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Transcriptional template activity of covalently modified DNA
Mutation Research 374 Ž1997. 139–143
Transcriptional template activity of covalently modified DNA Zofia Tołwinska-Stanczyk, Dorota Wilmanska, Kazimierz Studzian, ´ ´ ´ Marek Gniazdowski ),1 Department of General Chemistry, Medical UniÕersity of Łodz, ´ ´ Lindleya 6, 90-131 Łodz, ´ ´ Poland Received 2 April 1996; revised 9 July 1996; accepted 6 August 1996
Abstract The transcriptional template activity of covalent modified DNA is compared. 8-Methoxypsoralen ŽMOP., 3,4X dimethyl8-methoxypsoralen ŽDMMOP. and benzopsoralen ŽBP. forming with DNA covalent complexes upon UV irradiation and exhibiting preference to pyrimidines, mostly thymines, differ in their cross-linking potency. MOP and DMMOP form both monoadducts and diadducts while no cross-links are formed by BP. Nitracrine ŽNC. forms covalent complexes with DNA upon reductive activation with dithiothreitol exhibiting a preference to purines and low cross-linking potency. Semilogarithmic plots of the relative template activity against the number of the drugs molecules covalently bound per 10 3 DNA nucleotides fit to regression lines corresponding to one-hit inactivation characteristics. The number of drug molecules decreasing RNA synthesis to 37% differ from 0.25 to 1.26 depending on the template used and the base preference but no dependence on the cross-linking potency was found. Keywords: DNA; RNA polymerase; Furocoumarins; Psoralens; 8-Methoxypsoralen; Nitracrine; Adducts
1. Introduction Most of carcinogens and some cytotoxic drug exert their biological effects through covalent attachment to DNA. One of the properties of DNA, impaired by covalent modification, is transcriptional Abbreviations: CT, calf thymus; T7, phage T7; HA, hydroxylX apatite; MOP, 8-methoxy-psoralen; DMMOP, 3,4 -dimethyl-8methoxypsoralen; BP, benzopsoralen; NC, nitracrine, 1-nitro-9Ž3,3X-dimethylaminopropylamino.acridine, also known under its code C-283 and its trade name, ‘Ledakrin’ ) Corresponding author. Tel.: Ž42. 784277. Fax: Ž42. 322347. 1 This work was supported by the Medical University of Łodz ´ ´ within the project No. 502-11-81 and Foundation for Polish Science within the project BIMOL.
template activity. The decrease of the amount of RNA synthesized by DNA-dependent RNA polymerase on a modified template is measured by quantitating the incorporation of radioactive ribonucleotides into the acid-insoluble fraction. Covalent binding of furocoumarins poisons the transcriptional template activity depending on the binding level w1–3x. It was found in our earlier experiments with NC-CT DNA complexes and T7 DNA modified with two furocoumarin derivatives that the logarithmic plot of the percentage of RNA synthesis versus the number of covalent adducts on the template is a straight line w4–6x. This observation may be potentially of analytical importance, provided that we know to what extent the inhibition of RNA synthesis in vitro de-
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Z. Tołwinska-Stanczyk et al.r Mutation Research 374 (1997) 139–143 ´ ´
pends on the template used, the base specificity of the adduct formation and the adduct structure. Particularly a comparison between the monofunctionally binding drugs and the drugs forming interstrand cross-links seems of interest in this respect. The aim of these experiments is to evaluate the dependence of the inhibition on these parameters. Four drugs covalently binding to DNA are compared. Three psoralens form covalent complexes upon irradiation with near UV light w7,8x. All of them react with pyrimidines, mostly thymines, but they differ in their cross-linking potency. 8-Methoxypsoralen ŽMOP. w7x and 3,4-dimethyl-8-methoxypsoralen ŽDMMOP. w8x form with DNA both monoadducts and diadducts while almost no cross-links are formed by benzopsoralen ŽBP. w8x. Nitracrine ŽNC. forms covalent complexes with DNA upon reductive activation with sulfhydryl compounds w4x. In contrast to psoralens NC exhibits preferential binding to purines, particularly to guanines in DNA, its binding does not seem to depend on intercalation w9x and only few cross-links are detected in NC–DNA complexes w10x.
2. Materials and methods 2.1. Materials MOP w9x, NC w4,10x, calf thymus ŽCT. w4x and phage T7 w5x DNA, Escherichia coli DNA-dependent RNA polymerase ŽEC. 2.7.7.6. w5x and most of other materials are as described in the cited papers. DMMOP w7x and BP w8x were kindly donated by Dr O. Gia and Professor M. Palumbo ŽUniversity of Padova.. 2.2. Methods Covalent complexes of 3 H-labeled furocoumarins with either CT or T7 DNA were formed by irradiation at 365 nm, then purified and their stoichiometry was estimated as previously described w5,10x. NC– DNA complexes were formed by incubation of DNA with the 14 C-labeled drug in the presence of dithiothreitol Ž5 mM., then purified and subjected to stoichiometry estimation w4,5,10x. Transcriptional template activity of drug–DNA complexes estimated with E. coli DNA-dependent RNA polymerase was
expressed as percentages of the corresponding controls w4,5x. The fraction of cross-linked DNA was estimated by fluorescence assay according to Splengler and Singer w11x or by means of hydroxylapatite wHAx chromatography of heat-denaturated DNA w10x. It is expressed as percentage of the increase in renaturated DNA fraction in the drug–DNA complex relative to the corresponding heat-denaturated control DNA, i.e. DNA either irradiated or preincubated with dithiothreitol in the absence of a drug.
3. Results and discussion Stoichiometry of the complexes depends on drug concentration during the complex formation and it is similar for both the templates used Žsee the insets to Fig. 1.. The relative template activity of the complexes, plotted as a function of the number of the drug molecules covalently bound per 10 3 DNA nucleotides ŽN., is shown on Fig. 1. The semilogarithmic plots fit to the regression lines ŽFig. 1. described by the general equation:log 10 wrelative template activity Ž%.x s AN q B,with correlation coefficients 0.85–0.97 ŽTable 1.. This relationship corresponds to one-hit inactivation characteristics w12,13x. The number of adduct molecules per 10 3 DNA nucleotides which reduce the relative template activity to 37% ŽN37 . was obtained from the inhibition curves ŽTable 1.. As otherwise found these psoralen derivatives differ in their cross-linking potencies. MOP forms with DNA both monoadducts and diadducts. The latter induce renaturation following thermal denaturation and cooling which can be demonstrated by means of HA chromatography as a second peak ŽFig. 2A.. DMMOP exhibits lower cross-linking activity w7x and the second peak corresponding to renaturated DNA is eluted from the HA column ŽFig. 2B., while practically no cross-linked fraction is detected with BP ŽFig. 2C, see also Ref. w8x.. NC exhibits low cross-linking potency, the distinct renaturated DNA fraction is seen at the adduct density 10 drug molecules per 10 3 DNA nucleotides w10x but not at a ten-fold lower binding level ŽFig. 2D.. Similar results were obtained when the cross-links were esti-
Z. Tołwinska-Stanczyk et al.r Mutation Research 374 (1997) 139–143 ´ ´
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Fig. 1. Transcriptional template activity of covalent drug–DNA complexes. Transcriptional template activity calculated as percentages of the corresponding controls, i.e. DNA irradiated ŽA–C. or preincubated with dithiothreitol ŽD. in absence of drug and subsequently purified. The insets show dependence of stoichiometry of drug–DNA complexes on drug concentration during adduct formation. ŽA. 8-MOP, ŽB. DMMOP, ŽC. BP, ŽD. NC. N s number of drug molecules bound per 10 3 DNA nucleotides; CT–DNA ŽB.; T7–DNA ŽI..
mated by means of ethidium fluorescence assay ŽTable 2.. The number of adduct molecules which decrease RNA synthesis to 37% ŽN37 . is lower for a highly cross-linking agent, MOP, while DMMOP, exhibiting low cross-linking potency, or BP, binding only monofunctionally, inhibit RNA synthesis to 37% at a somewhat higher adduct density ŽTable 1.. These differences, however, are not dramatic. This is otherTable 1 Number of adduct molecules per 10 3 DNA nucleotides which reduce in vitro RNA synthesis to 37% ŽN37 . Drug
MOP DMMOP BP nC
T7 DNA
CT DNA
n
r
N37
n
r
N37
8 7 8 12
0.95 0.95 0.97 0.87
0.25 0.52 0.65 0.48
14 16 15 12
0.97 0.93 0.83 0.94
0.45 0.79 0.55 1.26
n is number of independent experiments whose data have been used to compute N37 and r is the correlation coefficient of the linear regression.
wise consistent with the notion, that monoadducts on the transcribed strand as well as diadducts of furocoumarines are equally efficient in terminating RNA synthesis w14,15x and with comparison of RNA synthesis inhibition by MOP and the monoadduct forming angelicin derivatives w5,6x. These results are substantially in agreement with other observations about the equivalence of monoadducts at cellular level Žsee reviews w3x and w16x.. The adduct structure andror specificity plays a role as NC, the only drug showing an affinity to purines w10x assayed here exhibits with CT DNA lower inhibitory effect which does not seem to be due exclusively to its low cross-linking efficiency ŽTable 1.. Compilation of the data obtained under various experimental conditions in different laboratories led to the conclusion that covalent binding of 0.2–0.8 drug molecules per 10 3 DNA nucleotides presenting polycyclic aromatic systems Žfurocoumarins, benzoŽ a.pyrene, 2-aminofluorene. reduce RNA synthesis to 37% w17x. The data here presented 0.25–1.26 drug molecules per 10 3 DNA nucleotides ŽTable 1. are
Z. Tołwinska-Stanczyk et al.r Mutation Research 374 (1997) 139–143 ´ ´
142
Fig. 2. Hydroxylapatite chromatography of denaturated complexes of CT DNA with ŽA. 8-MOP, ŽB. DMMOP, ŽC. BP, ŽD. NC. Drugs concentration at the complex formation was 5 m M. Termally denaturated drug–DNA complexes Ž`. were subjected to chromatography on the HA column as indicated in Section 2. Ultraviolet absorption of each fraction eluted at the indicated phosphate concentration is expressed as a percentage of the total absorbance recovered in the effluent. Dotted line, elution profiles of the control DNA.
not very different from those compiled w17x. Results may vary but the inhibition is relatively unsensitive to the template used and a cross-linking efficiency. Hence, the transcription assay may be a simple method to estimate stoichiometry of covalent complexes of DNA with polycyclic drugs. If we assume that a decrease of the template activity to 80% of the
controls is a limit of detection of covalent modification of DNA a sensitivity of the transcription assay is about 1 adduct per 10 4 DNA nucleotides. This sensitivity corresponds to that typical for conventional high-performance liquid chromatography combined with UV–visible detection although a detection level up to 1 adduct per 10 10 DNA nucleotides may be
Table 2 Double-stranded DNA fraction in drug–DNA complexes as measured by hydroxylapatite chromatography ŽHA. and fluorescence assay ŽF. Drug
HA
F
Relative template activity
MOP DMMOP BP NC
20.3 Ž n s 1. 13.1 Ž n s 1. 0.0 " 0.0 Ž n s 2. 0.0 Ž n s 1.
19.2 " 1.0 Ž n s 3. 14.6 Ž n s 1. 3.5 " 1.6 Ž n s 3. 1.9 " 2.0 Ž n s 4.
23.0 " 1.0 Ž n s 2. 31.0 " 3.0 Ž n s 2. 43 Ž n s 1. 51.9 " 4.2 Ž n s 3.
CT–DNA complexes were formed at 5 m M drug concentration by irradiation Žfurocoumarins. or in the presence of dithiothreitol Ž5 mM.. The number of MOP and NC molecules bound is 1.0, DMMOP 1.3 and BP 0.8 molecules per 10 3 DNA nucleotides under these conditions. The double-stranded DNA fraction is expressed as percentage of the increase relative to the corresponding controls Ži.e. the mock-treated and heat-denaturated. DNA whose double-stranded fraction was taken as 0%. Relative template activity is expressed as percentage of the corresponding controls. Numbers Ž n. of independent experiments are indicated by parentheses and "standard deviations or range values are indicated.
Z. Tołwinska-Stanczyk et al.r Mutation Research 374 (1997) 139–143 ´ ´
reached by means of more sophisticated variants of the method Žw18x, see also a recent paper of Anselmino et al. for analysis of psoralen adducts w19x.. The transcription assay may be used mainly for the analysis of in vitro incubations of drugs, alone or complexed with DNA proteins w20x. A similar adduct density was reached with experimental animals or in cell cultures and DNA of lowered transcriptional activity was isolated from the liver of rats pretreated with nitracrine. On the other hand lower molecular weight adducts exhibit similar inhibition at a 10- to 20-fold higher density of the binding w17x. References w1x Chandra, P. and A. Wacker Ž1966. Photodynamic effects on the template activity of nucleic acid, Z. Naturforsch., 21b, 663–666. w2x Rodighiero, G., P. Chandra and A. Wacker Ž1970. Structural specificity for the photo-inactivation of nucleic acids by furocoumarins, FEBS Lett., 10, 29–32. w3x Song, P-S. and K.J. Tapley Ž1979. Photochemistry and photobiology of psoralen, Photochem. Photobiol., 29, 1177– 1197. w4x Szmigiero, L. and M. Gniazdowski Ž1981. Complexes of nitracrine with DNA, Arzneim. Forsch. Drug Res., 31, 1875–1877. w5x Gniazdowski, M., M. Czyz, K. Studzian, M. ˙ D. Wilmanska, ´ Frasunek, A. Płucienniczak and L. Szmigiero Ž1988. Inhibition of DNA-dependent RNA synthesis by 8-methoxypsoralen, Biochim. Biophys. Acta, 950, 346–353. w6x Czyz, ˙ M., M. Piestrzeniewicz and M. Gniazdowski Ž1990. Comparison of inhibitory effects of mono- and diadducts of furocoumarins on the transcriptional template activity of phage T7 DNA, Stud. Biophys., 135, 147–154. w7x Palumbo, M., F. Baccichetti, C. Antonello, O. Gia, A. Capozzi, S. Marciani Magno Ž1990. Photobiological activity X of 3,4 -dimethyl-8-methoxypsoralen, a linear furocoumarin with unusual DNA-binding properties, Photochem. Photobiol., 52, 533–540. w8x Gia, O., S. Mobilio, M. Palumbo and M.A. Pathak Ž1993. Benzo- and tetrahydro-benzopsoralen congeners: DNA binding and photobiological properties, Photochem. Photobiol., 57, 497–503.
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w9x Wilmanska, D., E. Małagocka, L. Szmigiero and M. Gniaz´ dowski Ž1984. Effect of intercalating and groove-binding ligands on formation of covalent complexes between nitracrine ŽLedakrin, C-283. or 8-methoxypsoralen and DNA, Biochim. Biophys. Acta, 782, 285–294. w10x Gniazdowski, M., E. Ciesielska and L. Szmigiero Ž1981. Some properties of the irreversible complexes of nitracrine ŽLedakrin, C-283. with polynucleotides, Chem. Biol. Interact., 34, 355–366. w11x Splengler, S.J. and Singer B. Ž1988. Formation of interstrand cross-links in chloroacetaldehyde-treated DNA demonstrated by ethidium bromide fluorescence, Cancer Res., 48, 4884– 4886. w12x Glaser, R. Ž1975. Einfuhrung in die Biophysik ŽPolish trans¨ lation., PZWL, Warszawa, pp. 282–285. w13x Koch, K.S., R.G. Fletcher, M.P. Grond, A.J. Inyang, X.P. Lu, D.A. Brenner and H.L. Leffert Ž1993. Inactivation of plasmid report gene expression by one benzoŽ a.pyrene diolepoxide DNA adduct in adult rat hepatocytes, Cancer Res., 53, 2279–2286. w14x Shi, Y.-B., H. Gamper and J.E. Hearst Ž1987. The effects of covalent additions of a psoralen on transcription by E. coli RNA polymerase, Nucl. Acids Res., 15, 6843–6854. w15x Shi, Y.-B., H. Gamper and J.E. Hearst Ž1988. Interaction of T7 RNA polymerase with DNA in an elongation complex arrested at a specific psoralen adduct site, J. Biol. Chem., 263, 527–534. w16x Rodighiero, G., F. Dall’Acqua and M.A. Pathak Ž1984. Photobiological properties of monofunctional furocoumarin derivatives, in: K.C. Smith ŽEd.., Topics in Photomedicine, Plenum, New York, pp. 319–397. w17x Gniazdowski, M. and C. Cera Ž1996. The effects of DNA covalent adducts on in vitro transcription, Chem. Rev., 96, 619–634. w18x Cummings, J., R.C. French and J.F. Smyth Ž1993. Application of high-performance liquid chromatography for recognition of covalent nucleic acid modification with anticancer drugs, J. Chromatogr., 618, 251–276. w19x Anselmino, C., D. Averbeck and J. Cadet Ž1995. Photoreaction of 5-methoxypsoralen with thymidine and the thymine moiety of isolated and Saccharomyces cereÕisiae DNA. Characterization and measurement of the two cis-syn furanside monocycloadducts, Photochem. Photobiol., 62, 997– 1004. w20x Wilmanska, D., L. Szmigiero and M. Gniazdowski Ž1989. In ´ vitro binding of nitracrine to DNA in chromatin, Z. Naturforsch., 44c, 14–18.
Transcriptional template activity of covalently modified DNA Zofia Tołwinska-Stanczyk, Dorota Wilmanska, Kazimierz Studzian, ´ ´ ´ Marek Gniazdowski ),1 Department of General Chemistry, Medical UniÕersity of Łodz, ´ ´ Lindleya 6, 90-131 Łodz, ´ ´ Poland Received 2 April 1996; revised 9 July 1996; accepted 6 August 1996
Abstract The transcriptional template activity of covalent modified DNA is compared. 8-Methoxypsoralen ŽMOP., 3,4X dimethyl8-methoxypsoralen ŽDMMOP. and benzopsoralen ŽBP. forming with DNA covalent complexes upon UV irradiation and exhibiting preference to pyrimidines, mostly thymines, differ in their cross-linking potency. MOP and DMMOP form both monoadducts and diadducts while no cross-links are formed by BP. Nitracrine ŽNC. forms covalent complexes with DNA upon reductive activation with dithiothreitol exhibiting a preference to purines and low cross-linking potency. Semilogarithmic plots of the relative template activity against the number of the drugs molecules covalently bound per 10 3 DNA nucleotides fit to regression lines corresponding to one-hit inactivation characteristics. The number of drug molecules decreasing RNA synthesis to 37% differ from 0.25 to 1.26 depending on the template used and the base preference but no dependence on the cross-linking potency was found. Keywords: DNA; RNA polymerase; Furocoumarins; Psoralens; 8-Methoxypsoralen; Nitracrine; Adducts
1. Introduction Most of carcinogens and some cytotoxic drug exert their biological effects through covalent attachment to DNA. One of the properties of DNA, impaired by covalent modification, is transcriptional Abbreviations: CT, calf thymus; T7, phage T7; HA, hydroxylX apatite; MOP, 8-methoxy-psoralen; DMMOP, 3,4 -dimethyl-8methoxypsoralen; BP, benzopsoralen; NC, nitracrine, 1-nitro-9Ž3,3X-dimethylaminopropylamino.acridine, also known under its code C-283 and its trade name, ‘Ledakrin’ ) Corresponding author. Tel.: Ž42. 784277. Fax: Ž42. 322347. 1 This work was supported by the Medical University of Łodz ´ ´ within the project No. 502-11-81 and Foundation for Polish Science within the project BIMOL.
template activity. The decrease of the amount of RNA synthesized by DNA-dependent RNA polymerase on a modified template is measured by quantitating the incorporation of radioactive ribonucleotides into the acid-insoluble fraction. Covalent binding of furocoumarins poisons the transcriptional template activity depending on the binding level w1–3x. It was found in our earlier experiments with NC-CT DNA complexes and T7 DNA modified with two furocoumarin derivatives that the logarithmic plot of the percentage of RNA synthesis versus the number of covalent adducts on the template is a straight line w4–6x. This observation may be potentially of analytical importance, provided that we know to what extent the inhibition of RNA synthesis in vitro de-
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Z. Tołwinska-Stanczyk et al.r Mutation Research 374 (1997) 139–143 ´ ´
pends on the template used, the base specificity of the adduct formation and the adduct structure. Particularly a comparison between the monofunctionally binding drugs and the drugs forming interstrand cross-links seems of interest in this respect. The aim of these experiments is to evaluate the dependence of the inhibition on these parameters. Four drugs covalently binding to DNA are compared. Three psoralens form covalent complexes upon irradiation with near UV light w7,8x. All of them react with pyrimidines, mostly thymines, but they differ in their cross-linking potency. 8-Methoxypsoralen ŽMOP. w7x and 3,4-dimethyl-8-methoxypsoralen ŽDMMOP. w8x form with DNA both monoadducts and diadducts while almost no cross-links are formed by benzopsoralen ŽBP. w8x. Nitracrine ŽNC. forms covalent complexes with DNA upon reductive activation with sulfhydryl compounds w4x. In contrast to psoralens NC exhibits preferential binding to purines, particularly to guanines in DNA, its binding does not seem to depend on intercalation w9x and only few cross-links are detected in NC–DNA complexes w10x.
2. Materials and methods 2.1. Materials MOP w9x, NC w4,10x, calf thymus ŽCT. w4x and phage T7 w5x DNA, Escherichia coli DNA-dependent RNA polymerase ŽEC. 2.7.7.6. w5x and most of other materials are as described in the cited papers. DMMOP w7x and BP w8x were kindly donated by Dr O. Gia and Professor M. Palumbo ŽUniversity of Padova.. 2.2. Methods Covalent complexes of 3 H-labeled furocoumarins with either CT or T7 DNA were formed by irradiation at 365 nm, then purified and their stoichiometry was estimated as previously described w5,10x. NC– DNA complexes were formed by incubation of DNA with the 14 C-labeled drug in the presence of dithiothreitol Ž5 mM., then purified and subjected to stoichiometry estimation w4,5,10x. Transcriptional template activity of drug–DNA complexes estimated with E. coli DNA-dependent RNA polymerase was
expressed as percentages of the corresponding controls w4,5x. The fraction of cross-linked DNA was estimated by fluorescence assay according to Splengler and Singer w11x or by means of hydroxylapatite wHAx chromatography of heat-denaturated DNA w10x. It is expressed as percentage of the increase in renaturated DNA fraction in the drug–DNA complex relative to the corresponding heat-denaturated control DNA, i.e. DNA either irradiated or preincubated with dithiothreitol in the absence of a drug.
3. Results and discussion Stoichiometry of the complexes depends on drug concentration during the complex formation and it is similar for both the templates used Žsee the insets to Fig. 1.. The relative template activity of the complexes, plotted as a function of the number of the drug molecules covalently bound per 10 3 DNA nucleotides ŽN., is shown on Fig. 1. The semilogarithmic plots fit to the regression lines ŽFig. 1. described by the general equation:log 10 wrelative template activity Ž%.x s AN q B,with correlation coefficients 0.85–0.97 ŽTable 1.. This relationship corresponds to one-hit inactivation characteristics w12,13x. The number of adduct molecules per 10 3 DNA nucleotides which reduce the relative template activity to 37% ŽN37 . was obtained from the inhibition curves ŽTable 1.. As otherwise found these psoralen derivatives differ in their cross-linking potencies. MOP forms with DNA both monoadducts and diadducts. The latter induce renaturation following thermal denaturation and cooling which can be demonstrated by means of HA chromatography as a second peak ŽFig. 2A.. DMMOP exhibits lower cross-linking activity w7x and the second peak corresponding to renaturated DNA is eluted from the HA column ŽFig. 2B., while practically no cross-linked fraction is detected with BP ŽFig. 2C, see also Ref. w8x.. NC exhibits low cross-linking potency, the distinct renaturated DNA fraction is seen at the adduct density 10 drug molecules per 10 3 DNA nucleotides w10x but not at a ten-fold lower binding level ŽFig. 2D.. Similar results were obtained when the cross-links were esti-
Z. Tołwinska-Stanczyk et al.r Mutation Research 374 (1997) 139–143 ´ ´
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Fig. 1. Transcriptional template activity of covalent drug–DNA complexes. Transcriptional template activity calculated as percentages of the corresponding controls, i.e. DNA irradiated ŽA–C. or preincubated with dithiothreitol ŽD. in absence of drug and subsequently purified. The insets show dependence of stoichiometry of drug–DNA complexes on drug concentration during adduct formation. ŽA. 8-MOP, ŽB. DMMOP, ŽC. BP, ŽD. NC. N s number of drug molecules bound per 10 3 DNA nucleotides; CT–DNA ŽB.; T7–DNA ŽI..
mated by means of ethidium fluorescence assay ŽTable 2.. The number of adduct molecules which decrease RNA synthesis to 37% ŽN37 . is lower for a highly cross-linking agent, MOP, while DMMOP, exhibiting low cross-linking potency, or BP, binding only monofunctionally, inhibit RNA synthesis to 37% at a somewhat higher adduct density ŽTable 1.. These differences, however, are not dramatic. This is otherTable 1 Number of adduct molecules per 10 3 DNA nucleotides which reduce in vitro RNA synthesis to 37% ŽN37 . Drug
MOP DMMOP BP nC
T7 DNA
CT DNA
n
r
N37
n
r
N37
8 7 8 12
0.95 0.95 0.97 0.87
0.25 0.52 0.65 0.48
14 16 15 12
0.97 0.93 0.83 0.94
0.45 0.79 0.55 1.26
n is number of independent experiments whose data have been used to compute N37 and r is the correlation coefficient of the linear regression.
wise consistent with the notion, that monoadducts on the transcribed strand as well as diadducts of furocoumarines are equally efficient in terminating RNA synthesis w14,15x and with comparison of RNA synthesis inhibition by MOP and the monoadduct forming angelicin derivatives w5,6x. These results are substantially in agreement with other observations about the equivalence of monoadducts at cellular level Žsee reviews w3x and w16x.. The adduct structure andror specificity plays a role as NC, the only drug showing an affinity to purines w10x assayed here exhibits with CT DNA lower inhibitory effect which does not seem to be due exclusively to its low cross-linking efficiency ŽTable 1.. Compilation of the data obtained under various experimental conditions in different laboratories led to the conclusion that covalent binding of 0.2–0.8 drug molecules per 10 3 DNA nucleotides presenting polycyclic aromatic systems Žfurocoumarins, benzoŽ a.pyrene, 2-aminofluorene. reduce RNA synthesis to 37% w17x. The data here presented 0.25–1.26 drug molecules per 10 3 DNA nucleotides ŽTable 1. are
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Fig. 2. Hydroxylapatite chromatography of denaturated complexes of CT DNA with ŽA. 8-MOP, ŽB. DMMOP, ŽC. BP, ŽD. NC. Drugs concentration at the complex formation was 5 m M. Termally denaturated drug–DNA complexes Ž`. were subjected to chromatography on the HA column as indicated in Section 2. Ultraviolet absorption of each fraction eluted at the indicated phosphate concentration is expressed as a percentage of the total absorbance recovered in the effluent. Dotted line, elution profiles of the control DNA.
not very different from those compiled w17x. Results may vary but the inhibition is relatively unsensitive to the template used and a cross-linking efficiency. Hence, the transcription assay may be a simple method to estimate stoichiometry of covalent complexes of DNA with polycyclic drugs. If we assume that a decrease of the template activity to 80% of the
controls is a limit of detection of covalent modification of DNA a sensitivity of the transcription assay is about 1 adduct per 10 4 DNA nucleotides. This sensitivity corresponds to that typical for conventional high-performance liquid chromatography combined with UV–visible detection although a detection level up to 1 adduct per 10 10 DNA nucleotides may be
Table 2 Double-stranded DNA fraction in drug–DNA complexes as measured by hydroxylapatite chromatography ŽHA. and fluorescence assay ŽF. Drug
HA
F
Relative template activity
MOP DMMOP BP NC
20.3 Ž n s 1. 13.1 Ž n s 1. 0.0 " 0.0 Ž n s 2. 0.0 Ž n s 1.
19.2 " 1.0 Ž n s 3. 14.6 Ž n s 1. 3.5 " 1.6 Ž n s 3. 1.9 " 2.0 Ž n s 4.
23.0 " 1.0 Ž n s 2. 31.0 " 3.0 Ž n s 2. 43 Ž n s 1. 51.9 " 4.2 Ž n s 3.
CT–DNA complexes were formed at 5 m M drug concentration by irradiation Žfurocoumarins. or in the presence of dithiothreitol Ž5 mM.. The number of MOP and NC molecules bound is 1.0, DMMOP 1.3 and BP 0.8 molecules per 10 3 DNA nucleotides under these conditions. The double-stranded DNA fraction is expressed as percentage of the increase relative to the corresponding controls Ži.e. the mock-treated and heat-denaturated. DNA whose double-stranded fraction was taken as 0%. Relative template activity is expressed as percentage of the corresponding controls. Numbers Ž n. of independent experiments are indicated by parentheses and "standard deviations or range values are indicated.
Z. Tołwinska-Stanczyk et al.r Mutation Research 374 (1997) 139–143 ´ ´
reached by means of more sophisticated variants of the method Žw18x, see also a recent paper of Anselmino et al. for analysis of psoralen adducts w19x.. The transcription assay may be used mainly for the analysis of in vitro incubations of drugs, alone or complexed with DNA proteins w20x. A similar adduct density was reached with experimental animals or in cell cultures and DNA of lowered transcriptional activity was isolated from the liver of rats pretreated with nitracrine. On the other hand lower molecular weight adducts exhibit similar inhibition at a 10- to 20-fold higher density of the binding w17x. References w1x Chandra, P. and A. Wacker Ž1966. Photodynamic effects on the template activity of nucleic acid, Z. Naturforsch., 21b, 663–666. w2x Rodighiero, G., P. Chandra and A. Wacker Ž1970. Structural specificity for the photo-inactivation of nucleic acids by furocoumarins, FEBS Lett., 10, 29–32. w3x Song, P-S. and K.J. Tapley Ž1979. Photochemistry and photobiology of psoralen, Photochem. Photobiol., 29, 1177– 1197. w4x Szmigiero, L. and M. Gniazdowski Ž1981. Complexes of nitracrine with DNA, Arzneim. Forsch. Drug Res., 31, 1875–1877. w5x Gniazdowski, M., M. Czyz, K. Studzian, M. ˙ D. Wilmanska, ´ Frasunek, A. Płucienniczak and L. Szmigiero Ž1988. Inhibition of DNA-dependent RNA synthesis by 8-methoxypsoralen, Biochim. Biophys. Acta, 950, 346–353. w6x Czyz, ˙ M., M. Piestrzeniewicz and M. Gniazdowski Ž1990. Comparison of inhibitory effects of mono- and diadducts of furocoumarins on the transcriptional template activity of phage T7 DNA, Stud. Biophys., 135, 147–154. w7x Palumbo, M., F. Baccichetti, C. Antonello, O. Gia, A. Capozzi, S. Marciani Magno Ž1990. Photobiological activity X of 3,4 -dimethyl-8-methoxypsoralen, a linear furocoumarin with unusual DNA-binding properties, Photochem. Photobiol., 52, 533–540. w8x Gia, O., S. Mobilio, M. Palumbo and M.A. Pathak Ž1993. Benzo- and tetrahydro-benzopsoralen congeners: DNA binding and photobiological properties, Photochem. Photobiol., 57, 497–503.
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w9x Wilmanska, D., E. Małagocka, L. Szmigiero and M. Gniaz´ dowski Ž1984. Effect of intercalating and groove-binding ligands on formation of covalent complexes between nitracrine ŽLedakrin, C-283. or 8-methoxypsoralen and DNA, Biochim. Biophys. Acta, 782, 285–294. w10x Gniazdowski, M., E. Ciesielska and L. Szmigiero Ž1981. Some properties of the irreversible complexes of nitracrine ŽLedakrin, C-283. with polynucleotides, Chem. Biol. Interact., 34, 355–366. w11x Splengler, S.J. and Singer B. Ž1988. Formation of interstrand cross-links in chloroacetaldehyde-treated DNA demonstrated by ethidium bromide fluorescence, Cancer Res., 48, 4884– 4886. w12x Glaser, R. Ž1975. Einfuhrung in die Biophysik ŽPolish trans¨ lation., PZWL, Warszawa, pp. 282–285. w13x Koch, K.S., R.G. Fletcher, M.P. Grond, A.J. Inyang, X.P. Lu, D.A. Brenner and H.L. Leffert Ž1993. Inactivation of plasmid report gene expression by one benzoŽ a.pyrene diolepoxide DNA adduct in adult rat hepatocytes, Cancer Res., 53, 2279–2286. w14x Shi, Y.-B., H. Gamper and J.E. Hearst Ž1987. The effects of covalent additions of a psoralen on transcription by E. coli RNA polymerase, Nucl. Acids Res., 15, 6843–6854. w15x Shi, Y.-B., H. Gamper and J.E. Hearst Ž1988. Interaction of T7 RNA polymerase with DNA in an elongation complex arrested at a specific psoralen adduct site, J. Biol. Chem., 263, 527–534. w16x Rodighiero, G., F. Dall’Acqua and M.A. Pathak Ž1984. Photobiological properties of monofunctional furocoumarin derivatives, in: K.C. Smith ŽEd.., Topics in Photomedicine, Plenum, New York, pp. 319–397. w17x Gniazdowski, M. and C. Cera Ž1996. The effects of DNA covalent adducts on in vitro transcription, Chem. Rev., 96, 619–634. w18x Cummings, J., R.C. French and J.F. Smyth Ž1993. Application of high-performance liquid chromatography for recognition of covalent nucleic acid modification with anticancer drugs, J. Chromatogr., 618, 251–276. w19x Anselmino, C., D. Averbeck and J. Cadet Ž1995. Photoreaction of 5-methoxypsoralen with thymidine and the thymine moiety of isolated and Saccharomyces cereÕisiae DNA. Characterization and measurement of the two cis-syn furanside monocycloadducts, Photochem. Photobiol., 62, 997– 1004. w20x Wilmanska, D., L. Szmigiero and M. Gniazdowski Ž1989. In ´ vitro binding of nitracrine to DNA in chromatin, Z. Naturforsch., 44c, 14–18.