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Intact rat liver nuclei catalyze adriamycin irreversible interactions with dna and nuclear proteins
Toxicology Letters, 17 (1983) 343-348 Elsevier
343
INTACT RAT LIVER NUCLEI CATALYZE ADRIAMYCIN INTERACTIONS WITH DNA AND NUCLEAR PROTEINS (Adriamycin;
IRREVERSIBLE
covalent binding; DNA; proteins; rat liver nuclei)
ENRICO GARATTINI, PANTAROTTO*
MARIA
GRAZIA
DONELLI,
PAOLO
CATALAN1
and CLAUDIO
Istituto di Ricerche Farmacologiche ‘Mario Negri’, Via Eritrea 62, 20157 Milan (Italy) (Received February 4th, 1983) (Accepted February 17th, 1983)
SUMMARY Male rat liver intact nuclear preparations are able to metabolize adriamycin to reactive species that irreversibly interact with nuclear DNA and proteins in the presence of reduced NADPH. This process was not inhibited by 1 mM SKF-525A, suggesting that a nuclear monooxygenase enzymatic system was not involved.
INTRODUCTION
Adriamycin is an antineoplastic agent especially active in some animal and human malignancies [ 1,2]. This drug has been well studied in our Institute as regards its distribution [3] and pharmacodynamics [4-61. Its mechanism of action is not yet clear although some of its pharmacological properties are known, particularly the capacity to inter&ate within the nucleotide bases of double-stranded DNA [7]. Adriamycin’s cytotoxic activity has been interpreted in terms of this property. However, based especially on in vitro evidence, other hypotheses have now been proposed to explain the drug’s activity, stemming, for instance, from its stimulatory effect on DNase I [8] or inhibitory effect on DNA polymerase [7,9,10] and its capacity to produce toxic radicals such as semiquinones which can effect DNA breakages and increase the frequency of sister chromatid exchange [ 11,121. Recently the possibility that microsomal fractions from rat liver may produce electrophilic metabolites from adriamycin, which could then interact with nucleophilic sites of protein and isolated calf thymus DNA in vitro [13,14] has been proposed as well *To whom enquiries should be addressed. 0378-4274/83/0000-OOOO/$ 03.00 0 Elsevier Science Publishers
344
as the existence of a covalent interaction between adriamycin and DNA in vivo [ 151. Since adriamycin is known to accumulate in the nucleus of isolated cellular systems in vitro [ 16,171, the drug may be available in high local concentrations for nuclear enzymes to act on. We therefore decided to evaluate the capacity of intact nuclear preparations to produce adriamycin metabolites reacting with nuclear DNA and proteins. In this experimental system nuclei therefore act as both activating system and target. MATERIALS AND METHODS
Chemicals [14-14C] Adriamycin
(spec. act. 28.2 mCi/mmol) was obtained from FarmitaliaCarlo Erba, Nerviano, Milan (Italy). The radio-chemical purity of labelled adriamycin was checked by high-pressure liquid chromatography by the method of Israel et al. [18]. The compound was found to be > 99% pure. The following reagents were also used: reduced NADPH (Boehringer, Mannheim, FRG), /3diethylaminoethyl diphenyl propyl acetate, SKF-525A (Smith Klein and French Laboratories, Philadelphia, PA, USA). All other chemicals and solvents were of the purest grade commercially available. Preparation
of nuclei
Male CD-COBS rats (200-220 g) from Charles River Italy (Calco, Como, Italy) were used for the separation of liver nuclei according to the method of Bresnick et al. [19]. These nuclear preparations were free of any significant microsomal contamination [20]. Incubation
Nuclear proteins (4 mg) were suspended in 0.05 M sodium phosphate 7.4 + 0.15 M KC1 incubated at 37°C with 2.5 PM adriamycin in the NADPH (2.4 mM) for 2 h in subdued light. The reaction was stopped and centrifuging at 4°C in a J-21B refrigerated Beckman centrifuge at for 10 min. The radioactive supernatant was aspirated and discarded.
buffer pH presence of by chilling 35 000 x g
Separation of nuclear DNA and proteins
DNA and proteins were separated by a modification of the Marmur method [21]. The nuclear pellet was resuspended in 4.5 ml of an aqueous solution containing 1.2% SDS, 0.15 M NaCl, 0.1 M EDTA. Lysis was performed overnight in the dark. A 300 ~1 portion of 5 M NaC104 was added, followed by 5 ml of chloroform-isoamyl alcohol (24:l,v/v). The mixture was shaken for 10 min, then centrifuged at 30000 x g for another 10 min. The aqueous phase containing DNA was separated and the interface protein fraction was isolated and processed for the determination of bound radioactivity. The DNA phase was washed a second time
345
with the chloroform-isoamyl alcohol mixture, the nucleic acid was precipitated with 2 ~01s. of ethanol and removed with a glass rod. The protein fraction was practically free of any nucleic acid contamination 1221 and the DNA contained less than 1% proteins as determined by applying the Lowry reaction [23] to the isolated nucleic acid. RNA contamination of DNA was not taken into consideration because treatment of DNA samples with rubonuclease did not alter our results, indicating very little contamination. Determination of radioactivity bound to DNA and proteins Protein-bound radioactivity was determined as described for microsomal proteins [ 131. DNA bound radioactivity was determined after 5 washings in 5 ml of ethanol and 5 washings in 5 ml of methanol. The DNA was then dissolved in 600 ~1 of 5 mM NaOH. 20 ~1 were used for the determination of DNA [22]. To the remaining DNA solution 50 ~1 of concentrated sulphuric acid were added. The mixture was heated at 60°C until complete dissolution of DNA and then transferred to a vial containing 10 ml of Lumagel (Lumac Systems AC, Basel, Switzerland). Radioactivity was measured in a Nuclear Chicago Isocap 300 liquid scintillation counter. The values were corrected for quenching by the external standardization method. RESULTS AND DISCUSSION
Table I shows the capacity of adriamycin to interact with nuclear DNA and proteins in the presence or absence of NADPH and of SKF-525A, a known inhibitor of microsomal and nuclear P-450-dependent monooxygenase enzymatic activity 119,241. The radioactivity bound to DNA and proteins was significantly increased when NADPH was added to the incubation mixture. The effect was much more evident for DNA than for nuclear proteins, with an approximate ratio of 5:l. TABLE I IRREVERSIBLE INTERACTION OF [i4C-]ADRIAMYCINa AND NUCLEAR PROTEINS AND DNA IN PRESENCE OF SKF-525A Experimental conditions
SKF-525A’
- NADPH + NADPH - NADPH + NADPH
WITH
Irreversible interaction of adriamycin and metabolites with 1 mg of DNA or proteins (pmol f S.EJb Proteins
Control
METABOLITES
22.7 36.1 25.9 37.0
+ * zt zt
DNA 0.7 1.2, 1.6 1.8’
17.7 90.1 18.3 81.7
+ f k k
1.0 5.9* 1.9 2.8*
a[‘4C]adriamycin was added to the incubation mixture at a final concentration of 2.5 pM. bEach value is the mean f S.E. of four determinations. ’ SKF-525A was added to the incubation mixture at a final concentration of 1 mM. * P < 0.01 compared to nuclei-NADPH.
346
This phenomenon is the expression of a real nuclear NADPH-dependent enzymatic activity and not of a simple chemical interaction between NADPH and the anthracycline drug resulting in the formation of reactive molecular species. As regards the adriamycin-DNA interaction, when nucleic acid from calf thymus alone was incubated with radiolabelled adriamycin in the presence or absence of NADPH no such effect was observed, the radioactivity determined in both these conditions not differing from the background levels of the vials. The same can be said for the adriamycin-protein interaction on the basis of inference from similarly designed experiments with solvent-denaturated microsomal preparations [ 131. Table I also shows that SKF-525A did not quantitatively change the pattern of adriamycin interaction with DNA and proteins. The effect of SKF-525A on the nuclear metabolism of adriamycin was tested since such evidence would help in the elucidation of the molecular species of the anthracycline drug involved in the interaction with DNA and protein. To verify the validity of the method used for the isolation and purification of DNA, in a second set of experiments, identical to that described above, the nucleic acid was extracted and processed through a hydroxylapatite column as described by Viviani and Lutz [25]. The results of this procedure were superimposable on those reported in Table I and therefore further demonstrated the high degree of purity of the DNA isolated by the Marmur method [21]. This study shows that in rat liver not only microsomal fractions [ 131 but also intact nuclear preparations can metabolize adriamycin to reactive species capable of irreversibly interacting with cellular macromolecules such as DNA and proteins. No definite hypothesis as to the nature of this interaction can be advanced. Our data nevertheless seem to help clarify how adriamycin exerts its cytotoxic activity in that they suggest a further mechanism of action, i.e. irreversible interaction of the anthracycline metabolites with nuclear DNA and proteins after membrane enzymatic activation. As regards the enzymatic system determining this interaction, our data suggest that a cytochrome P-450-dependent monooxygenase is not involved and might indeed support the idea already suggested [26] that cytochrome P-450 reductase activity, producing highly reactive radicals, is important, though further study is needed to clarify this. ACKNOWLEDGEMENTS
The authors wish to thank Prof. F. Arcamone and Dr. G. Vicario from Farmitalia-Carlo Erba, Milan, Italy, for the kind gift of [14C-]adriamycin. This work was supported by the Italian National Council for Research, Rome, Project: ‘Control of Neoplastic Growth’, Contract No. 80.01539.96.
347
REFERENCES 1 S.K. Carter, A. Di Marco, M. Ghione, I.H. Krakoff and G. Math6 (Eds.), International Symposium on Adriamycin, Springer-Verlag, Berlin, 1972. 2 A. Di Marco, F. Arcamone and F. Zunino, Daunomycin (daunorubicin), adriamycin and structural analogues: Biological activity and mechanism of action, in J.W. Corcoran and F.E. Hahn (Eds.), Antibiotics. Mechanism of Action of Antimicrobial and Antitumor Agents, Vol. 3, Springer-Verlag, Berlin, 1975 pp. 101-128. 3 M.A. Pacciarini, B. Barbieri, T. Colombo, M. Broggini, S. Garattini and M.G. Donelli, Distribution and antitumor activity of adriamycin given by a high-dose and a repeated low-dose schedule to mice, Cancer Treat. Rep., 62 (1978) 791-800. 4 A. Vecchi, A. Mantovani, A. Tagliabue and F. Spreafico, A characterization of the immunosuppressive activity of adriamycin and daunomycin on humoral antibody production and tumor allograft rejection, Cancer Res., 36 (1976) 1222-1227. 5 A. Mantovani, A. Vecchi, A. Tagliabue and F. Spreafico, The effects of adriamycin and daunomycin on antitumoral immune effector mechanisms in an allogeneic system, Eur. J. Cancer, 12 (1976) 371-379. 6 A. Poggi, L. Kornblihtt, F. Delaini, T. Colombo, L. Mussoni, I. Reyers and M.B. Donati, Delayed hypercoagulability after a single dose of adriamycin to normal rats, Thromb. Res., 16 (1979) 639-650. 7 H.S. Schwartz, Mechanisms and selectitivity of anthracycline aminoglycosides and other intercalating agents, Biomedicine, 24 (1976) 317-323. 8 T. Facchinetti, A. Mantovani, R. Cantoni, L. Cantoni, C. Pantarotto and M. Salmona, Effect of daunomycin, adriamycin and its congener AD32 on the activity of DNase I from bovine pancreas, Biochem. Pharmacol., 26 (1977) 1953-1954. 9 W.J. Pigram, W. Fuller and L.D. Hamilton, Stereochemistry of intercalation: Interaction of daunomycin with DNA, Nature New Biol., 235 (1972) 17-19. 10 K. Yamamoto, E.M. Acton and D.W. Henry, Antitumor activity of some derivatives of daunorubicin at the amino and methyl ketone functions, J. Med. Chem., 15 (1972) 872-875. 11 J.W. Lown, S.K. Sim, K.C. Majumdar and R.Y. Chang, Strand scission of DNA by bound adriamycin and daunorubicin in the presence of reducing agents, Biochem. Biophys. Res. Commun., 76 (1977) 705-719. 12 Y. Nakanishi and E.L. Schneider, In vivo sister-chromatic exchange: A sensitive measure of DNA damage, Mutation Res., 60 (1979) 329-337. 13 P. Ghezzi, M.G. Donelli, C. Pantarotto, T. Facchinetti and S. Garattini, Evidence for covalent binding of adriamycin to rat liver microsomal proteins, Biochem. Pharmacol., 30 (1981) 175-177. 14 B.K. Sinha and J.L. Gregory, Role of one-electron and two-electron reduction products of adriamycin and daunomycin in deoxyribonucleic acid binding, Biochem. Pharmacol., 30 (1981) 2626-2629. 15 B.K. Sinha and R.H. Sik, Binding of [‘%]adriamycin to cellular macromolecules in vivo, Biochem. Pharmacol., 29 (1980) 1867-1868. 16 M.J. Egorin, R.C. Hildebrand, E.F. Cimino and N.R. Bachur, Cytofluorescence localization of adriamycin and daunorubicin, Cancer Res., 34 (1974) 2243-2245. 17 A. Krishan, R.N. Ganapathi and M. Israel, Effect of adriamycin and analogs on the nuclear fluorescence of propidium iodide-stained cells, Cancer Res., 38 (1978) 3656-3662. 18 M. Israel, W.J. Pegg, P.M. Wilkinson and M.B. Garnick, Liquid chromatographic analysis of adriamycin and metabolites in biological fluids, J. Liquid Chromatogr., 1 (1978) 795-809. 19 E. Bresnick, J.B. Vaught, A.H.L. Chuang, T.A. Stoming, D. Bockman and H. Mukhtar, Nuclear aryl hydrocarbon hydroxylase and interaction of polycyclic hydrocarbons with nuclear components, Arch. Biochem. Biophys., 181 (1977) 257-269.
348 20. E. Garattini, G. Gazzotti and M. Salmona, Is nuclear styrene monooxygenase activity a microsomal artifact? Chem.-Biol. Interact., 31 (1980) 341-346. 21 J. Marmur, A procedure for the isolation of deoxyribonucleic acid from micro-organisms, J. Mol. Biol., 3 (1961) 208-218. 22 K. Burton, The conditions and mechanism of the diphenylamine reaction for the calorimetric estimation of deoxyribonucleic acid, Biochem. J., 62 (1956) 315-323. 23 O.H. Lowry, N.J. Rosebrough, A.L. Farr and R.J. Randall, Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. 24 G. Gazzotti, E. Garattini and M. Salmona, Nuclear metabolism, I. Determination of styrene monooxygenase activity in rat liver nuclei, Chem.-Biol. Interact, 29 (1980) 189-195. 25 A. Viviani and W.K. Lutz, Modulation of the binding of the carcinogen benzo(a)pyrene to rat liver DNA in vivo by selective induction of microsomal and nuclear aryl hydrocarbon hydroxylase activity, Cancer Res., 38 (1978) 4640-4644. 26 N.R. Bachur, S.L. Gordon and M.V. Gee, A general mechanism for microsomal activation of quinone anticancer agents to free radicals, Cancer Res., 38 (1978) 1745-1750.
343
INTACT RAT LIVER NUCLEI CATALYZE ADRIAMYCIN INTERACTIONS WITH DNA AND NUCLEAR PROTEINS (Adriamycin;
IRREVERSIBLE
covalent binding; DNA; proteins; rat liver nuclei)
ENRICO GARATTINI, PANTAROTTO*
MARIA
GRAZIA
DONELLI,
PAOLO
CATALAN1
and CLAUDIO
Istituto di Ricerche Farmacologiche ‘Mario Negri’, Via Eritrea 62, 20157 Milan (Italy) (Received February 4th, 1983) (Accepted February 17th, 1983)
SUMMARY Male rat liver intact nuclear preparations are able to metabolize adriamycin to reactive species that irreversibly interact with nuclear DNA and proteins in the presence of reduced NADPH. This process was not inhibited by 1 mM SKF-525A, suggesting that a nuclear monooxygenase enzymatic system was not involved.
INTRODUCTION
Adriamycin is an antineoplastic agent especially active in some animal and human malignancies [ 1,2]. This drug has been well studied in our Institute as regards its distribution [3] and pharmacodynamics [4-61. Its mechanism of action is not yet clear although some of its pharmacological properties are known, particularly the capacity to inter&ate within the nucleotide bases of double-stranded DNA [7]. Adriamycin’s cytotoxic activity has been interpreted in terms of this property. However, based especially on in vitro evidence, other hypotheses have now been proposed to explain the drug’s activity, stemming, for instance, from its stimulatory effect on DNase I [8] or inhibitory effect on DNA polymerase [7,9,10] and its capacity to produce toxic radicals such as semiquinones which can effect DNA breakages and increase the frequency of sister chromatid exchange [ 11,121. Recently the possibility that microsomal fractions from rat liver may produce electrophilic metabolites from adriamycin, which could then interact with nucleophilic sites of protein and isolated calf thymus DNA in vitro [13,14] has been proposed as well *To whom enquiries should be addressed. 0378-4274/83/0000-OOOO/$ 03.00 0 Elsevier Science Publishers
344
as the existence of a covalent interaction between adriamycin and DNA in vivo [ 151. Since adriamycin is known to accumulate in the nucleus of isolated cellular systems in vitro [ 16,171, the drug may be available in high local concentrations for nuclear enzymes to act on. We therefore decided to evaluate the capacity of intact nuclear preparations to produce adriamycin metabolites reacting with nuclear DNA and proteins. In this experimental system nuclei therefore act as both activating system and target. MATERIALS AND METHODS
Chemicals [14-14C] Adriamycin
(spec. act. 28.2 mCi/mmol) was obtained from FarmitaliaCarlo Erba, Nerviano, Milan (Italy). The radio-chemical purity of labelled adriamycin was checked by high-pressure liquid chromatography by the method of Israel et al. [18]. The compound was found to be > 99% pure. The following reagents were also used: reduced NADPH (Boehringer, Mannheim, FRG), /3diethylaminoethyl diphenyl propyl acetate, SKF-525A (Smith Klein and French Laboratories, Philadelphia, PA, USA). All other chemicals and solvents were of the purest grade commercially available. Preparation
of nuclei
Male CD-COBS rats (200-220 g) from Charles River Italy (Calco, Como, Italy) were used for the separation of liver nuclei according to the method of Bresnick et al. [19]. These nuclear preparations were free of any significant microsomal contamination [20]. Incubation
Nuclear proteins (4 mg) were suspended in 0.05 M sodium phosphate 7.4 + 0.15 M KC1 incubated at 37°C with 2.5 PM adriamycin in the NADPH (2.4 mM) for 2 h in subdued light. The reaction was stopped and centrifuging at 4°C in a J-21B refrigerated Beckman centrifuge at for 10 min. The radioactive supernatant was aspirated and discarded.
buffer pH presence of by chilling 35 000 x g
Separation of nuclear DNA and proteins
DNA and proteins were separated by a modification of the Marmur method [21]. The nuclear pellet was resuspended in 4.5 ml of an aqueous solution containing 1.2% SDS, 0.15 M NaCl, 0.1 M EDTA. Lysis was performed overnight in the dark. A 300 ~1 portion of 5 M NaC104 was added, followed by 5 ml of chloroform-isoamyl alcohol (24:l,v/v). The mixture was shaken for 10 min, then centrifuged at 30000 x g for another 10 min. The aqueous phase containing DNA was separated and the interface protein fraction was isolated and processed for the determination of bound radioactivity. The DNA phase was washed a second time
345
with the chloroform-isoamyl alcohol mixture, the nucleic acid was precipitated with 2 ~01s. of ethanol and removed with a glass rod. The protein fraction was practically free of any nucleic acid contamination 1221 and the DNA contained less than 1% proteins as determined by applying the Lowry reaction [23] to the isolated nucleic acid. RNA contamination of DNA was not taken into consideration because treatment of DNA samples with rubonuclease did not alter our results, indicating very little contamination. Determination of radioactivity bound to DNA and proteins Protein-bound radioactivity was determined as described for microsomal proteins [ 131. DNA bound radioactivity was determined after 5 washings in 5 ml of ethanol and 5 washings in 5 ml of methanol. The DNA was then dissolved in 600 ~1 of 5 mM NaOH. 20 ~1 were used for the determination of DNA [22]. To the remaining DNA solution 50 ~1 of concentrated sulphuric acid were added. The mixture was heated at 60°C until complete dissolution of DNA and then transferred to a vial containing 10 ml of Lumagel (Lumac Systems AC, Basel, Switzerland). Radioactivity was measured in a Nuclear Chicago Isocap 300 liquid scintillation counter. The values were corrected for quenching by the external standardization method. RESULTS AND DISCUSSION
Table I shows the capacity of adriamycin to interact with nuclear DNA and proteins in the presence or absence of NADPH and of SKF-525A, a known inhibitor of microsomal and nuclear P-450-dependent monooxygenase enzymatic activity 119,241. The radioactivity bound to DNA and proteins was significantly increased when NADPH was added to the incubation mixture. The effect was much more evident for DNA than for nuclear proteins, with an approximate ratio of 5:l. TABLE I IRREVERSIBLE INTERACTION OF [i4C-]ADRIAMYCINa AND NUCLEAR PROTEINS AND DNA IN PRESENCE OF SKF-525A Experimental conditions
SKF-525A’
- NADPH + NADPH - NADPH + NADPH
WITH
Irreversible interaction of adriamycin and metabolites with 1 mg of DNA or proteins (pmol f S.EJb Proteins
Control
METABOLITES
22.7 36.1 25.9 37.0
+ * zt zt
DNA 0.7 1.2, 1.6 1.8’
17.7 90.1 18.3 81.7
+ f k k
1.0 5.9* 1.9 2.8*
a[‘4C]adriamycin was added to the incubation mixture at a final concentration of 2.5 pM. bEach value is the mean f S.E. of four determinations. ’ SKF-525A was added to the incubation mixture at a final concentration of 1 mM. * P < 0.01 compared to nuclei-NADPH.
346
This phenomenon is the expression of a real nuclear NADPH-dependent enzymatic activity and not of a simple chemical interaction between NADPH and the anthracycline drug resulting in the formation of reactive molecular species. As regards the adriamycin-DNA interaction, when nucleic acid from calf thymus alone was incubated with radiolabelled adriamycin in the presence or absence of NADPH no such effect was observed, the radioactivity determined in both these conditions not differing from the background levels of the vials. The same can be said for the adriamycin-protein interaction on the basis of inference from similarly designed experiments with solvent-denaturated microsomal preparations [ 131. Table I also shows that SKF-525A did not quantitatively change the pattern of adriamycin interaction with DNA and proteins. The effect of SKF-525A on the nuclear metabolism of adriamycin was tested since such evidence would help in the elucidation of the molecular species of the anthracycline drug involved in the interaction with DNA and protein. To verify the validity of the method used for the isolation and purification of DNA, in a second set of experiments, identical to that described above, the nucleic acid was extracted and processed through a hydroxylapatite column as described by Viviani and Lutz [25]. The results of this procedure were superimposable on those reported in Table I and therefore further demonstrated the high degree of purity of the DNA isolated by the Marmur method [21]. This study shows that in rat liver not only microsomal fractions [ 131 but also intact nuclear preparations can metabolize adriamycin to reactive species capable of irreversibly interacting with cellular macromolecules such as DNA and proteins. No definite hypothesis as to the nature of this interaction can be advanced. Our data nevertheless seem to help clarify how adriamycin exerts its cytotoxic activity in that they suggest a further mechanism of action, i.e. irreversible interaction of the anthracycline metabolites with nuclear DNA and proteins after membrane enzymatic activation. As regards the enzymatic system determining this interaction, our data suggest that a cytochrome P-450-dependent monooxygenase is not involved and might indeed support the idea already suggested [26] that cytochrome P-450 reductase activity, producing highly reactive radicals, is important, though further study is needed to clarify this. ACKNOWLEDGEMENTS
The authors wish to thank Prof. F. Arcamone and Dr. G. Vicario from Farmitalia-Carlo Erba, Milan, Italy, for the kind gift of [14C-]adriamycin. This work was supported by the Italian National Council for Research, Rome, Project: ‘Control of Neoplastic Growth’, Contract No. 80.01539.96.
347
REFERENCES 1 S.K. Carter, A. Di Marco, M. Ghione, I.H. Krakoff and G. Math6 (Eds.), International Symposium on Adriamycin, Springer-Verlag, Berlin, 1972. 2 A. Di Marco, F. Arcamone and F. Zunino, Daunomycin (daunorubicin), adriamycin and structural analogues: Biological activity and mechanism of action, in J.W. Corcoran and F.E. Hahn (Eds.), Antibiotics. Mechanism of Action of Antimicrobial and Antitumor Agents, Vol. 3, Springer-Verlag, Berlin, 1975 pp. 101-128. 3 M.A. Pacciarini, B. Barbieri, T. Colombo, M. Broggini, S. Garattini and M.G. Donelli, Distribution and antitumor activity of adriamycin given by a high-dose and a repeated low-dose schedule to mice, Cancer Treat. Rep., 62 (1978) 791-800. 4 A. Vecchi, A. Mantovani, A. Tagliabue and F. Spreafico, A characterization of the immunosuppressive activity of adriamycin and daunomycin on humoral antibody production and tumor allograft rejection, Cancer Res., 36 (1976) 1222-1227. 5 A. Mantovani, A. Vecchi, A. Tagliabue and F. Spreafico, The effects of adriamycin and daunomycin on antitumoral immune effector mechanisms in an allogeneic system, Eur. J. Cancer, 12 (1976) 371-379. 6 A. Poggi, L. Kornblihtt, F. Delaini, T. Colombo, L. Mussoni, I. Reyers and M.B. Donati, Delayed hypercoagulability after a single dose of adriamycin to normal rats, Thromb. Res., 16 (1979) 639-650. 7 H.S. Schwartz, Mechanisms and selectitivity of anthracycline aminoglycosides and other intercalating agents, Biomedicine, 24 (1976) 317-323. 8 T. Facchinetti, A. Mantovani, R. Cantoni, L. Cantoni, C. Pantarotto and M. Salmona, Effect of daunomycin, adriamycin and its congener AD32 on the activity of DNase I from bovine pancreas, Biochem. Pharmacol., 26 (1977) 1953-1954. 9 W.J. Pigram, W. Fuller and L.D. Hamilton, Stereochemistry of intercalation: Interaction of daunomycin with DNA, Nature New Biol., 235 (1972) 17-19. 10 K. Yamamoto, E.M. Acton and D.W. Henry, Antitumor activity of some derivatives of daunorubicin at the amino and methyl ketone functions, J. Med. Chem., 15 (1972) 872-875. 11 J.W. Lown, S.K. Sim, K.C. Majumdar and R.Y. Chang, Strand scission of DNA by bound adriamycin and daunorubicin in the presence of reducing agents, Biochem. Biophys. Res. Commun., 76 (1977) 705-719. 12 Y. Nakanishi and E.L. Schneider, In vivo sister-chromatic exchange: A sensitive measure of DNA damage, Mutation Res., 60 (1979) 329-337. 13 P. Ghezzi, M.G. Donelli, C. Pantarotto, T. Facchinetti and S. Garattini, Evidence for covalent binding of adriamycin to rat liver microsomal proteins, Biochem. Pharmacol., 30 (1981) 175-177. 14 B.K. Sinha and J.L. Gregory, Role of one-electron and two-electron reduction products of adriamycin and daunomycin in deoxyribonucleic acid binding, Biochem. Pharmacol., 30 (1981) 2626-2629. 15 B.K. Sinha and R.H. Sik, Binding of [‘%]adriamycin to cellular macromolecules in vivo, Biochem. Pharmacol., 29 (1980) 1867-1868. 16 M.J. Egorin, R.C. Hildebrand, E.F. Cimino and N.R. Bachur, Cytofluorescence localization of adriamycin and daunorubicin, Cancer Res., 34 (1974) 2243-2245. 17 A. Krishan, R.N. Ganapathi and M. Israel, Effect of adriamycin and analogs on the nuclear fluorescence of propidium iodide-stained cells, Cancer Res., 38 (1978) 3656-3662. 18 M. Israel, W.J. Pegg, P.M. Wilkinson and M.B. Garnick, Liquid chromatographic analysis of adriamycin and metabolites in biological fluids, J. Liquid Chromatogr., 1 (1978) 795-809. 19 E. Bresnick, J.B. Vaught, A.H.L. Chuang, T.A. Stoming, D. Bockman and H. Mukhtar, Nuclear aryl hydrocarbon hydroxylase and interaction of polycyclic hydrocarbons with nuclear components, Arch. Biochem. Biophys., 181 (1977) 257-269.
348 20. E. Garattini, G. Gazzotti and M. Salmona, Is nuclear styrene monooxygenase activity a microsomal artifact? Chem.-Biol. Interact., 31 (1980) 341-346. 21 J. Marmur, A procedure for the isolation of deoxyribonucleic acid from micro-organisms, J. Mol. Biol., 3 (1961) 208-218. 22 K. Burton, The conditions and mechanism of the diphenylamine reaction for the calorimetric estimation of deoxyribonucleic acid, Biochem. J., 62 (1956) 315-323. 23 O.H. Lowry, N.J. Rosebrough, A.L. Farr and R.J. Randall, Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. 24 G. Gazzotti, E. Garattini and M. Salmona, Nuclear metabolism, I. Determination of styrene monooxygenase activity in rat liver nuclei, Chem.-Biol. Interact, 29 (1980) 189-195. 25 A. Viviani and W.K. Lutz, Modulation of the binding of the carcinogen benzo(a)pyrene to rat liver DNA in vivo by selective induction of microsomal and nuclear aryl hydrocarbon hydroxylase activity, Cancer Res., 38 (1978) 4640-4644. 26 N.R. Bachur, S.L. Gordon and M.V. Gee, A general mechanism for microsomal activation of quinone anticancer agents to free radicals, Cancer Res., 38 (1978) 1745-1750.