxidative level. A second group of chemicals, such as reduced glutathione, semicarbazide, and NTU, inhibit the binding by acting after the oxidative stage at the carbonium ion level. ACKNOWLEDGEMENTS
The research was supported by grants CA-10604 and CA-12227 National Cancer Institute, United States Public Health Service.
from the
REFERENCES 1 S.E. Abanobi, J.A. Popp, S.K. Chang, G.W. Harrington, P.D. Lotlikar, D. Hadjiolov, M. Levitt, S. Rajalakshmi and D.S.R. Sarma, Inhibition of dimethylnitrosamine-induced strand breaks in liver DNA and liver cell necrosis by diethyldithiocarbamate, J. Natl. Cancer Inst., 58 (1977) 263-271. 2 M.W. Anders, Enhancement and inhibition of drug metabolism, Annu. Rev. Pharmacol. 11 (1971) 37-56. 3 M.F. Argus, J.C. Amos, K.M. Pastor, B.C. Wu and N. Venkatesan, Dimethylnitrosaminedemethylase: absence of increased enzyme catabolism and multiplicity of effector sites in repression. Hemoprotein involvement, Chem.-Biol. Interact., 13 (1976) 127-140. 4 A.E. Chin and H.B. Bosmann, Microsome-mediated methylation of DNA by N,Ndimethylnitrosamine in vitro, Biochem. Pharmacol., 25 (1976) 1921-1926. 5 P. Czygan, H. Greim, A.J. Garro, F. Hutterer, F. Schaffner, H. Popper, 0. Rosenthal and D. Cooper, Microsomal metabolism of dimethylnitrosamine and cytochrome P-450 dependency of its activation to a mutagen, Cancer Res., 33 (1973) 2983-2986. 6 H. Druckrey, R. Preusmann, S. Ivankovic and D. Schmal, Organotrope carcinogene wirkungen bei 65 verschiedenen N-nitroso-verbindungen an BD-ratten, Z. Krebsforsch., 69 (1967) 103-201. 7 L. Fiume, G. Campadelli-Fiume, P.N. Magee and J. Holsman, Cellular injury and carcinogenesis: Inhibition of metabolism of dimethylnitrosamine by aminoacetonitrile, Biochem. J. 120 (1970) 601-605. 8 W.R. Frisell and C.G. MacKenzie, The determination of formaldehyde and serine in biological systems, in: D. Glick (Ed.) Methods of Biochemical Analysis, vol 6, Interscience, New York, 1958, pp. 63-77. 9 H.M. Godoy, MI. Diaz Gomez and J.A. Castro, Mechanism of dimethylnitrosamine metabolism and activation in rats, J. Natl. Cancer Inst., 61 (1978) 1285-1289. 10 F.P. Guengerich, Separation and purification of multiple forms of cytochrome P-450. Activities of different forms of cytochrome P-450 toward several compounds of environmental interest, J. Biol. Chem., 252 (1977) 3970-3979. 11 D. Hadjiolov and D. Mundt, Effect of aminoacetonitrile on the metabolism of dimethylnitrosamine and methylation of RNA during liver carcinogenesis, J. Natl. Cancer Inst., 52 (1974) 753-756. 12 M. Kiese, The biochemical production of ferrihemoglobin forming derivatives from aromatic amines, and mechanisms of ferrihemoglobin formation, Pharmacol. Rev., 18 (1966) 1091-1161. 13 S. Kim, P.D. Lotlikar, W. Chin and P.N. Magee, Protein bound carboxyl-methyl ester as a precursor of methanol formation during oxidation of dimethylnitrosamine in vitro, Cancer Lett., 2 (1977) 279-284.
351 14
15
16 17
18
19 20 21 22 23 24
25
26
27
B.G. Lake, M.J. Minski, J.C. Phillips, S.D. Gangolli and A.G. Lloyd, Investigations into the hepatic metabolism of dimethylnitrosamine in the rat, Life Sci., 17 (1976) 1599-1606. B.G. Lake, J.C. Phillips, S.D. Gangolli and A.G. Lloyd, Further studies on the inhibition of rat hepatic dimethylnitrosamine metabolism in vitro, Biochem. SOC. Trans., 4 (1976) 684-685. B.G. Lake, J.C. Phillips, C.E. Heading and SD. Gangolli, Studies on the in vitro metabolism of dimethylnitrosamine by rat liver, Toxicology, 5 (1976) 297-309. P.D. Lotlikar, W.J. Baldy Jr. and E.N. Dwyer, Dimethylnitrosamine demethylation by reconstituted liver microsomal cytochrome P-450, Biochem. J., 152 (1975) 705-708. P.D. Lotlikar, L. Luha and K. Zaleski, Reconstituted hamster liver microsomal enzyme system for N-hydroxylation of the carcinogen 2-acetylaminofluorene, Biochem. Biophys. Res. Commun., 59 (1974) 1349-1355. P.N. Magee and J.M. Barnes, Carcinogenic nitroso compounds, Adv. Cancer Res., 10 (1967) 163-246. P.N. Magee, A.E. Pegg and P.F. Swann, Molecular mechanisms of chemical carcinogenesis. Handb. Alleg. Pathol., 6 (1975) 329-420. J.A. Miller, Carcinogenesis by chemicals: An overview - G.H.A. Clowes memorial lecture, Cancer Res., 30 (1970) 559-576. T. Omura and R. Sato, The carbon monoxide-binding pigment of liver microsomes. I. Evidence for its hemoprotein nature, J. Biol. Chem., 239 (1964) 2370-2378. W.K. Paik and S. Kim, e-Alkyllysinase: New assay method, purification and biological significance, Arch. Biochem. Biophys., 165 (1974) 369-378. C.J. Parli, N.W. Lee and R.E. McMahon, The relationship between the metabolism of 2,4-dichloro-6-phenylphenoxyethylamine (DPEA) and related compounds and their activities as microsomal mono-oxygenase inhibitors, Drug Metab. Disp., 1 (1973) 628-633. J.C. Phillips, B.G. Lake, SD, Gangolli, P. Grass0 and A.G. Lloyd, Effects of pyrazole and 3-amino-1,2,4-triazole on the metabolism and toxicity of dimethylnitrosamine in the rat, J. Natl. Cancer Inst., 58 (1977) 629-633. D.M. Ziegler and F.H. Pettit, Formation of an intermediate N-oxide in the oxidative demethylation of N,N-dimethylaniline catalyzed by liver microsomes, Biochem. Biophys. Res. Commun., 15 (1964) 188-193. D.M. Ziegler and C.H. Mitchell, Microsomal oxidase IV: Properties of a mixed-function amine oxidase isolated from pig liver microsomes, Arch. Biochem. Biophys., 150 (1972) 116-125.