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DNA damage fey anthracyclines and their copper(II) complexes
1528
major metabolic pathways and detoxication of arsenic trioxide. A sublethal dose of arsenic trioxide may increase methyltransferase activity so that may reduce the lethal toxicity of arsenic trioxide. So, we made clear whether periodate-oxidized adenosin (PAD), an inhibitor of ~ethyltransferase activity (Marafante and Vahter, 1984), reduced As-detoxication effect by a sublethal dose of arsenic trioxide. The injection of PAD (50 pmol/kg i.p.) greatly enhanced the mortality by both lethal doses of arsenic trioxide and partly diminished the detoxication effect by a sublethal dose of arsenic trioxide. Our findings indicate that arsenic trioxide does not induce only methyltransferase but also a metallothionein-likeprotein which is involved in As- or Zn-detoxication. Furthermore, the inductive synthesis of a As-metallothionein-like or Z n - ~ was investigated by measuring the amount of the protein-binding As and of the incorporated teine (~S.cys) in the fractionated protein in the liver of mice given a sublethal dose of arsenic trioxide or zinc sulfate + / - a 50~ lethal dose of arsenic trioxide. 3 ~ z y s (6.29 MBq/kg) was intravenously injected into mice 2 hours before excision of the liver. The fiver was homogenized in 4 vol. of ice cold 0.25 M-sucrose in 10 mM-Tris/acetate (pH 8.2) with a glass/Teflon homogenizer. Cytosol fractions were obtained by centrifuging the liver homogenate at 105000 x g for 60 rain at 4°C and 1 or 3 ml of which was applied to a Sephadex G-75 column (60 cm × 2.5 cm) equilibr~t~-~_ ~ g h 10 mM-Tris/acetate (pH 8.2) containing 0.02% of sodium azide. Fractions were used for measuring the amount of As and of 3"~cys. As-cotaining peak was detected in the high molecule (approx. 60000) region and in the low molecule (below 10000) region which were labeled with 3~S-cys. The total amount of As binding with the low mol~metallothionein-like protein or with the 3h~gh molecule protein induced by arsenic trioxide was 80~ less or 60~ more, respectively, than those by zinc sulfate. 5S-cys incorporation in the low molecu~ protein by arsenic trioxide increased 120~ compared with that of untreated mice, and kept at 84% of the level by zinc sulfate. Condnsion: Arsenic trioxide has inductive activity of metallothionein and of a hiSh molecule, cysteine-rich protein, both which are involved in short term tolerance.
~
Refegwees An~ersaa, R.D., Winter, W.P., Maher, JJ~ and lkrnstein, I.A., 1978, Biochem. J. 174, 327. Marafante, E. and Vaht~, M., 1984, Chen~ Biol. Interact. 50, 49. Nor~.4be~ G.F., Nordber8, M., Piscator, M. and Vesterbegg, O., 1972, Biochem. J. 126, 491.
P.we.423 ]
DNA damage by ~ ~ d b l e s
and their copper(H) complexes
Piccinini, F., Cova *, D., Sassano *, M. and Monfi, E. Institute of Plmmmcology, v. Celoria 2(b 120133 Milano and * CNR Center of Cytopharmacology, Dept. of Pharmacology, v. Vanvitelli 32, 120129 Milano, Italy
The enzymatic generation of carbon-centered and oxygen-derived free radicals has been proposed as the principal mechanism for anthracycline-induced DNA strand scission (Berlin and Haseltine, 1981). Transition metals have been implicated as critical cofactors in these reactions (~L:ot et al., 1984). More recently, a metal-dependent non-enzymatic mechanism of activation has ~ proposed for doxorubicin (DXR) (Wallace, 1986). In the present study, the damage induced by DXR, 4"epidoxorubicin (4'epiDXg) and their Cu(ll) complexes was evaluated on plasmidic DNA (pUC-434). Both DXR and 4'epiDXR were found to produce dose-dependent alterations of the electrophoretic pattern on 0.8~ agarose gel, suggesting formation of single strand nicks in DNA. This effect showed an absolute requirement for a reducing agent and ~as de'ec, ted opJy when 10 Mm sodium borohy&-ide O~aBH4) was added to the reaction mixtures. Therefore, it was concluded that reduction of the anthracyclines to the corresponding semiquinone forms is a prerequisite for inter~tion with DNA. Cu(II)-anthracycline complexes formed at a drug:metal ratio r ffi 2 produced a more severe derangement of the e]ectrophoretic pattern as compared to equimolar concentrations of the corresponding free drugs. On the other hand, when Cu(ll) ions were tested for their intrinsic ability to induce DNA strand scission on this same experimental model it was observed that, in the presence of NaBH 4, only very high metal ion concentrations were able to produce
1529 electrophoretic alterations. It seems therefore that the increased DNA-damaging activities displayed by the complexes do not depend on a mere copper-carrying action, but ~hat formation of a DNA-Cu(ll)-anthracycline ternary complex is involved in this effect. In this experimental model, Cu(ll)4'epiDXR proved to be more potent than Cu(II)DXR. This fmding might be acco,mted for by the different structural peculiarities of the two Cu(ll)-anthracycline complexes. In fact, at the drug:metal ratio adopted for the present study DXR was shown to form self-aggregating polymeric complexes, whereas 4'epiDXR retains its theoretical structure, with two drug molecules bound to one Cu(ll) ion. On the whole, the pre~ent fmdings support the existence of a non-enzymatic mechanism of activation for the anthracyclines tested, whicl~ seems to be particularly efficient when transition metal ions are complexed to the drugs. Supported by C.N.R., Speoal Project "Oncology", grant n. 88.00811.44. References Berlin, V. and Haselfine, W.A., 1981, J. Biol. Chem. 256, 4"/4"/. Eliot, H., et al., 1984, Biochemistry 23, 928. Wallace, K.B., 1986, Toxicol. Appl. Pharmacol. 86, 69.
P.we.4241 The influence of cerium-induced liver injury on coumarin metabolism in D B A / 2 N and C57BL/6N mice Arvela, P., Kraul *, H. and Pelkonen, O. Department of Pharmacology and Toxicology, University of Oulu, Finland and * Institute of Clinical Pharmacology, Friedrich Schiller University, ,lena, G.D.R.
Cerium (Ce) which belongs to the light lanthmion elements is known to cause severe river injury when administered i.v. to rats and mice (Arvela, 1979). This leads usually to a decreased activity of river microsomal monoxygenases. One of these microsomai oxidases is coumarin 7-hydroxylase (COH) which is genetically regulated by the Coh-locus. Its activity varies among animal species and it is differently regulated from other microsomal 1'450 dependent reactions. It has also been shown that treatments with substances such as pyrazole or cobalt which usually decrease the river metabolic capacity in mice, have an opposite effect on the activity of COH. In the present study we tested the influence of Ce on COH activity in two inbread strains of male mice, DBA/2N (D2) which is known to exhibit high river microsomal COH activity and C57BL/6N (B6) with much lower COH activity. Ce was administered as a saline solution i.v. 0.5, 1.0 and 2.0 mg/kg and the animals were killed 24 h and 72 h after the injection. Livers were removed and microsomes prepared in Tris-HC! buffer of pH 7.4. The activities of COH, 7-etboxyresorufin O-deethylase (ERDE) and 7-ethoxy-coumarin O-deethylase (ECDE) as well as the content of cytochrome P450 (P450) were measured. The B6 mice were more resistant to the hepatotoxic effect of Ce. A clear increase in all three enzymes studied was seen with the highest dose and longest exposure of Ce. In D2 mice this inducing effect was only seen with the two lower doses of Ce but with the dose of 2 mg/kg and 72 h exposmc a clear inhibitory effect was seen. Macroscopically these livers exhibited typical Ce-induced fatty changes. It is known that the light ianthanons including Ce needs about 72 h to reach its maximal toxic effects. Our preliminary studies using radioactive Ce have shown that the accumulation of Ce into the liver, kidney and spleen was less in the B6 strain mice but if this distribution difference is the only factor causing different effect on coumarin metabolism in these two mouse s t r ~ s needs further studies. Reference Arvela, P., ~979, Progress in Pharmacology, G. Fischer Verlag, pp. 69-114.
major metabolic pathways and detoxication of arsenic trioxide. A sublethal dose of arsenic trioxide may increase methyltransferase activity so that may reduce the lethal toxicity of arsenic trioxide. So, we made clear whether periodate-oxidized adenosin (PAD), an inhibitor of ~ethyltransferase activity (Marafante and Vahter, 1984), reduced As-detoxication effect by a sublethal dose of arsenic trioxide. The injection of PAD (50 pmol/kg i.p.) greatly enhanced the mortality by both lethal doses of arsenic trioxide and partly diminished the detoxication effect by a sublethal dose of arsenic trioxide. Our findings indicate that arsenic trioxide does not induce only methyltransferase but also a metallothionein-likeprotein which is involved in As- or Zn-detoxication. Furthermore, the inductive synthesis of a As-metallothionein-like or Z n - ~ was investigated by measuring the amount of the protein-binding As and of the incorporated teine (~S.cys) in the fractionated protein in the liver of mice given a sublethal dose of arsenic trioxide or zinc sulfate + / - a 50~ lethal dose of arsenic trioxide. 3 ~ z y s (6.29 MBq/kg) was intravenously injected into mice 2 hours before excision of the liver. The fiver was homogenized in 4 vol. of ice cold 0.25 M-sucrose in 10 mM-Tris/acetate (pH 8.2) with a glass/Teflon homogenizer. Cytosol fractions were obtained by centrifuging the liver homogenate at 105000 x g for 60 rain at 4°C and 1 or 3 ml of which was applied to a Sephadex G-75 column (60 cm × 2.5 cm) equilibr~t~-~_ ~ g h 10 mM-Tris/acetate (pH 8.2) containing 0.02% of sodium azide. Fractions were used for measuring the amount of As and of 3"~cys. As-cotaining peak was detected in the high molecule (approx. 60000) region and in the low molecule (below 10000) region which were labeled with 3~S-cys. The total amount of As binding with the low mol~metallothionein-like protein or with the 3h~gh molecule protein induced by arsenic trioxide was 80~ less or 60~ more, respectively, than those by zinc sulfate. 5S-cys incorporation in the low molecu~ protein by arsenic trioxide increased 120~ compared with that of untreated mice, and kept at 84% of the level by zinc sulfate. Condnsion: Arsenic trioxide has inductive activity of metallothionein and of a hiSh molecule, cysteine-rich protein, both which are involved in short term tolerance.
~
Refegwees An~ersaa, R.D., Winter, W.P., Maher, JJ~ and lkrnstein, I.A., 1978, Biochem. J. 174, 327. Marafante, E. and Vaht~, M., 1984, Chen~ Biol. Interact. 50, 49. Nor~.4be~ G.F., Nordber8, M., Piscator, M. and Vesterbegg, O., 1972, Biochem. J. 126, 491.
P.we.423 ]
DNA damage by ~ ~ d b l e s
and their copper(H) complexes
Piccinini, F., Cova *, D., Sassano *, M. and Monfi, E. Institute of Plmmmcology, v. Celoria 2(b 120133 Milano and * CNR Center of Cytopharmacology, Dept. of Pharmacology, v. Vanvitelli 32, 120129 Milano, Italy
The enzymatic generation of carbon-centered and oxygen-derived free radicals has been proposed as the principal mechanism for anthracycline-induced DNA strand scission (Berlin and Haseltine, 1981). Transition metals have been implicated as critical cofactors in these reactions (~L:ot et al., 1984). More recently, a metal-dependent non-enzymatic mechanism of activation has ~ proposed for doxorubicin (DXR) (Wallace, 1986). In the present study, the damage induced by DXR, 4"epidoxorubicin (4'epiDXg) and their Cu(ll) complexes was evaluated on plasmidic DNA (pUC-434). Both DXR and 4'epiDXR were found to produce dose-dependent alterations of the electrophoretic pattern on 0.8~ agarose gel, suggesting formation of single strand nicks in DNA. This effect showed an absolute requirement for a reducing agent and ~as de'ec, ted opJy when 10 Mm sodium borohy&-ide O~aBH4) was added to the reaction mixtures. Therefore, it was concluded that reduction of the anthracyclines to the corresponding semiquinone forms is a prerequisite for inter~tion with DNA. Cu(II)-anthracycline complexes formed at a drug:metal ratio r ffi 2 produced a more severe derangement of the e]ectrophoretic pattern as compared to equimolar concentrations of the corresponding free drugs. On the other hand, when Cu(ll) ions were tested for their intrinsic ability to induce DNA strand scission on this same experimental model it was observed that, in the presence of NaBH 4, only very high metal ion concentrations were able to produce
1529 electrophoretic alterations. It seems therefore that the increased DNA-damaging activities displayed by the complexes do not depend on a mere copper-carrying action, but ~hat formation of a DNA-Cu(ll)-anthracycline ternary complex is involved in this effect. In this experimental model, Cu(ll)4'epiDXR proved to be more potent than Cu(II)DXR. This fmding might be acco,mted for by the different structural peculiarities of the two Cu(ll)-anthracycline complexes. In fact, at the drug:metal ratio adopted for the present study DXR was shown to form self-aggregating polymeric complexes, whereas 4'epiDXR retains its theoretical structure, with two drug molecules bound to one Cu(ll) ion. On the whole, the pre~ent fmdings support the existence of a non-enzymatic mechanism of activation for the anthracyclines tested, whicl~ seems to be particularly efficient when transition metal ions are complexed to the drugs. Supported by C.N.R., Speoal Project "Oncology", grant n. 88.00811.44. References Berlin, V. and Haselfine, W.A., 1981, J. Biol. Chem. 256, 4"/4"/. Eliot, H., et al., 1984, Biochemistry 23, 928. Wallace, K.B., 1986, Toxicol. Appl. Pharmacol. 86, 69.
P.we.4241 The influence of cerium-induced liver injury on coumarin metabolism in D B A / 2 N and C57BL/6N mice Arvela, P., Kraul *, H. and Pelkonen, O. Department of Pharmacology and Toxicology, University of Oulu, Finland and * Institute of Clinical Pharmacology, Friedrich Schiller University, ,lena, G.D.R.
Cerium (Ce) which belongs to the light lanthmion elements is known to cause severe river injury when administered i.v. to rats and mice (Arvela, 1979). This leads usually to a decreased activity of river microsomal monoxygenases. One of these microsomai oxidases is coumarin 7-hydroxylase (COH) which is genetically regulated by the Coh-locus. Its activity varies among animal species and it is differently regulated from other microsomal 1'450 dependent reactions. It has also been shown that treatments with substances such as pyrazole or cobalt which usually decrease the river metabolic capacity in mice, have an opposite effect on the activity of COH. In the present study we tested the influence of Ce on COH activity in two inbread strains of male mice, DBA/2N (D2) which is known to exhibit high river microsomal COH activity and C57BL/6N (B6) with much lower COH activity. Ce was administered as a saline solution i.v. 0.5, 1.0 and 2.0 mg/kg and the animals were killed 24 h and 72 h after the injection. Livers were removed and microsomes prepared in Tris-HC! buffer of pH 7.4. The activities of COH, 7-etboxyresorufin O-deethylase (ERDE) and 7-ethoxy-coumarin O-deethylase (ECDE) as well as the content of cytochrome P450 (P450) were measured. The B6 mice were more resistant to the hepatotoxic effect of Ce. A clear increase in all three enzymes studied was seen with the highest dose and longest exposure of Ce. In D2 mice this inducing effect was only seen with the two lower doses of Ce but with the dose of 2 mg/kg and 72 h exposmc a clear inhibitory effect was seen. Macroscopically these livers exhibited typical Ce-induced fatty changes. It is known that the light ianthanons including Ce needs about 72 h to reach its maximal toxic effects. Our preliminary studies using radioactive Ce have shown that the accumulation of Ce into the liver, kidney and spleen was less in the B6 strain mice but if this distribution difference is the only factor causing different effect on coumarin metabolism in these two mouse s t r ~ s needs further studies. Reference Arvela, P., ~979, Progress in Pharmacology, G. Fischer Verlag, pp. 69-114.