Binding of platinum-diaminonitroxyl complexes to animal DNA

Journal of Inorganic Biochemistry 69 (1998) 67±77

Binding of platinum-diaminonitroxyl complexes to animal DNA Alexander V. Shugalii, Alexander V. Kulikov, Margarita V. Lichina, Valery A. Golubev, Vasily D. Sen' * Institute of Chemical Physics in Chernogolovka, Russian Academy of Sciences, Chernogolovka, 142432 Moscow Region, Russian Federation Received 7 November 1996; received in revised form 16 September 1997; accepted 17 September 1997

Abstract Reaction of PtII (DAPO)X2 complexes (where DAPO is trans-3,4-diamino-2,2,6,6-tetramethylpiperidine-1-oxyl, X2 ˆ (NO3 )2 , oxalato (Ox) or 1,1-cyclobutanedicarboxylato (Cbdca)) with a bovine spleen DNA in 0.01 M NaHCO3 at 37°C for 24 h gives rise to formation of platinated DNA. The [bound PtII (DAPO)]/[nucleotide] ratio (r) depends on the initial ratio of the reagents and on the nature of leaving ligands X. Nitroxyl±nitroxyl distances in platinated DNA determined by the ESR suggest that at r P 0.1 PtII (DAPO) fragments are uniformly attached to DNA. But at lower r, the thermal characteristics of modi®ed DNA (melting temperature Tm , melting range width DT) and the guanine-to-adenine platination degree ratios GPt /APt imply that the nature of leaving ligands X a€ect the selectivity of DNA platination. At r P 0.1, nitroxyl groups can approach each other so close that, in an acidic medium, the electron transfer from one nitroxyl group to another becomes possible, and the nitroxyls readily disproportionate to diamagnetic products. Correlation time of nitroxyl rotation in PtII (DAPO)±DNA adducts is 10ÿ8 s, which is related to predominantly bifunctional bonding of PtII (DAPO) with DNA. Platination-induced distortion of DNA was evidenced by changes in Tm , DT and degree of hyperchromicity H. The major part of adducts form the intrastrand cross-links which destabilize the structure of DNA duplex. The interstrand PtII (DAPO) cross-linking (1% of the adducts) facilitates renaturation of despiralized DNA molecules upon cooling. Two types of PtII (DAPO)±DNA adducts are revealed, which di€er substantially in their rates of deplatination with NaCN. ESR, electron spin resonance; r, degree of modi®cation; cisplatin, cis-diamminedichloroplatinum(II); Tm , melting temperature; DT, melting range width; H, degree of hyperchromicity; R, degree of renaturation; AAS, atomic absorption spectroscopy; HPLC, high performance liquid chromatography. Ó 1998 Elsevier Science Inc. All rights reserved. Keywords: Platinum; Nitroxyl radicals; DNA; Platination; Cisplatin

1. Introduction Nitroxyl radicals are known to possess a cytostatic activity against leukemia La in vivo [1] and against cancer cells HeLa in vitro [2]. It has been shown in vivo [3] that the general toxicity of some cytostatics is decreased when they are administered together with nitroxyl radicals. Modifying antitumor compounds with nitroxyl radicals improves their chemotherapeutic properties [4]. Mechanism of biological activity of nitroxyls remains unclear so far. Some authors [5,6] explain the modifying e€ect of nitroxyls by their ability to deacti-

*

Corresponding author. Fax: +7 096 5153588; e-mail: [email protected]. 0162-0134/98/$19.00 Ó 1998 Elsevier Science Inc. All rights reserved. PII: S 0 1 6 2 - 0 1 3 4 ( 9 7 ) 1 0 0 2 1 - 6

vate highly reactive harmful species such as Oÿ´ 2 , which are actively generated in many disease pathologies and under the action of xenobiotics. We synthesized PtII (DAPO)X2 complexes with a DAPO nitroxyl radical as a carrying ligand and di€erent leaving ligands X and studied their toxicity and antitumor activity in vivo [7]. New complexes appear to be less toxic than their structural analogs PtII (DACH)X2 (DACH ˆ 1,2-diaminocyclohexane) having the same leaving ligands [8]. Activity of PtII (DAPO)Cl2 , PtII (DAPO)Ox, and PtII (DAPO)Cbdca against mice tumors was shown to be comparable to that of cisplatin.

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2. Materials and methods Cisplatin was prepared and puri®ed as described elsewhere [19], synthesis of PtII (DAPO)X2 is described in [7]. The HPLC of starting complexes and their hydrolysis products was carried out on a Milichrom chromatograph with a 2 mm ´ 64 mm column packed with 5 lm Separone C18, at k ˆ 240 nm. Hydrolysis rate of these complexes to PtII (DAPO)(OH)2 was determined in 0.08 M NaOH at initial concentration of the complexes (1.5±2) ´ 10ÿ4 M and temperatures 25±90°C. Current concentrations of the complexes were determined from the system of equations: Cisplatin and its analogs bind rapidly to numerous accessible nucleophilic centers of biomolecules in vivo [9,10]. However, formation of their adducts with DNA and, as a consequence, distortion of replication is considered to be the main cause of antitumor activity of platinum complexes [11,12]. E€ectiveness of DNA platination and properties of formed adducts depend on the volume and hydrophobicity of carrying ligands [10]. Quantum-mechanical calculations showed [13] that binding of even a single methyl group to 3a- (a means axial) or 5a-position of the trans-DACH hinders formation of PtII [3a(5a)-MeDACH]±DNA adducts due to a signi®cant increase in steric energy. Crystal structures of PtII (DACH)Ox and PtII (DAPO)Ox are very similar [14,15]. The only di€erence is that respective 3- and 5-positions in DACH are occupied by four CH3 groups in DAPO. This implies that there are steric hindrances for platination of DNA by PtII (DAPO)X2 complexes. Reactivity of platinum-diaminonitroxyl complexes signi®cantly depends on the nature of leaving ligands X. Available data suggest that substitution of X by water molecules or by S-containing ligands such as cysteine, methionine in living cells precedes DNA platination [10±12]. However, DNA bases can substitute for strongly coordinated ligand Cbdca prior to its hydrolysis [12,16]. Therefore, it is reasonable to expect a di€erence in properties of DNA modi®ed by complexes containing slowly or rapidly hydrolyzed X ligands. Introduction of nitroxyls to anticancer agents allows the ESR method to be applied for investigation of their reactions with biological targets [17]. Recently, a mixedligand cis-[PtII (4-H2 N-TEMPO)(NH3 )ClI] complex has been used for determination of a long-range interaction in a platinated dinucleotide [18]. In this work, we studied platination of DNA from bovine spleen by cisplatin and PtII (DAPO)X2 complexes (where X2 ˆ (NO3 )2 , Ox, or Cbdca) and integral thermodynamic characteristics of corresponding modi®ed DNA (melting temperature Tm , melting range width DT, and degree of hyperchromicity H). Degree of DNA modi®cation, distances between nitroxyl groups, and deplatination of PtII (DAPO)±DNA with NaCN were studied by ESR.

c1 ‡ c2 ˆ c0 ;

e11 c1 ‡ e12 c2 ˆ D;

…1†

where c1 and c0 are current and initial concentrations of PtII (DAPO)X2 respectively, c2 is current concentration of PtII (DAPO)(OH)2 , e11 and e12 are extinction coecients for PtII (DAPO)X2 and PtII (DAPO)(OH)2 , respectively, D is optical density of the reaction solution. The measurements were carried out with a Specord UV±VIS spectrophotometer (Carl Zeiss, Jena) at m ˆ 39000 cmÿ1 for PtII (DAPO)Ox (e11 ˆ 4370 and e12 ˆ 2170 Mÿ1 cmÿ1 ) and at m ˆ 42000 cmÿ1 for PtII (DAPO)Cbdca (e11 ˆ 4300 and e12 ˆ 3420 Mÿ1 cmÿ1 ). Bovine spleen DNA (Reakhim, Russia) was fragmented by squeezing the DNA solution (1 mg/ml) through a 0.1 mm syringe needle. The mean molecular weight of fragmented DNA molecules calculated from the intrinsic viscosity of solution [20] was 1.65 ´ 106 (2500 nucleotide pairs). Concentration of DNA nucleotides was determined from optical density at k ˆ 260 nm using the average extinction coecient e ˆ 6.6 ´ 103 Mÿ1 cmÿ1 . The fragmented DNA was modi®ed by the platinum complexes as follows. The mixture of DNA (0.5 mg/ml) with complexes in 0.01 M NaHCO3 bu€er solution was incubated at 37°C. NaHCO3 was chosen in order to diminish the e€ect and HCOÿ of bu€er on the reaction, because CO2ÿ 3 3 ions poorly react with platinum under the experimental conditions. The unreacted complex was removed by dialysis for 5 days at 4°C or by dialysis for 12 h at 4°C with subsequent chromatography on a Sephadex G-50 column. For both methods, the characteristics of modi®ed DNA were the same within the accuracy of measurements. The initial [complex]/[nucleotide] ratio (rin ) in the reaction mixture was 0.005±0.8. Degree of modi®cation r was determined after removal of unreacted complexes. Concentration of PtII (DAPO)±DNA adducts was determined from ESR data as concentration of >N±O´ groups, or from AAS data (AAS-3 spectrometer) as platinum concentration, or from acidic hydrolysis data of platinated DNA (vide infra). To determine the degree of modi®cation for di€erent DNA bases, the samples of modi®ed DNA were lyophilized and hydrolyzed with 12 M HClO4 at 100°C for 50 min [21]. Under these conditions, platinated DNA bases decompose, while unmodi®ed bases remain in hydrolyz-

A.V. Shugalii et al. / J. Inorg. Biochem. 69 (1998) 67±77

ate. The neutralized hydrolyzate was studied by paper chromatography (3MM paper, butanol-30% aqueous ammonia (6:1 v/v)). The spots were extracted for 12 h by 0.1 M HCl. The unreacted base content was determined spectrophotometrically. Fraction of platinated bases (BPt ) was calculated according to the equation BPt ˆ 100…B ÿ BM †=B (%), where B and BM are the base content in initial and modi®ed DNA, respectively. Degree of platination was calculated from the equation r ˆ …0:2  GPt ‡ 0:3  APt †=2  100, where 0.2 and 0.3 coecients are molar fractions of guanine and adenine in DNA, coecient 2 accounts for binding of a platinum predominantly with two bases. Degree of modi®cation values determined by ESR, AAS or chromatography agree well. For example, for platination of DNA by PtII (DAPO) Ox for 24 h at rin ˆ 0.8, 37°C, the values of r (‹0.01) obtained by these methods are 0.21, 0.20 and 0.20, respectively. Integral thermodynamic characteristics (Tm , DT, H), and degree of renaturation R were determined from temperature dependence of optical density of DNA solutions at k ˆ 260 nm in the range 25±95°C at constant heating and cooling rates 10°C/h. Measurements were performed before heating, at 95°C, and after cooling the samples to 25°C (D025 , D95 and D25 , respectively). D95 corresponds to the optical density of completely denaturated duplex. R was calculated as follows: D95 ÿ D25 Rˆ  100 …%†: …2† D95 ÿ D025 Degree of hyperchromicity H was calculated from the formula: D95 ÿ D025  100 …%†: …3† D025 Measurements were carried out with the accuracy ‹0.2°C, ‹0.3°C, ‹1% and ‹3% for Tm , DT, H and R, respectively. To determine degree of despiralization of DNA with formaldehyde m1 , we studied variation of optical density of reaction mixture containing 20 lg/ml DNA and 0.8 M formaldehyde. m1 was calculated as follows: Dt ÿ D0 m1 ˆ  100 …%†; …4† Dmax ÿ D0 Hˆ

where D0 , Dt and Dmax are initial, current and ®nal optical densities of solution measured at k ˆ 260 nm, respectively. ESR spectra were recorded with a SE/X 2544 radiospectrometer (Radiopan, Poznan). 1- and 4-mm diameter glass tubes were used for ESR measurements at room temperatures and )196°C, respectively. Concentration of >N±O´ groups was determined by comparison of the second integrals of ESR spectra of the samples (at 20°C or )196°C) with that of a reference sample 20 lM 2,2,6,6-tetramethylpiperidine-1-oxyl. At low r, concentration of nitroxyls was determined by comparison of ESR line amplitudes at )196°C with those of the reference sample; this method proved to be the most sensitive (10ÿ6 M). Measurements were carried out with the accu-

69

racy of 5±20% depending on the concentration of the complexes (the higher value corresponds to the lower concentration). Deplatination kinetics of PtII (DAPO)±DNA with NaCN or thiourea was studied by an increase in the amplitudes of narrow ESR lines corresponding tentatively to PtII (DAPO)(CN)2 . 3. Results 3.1. Hydrolytic properties of PtII (DAPO)X2 Degree of PtII (DAPO)X2 hydrolysis depends on pH of the reaction medium and the nature of leaving ligand X. PtII (DAPO)(NO3 )2 is strongly hydrolyzed in aqueous solution and is in the equilibrium with respective aquaand hydroxocomplexes (see Scheme 1). The equilibrium can be shifted either to starting PtII (DAPO)(NO3 )2 , or to diaquacomplex [PtII (DAPO) (H2 O)2 ]2‡ , or to dihydroxocomplex PtII (DAPO)(OH)2 by variation of ion composition and pH of reaction medium. We managed to detect these complexes by HPLC on C18-reversed phase and to characterize them by ESR and UV-spectroscopy. Retention volumes and UV-characteristics of the species are given in Table 1. The HPLC fractions, containing the DAPO species, give three-line ESR spectra with splitting on nitrogen nuclei aN ˆ 1.68 mT. Only one broadened peak of starting PtII (DAPO)(NO3 )2 with a retention volume Vr ˆ 540 ll is revealed chromatographically in 5% MeOH+95% 0.1 M NaNO3 . In acidic medium (50% 0.1 M H3 PO4 +50% 0.1 M KH2 PO4 or 0.01 M HClO4 ), the equilibrium is shifted to the diaquacomplex, and two peaks of [PtII (DAPO)(H2 O)2 ]2‡ and NOÿ 3 species with Vr ˆ 275 and 160 ll are revealed. In alkaline medium (5% MeOH+95% 0.1 M Na3 PO4 ), the equilibrium is shifted to the dihydroxocomplex, and only peaks related to PtII (DAPO)(OH)2 and NOÿ 3 with Vr ˆ 340 and 145 ll are revealed. The injection of neutral, acidic or alkaline solutions of PtII (DAPO)(NO3 )2 or its solution in 0.1 M NaNO3 into the column produce no changes in chromatograms, thus implying rapid establishment of equilibrium (Scheme 1). Dichlorocomplexes hydrolyze also fairly easy. For cisplatin, in neutral and acidic aqueous media, the equilibrium is shifted almost completely to the cis[Pt(NH3 )2 Cl(H2 O)]‡ monoaquacomplex [22,23]. By contrast, under the identical conditions, the equilibrium for dicarboxylate complexes (X2 ˆ Ox or Cbdca) is shifted to initial complexes (Scheme 2).

Scheme 1.

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A.V. Shugalii et al. / J. Inorg. Biochem. 69 (1998) 67±77

Table 1 Retention volumes Vr and UV-characteristics of PtII (DAPO)X2 complexes and products of their hydrolysis Complex

Solvent

Vr (ll)

UV-spectrum, kmax (nm) (e, Mÿ1 cmÿ1 )

PtII (DAPO)(NO3 )2

H2 O 5% MeOH+95% 0.1 M NaNO3 50% 0.1 M H3 PO4 + 50% 0.1 M KH2 PO4 H2 O H2 O 50% 0.1 M H3 PO4 + 50% 0.1 M KH2 PO4 50% 0.1 M H3 PO4 + 50% 0.1 M KH2 PO4 5% MeOH+95% 0.1 M Na3 PO4 0.08 M NaOH 5% MeOH+95% 0.1 M Na3 PO4

) 540

248 sh (2140) )

275

242

) 605 605

241 (4780) 241 b 240 b

370

)

200

240 sh

) 340

244 sh (3430) 244 sh b

[PtII (DAPO)(H2 O)2 ]2‡ PtII (DAPO)Ox

[PtII (DAPO)(C2 O4 H)H2 O]‡ [PtII (DAPO)(C2 O4 )OH]ÿ PtII (DAPO)(OH)2

a b

a

b

a

b

a

Spectra were recorded with a Specord UV±VIS spectrophotometer; sh stands for shoulder. UV-spectra were recorded with a Milichrom chromatograph at the maximums of chromatographic peaks.

Scheme 2.

Chromatography of aqueous solution of PtII (DAPO)Ox in 50% 0.1 M H3 PO4 +50% 0.1 M KH2 PO4 reveals two peaks of initial complex PtII (DAPO)Ox and [PtII (DAPO)(C2 O4 H)(H2 O)]‡ with the 25:1 height ratio and with Vr ˆ 605 and 370 ll, respectively. The equilibrium of PtII (DAPO)Ox hydrolysis is attained for 1 h. The PtII (DAPO)Cbdca complex is more stable; therefore, its hydrolysis products are not revealed chromatographically. The hydrolysis rate of carboxylate complex can be measured only in alkaline media when the equilibrium is shifted to PtII (DAPO)(OH)2 . The alkaline hydrolysis of PtII (DAPO)Ox proceeds via [PtII (DAPO)(C2 O4 )(OH)]ÿ intermediate. In 5% MeOH+95% 0.1 M Na3 PO4 , Vr for PtII (DAPO)Ox, PtII (DAPO)(OH)2 , [PtII (DAPO)(C2 O4 )(OH)]ÿ , and C2 O2ÿ 4 are 450, 340, 200, and 140 ll, respectively. However, instability of reversed C18 phase in alkaline media complicates the study of the reaction kinetics by HPLC. Small di€erences in UV spectra of ®nal PtII (DAPO)(OH)2 and intermediate [PtII (DAPO)(C2 O4 ) (OH)]ÿ do not allow tracing of the intermediate spectrophotometrically. Therefore, the determined rate constants characterize the overall process of PtII (DAPO)X2 hydrolysis to PtII (DAPO)(OH)2 (see Scheme 2). For the hydrolysis of PtII (DAPO)Cbdca

and PtII (DAPO)Ox at 25°C in 0.08 M NaOH, pseudomonomolecular rate constants are 2:9  10ÿ7 and 1:2  10ÿ4 sÿ1 , activation energies being 110 and 43 kJ/ mol, respectively. Under the same conditions, PtII (DAPO)(NO3 )2 hydrolyzes to PtII (DAPO)(OH)2 during the alkalization of solution; therefore, the hydrolysis rate constant exceeds 10ÿ2 sÿ1 . The alkaline hydrolysis rate constant for cisplatin is independent of the alkali concentration; at 25°C, it is 1.9 ´ 10ÿ5 sÿ1 , activation energy being 86.8 kJ/mol [22]. 3.2. Platination of DNA Fig. 1 shows ESR spectra of starting PtII (DAPO)Ox and that of PtII (DAPO)Ox-modi®ed DNA. At room temperature, the ESR spectrum of PtII (DAPO)Ox (Fig. 1(a)) contains three narrow lines, which indicates fast rotation of nitroxyl groups. The correlation time of rotation s can be estimated from the comparison of experimental and calculated spectra, assuming rotation to be isotropic [17], ch. 1. For unbound complex s 10ÿ11 s. Platination of DNA by PtII (DAPO)Ox gives rise to PtII (DAPO)±DNA adducts. ESR spectrum of PtII (DAPO)±DNA (Fig. 1(b)) indicates considerable decrease in rotation rate. DNA modi®ed by di€erent PtII (DAPO)X2 show the same shape of ESR spectra independently of leaving ligand X and r value. Nitroxyl groups bound to DNA are involved in two anisotropic motions: (1) motion of nitroxyl with respect to DNA molecule and (2) overall motion of DNA molecule. To distinguish these motions, experimental and theoretical methods were developed (see, for example, [24,25]). We failed to detect both motions owing to signi®cant dispersion of dynamic characteristics for PtII (DAPO) fragments attached to di€erent sites of

A.V. Shugalii et al. / J. Inorg. Biochem. 69 (1998) 67±77

71

Table 2 Degree of guanine and adenine platination after DNA modi®cation by PtII (DAPO)X2 complexes for 1 h at 37°C and rin ˆ 0.8

Fig. 1. ESR spectra of free unbound PtII (DAPO)Ox and DNA modi®ed by this complex. (a) PtII (DAPO)Ox in water, 20°C; (b) DNA modi®ed by PtII (DAPO)Ox, r ˆ 0.155, 20°C; (c) PtII (DAPO)Ox in ethanol, )196°C; (d) DNA modi®ed by PtII (DAPO)Ox, r ˆ 0.155, )196°C. Spectra were recorded at magnetic modulation 0.32 mT, microwave power 5 (20°C) and 0.5 mW ()196°C), ®eld scan 20 mT, scan time 8 min, time constant 1 s.

DNA. Therefore, we cannot use these methods. Therefore, rough estimation of the e€ective correlation time s 1 ´ 10ÿ8 s was performed on the basis of isotropic motion model [17]. This value of s is mainly related to motion of nitroxyls with respect to DNA molecule, because the latter rotates around its axis with s > 10ÿ8 s [25,26]. Degree of DNA modi®cation depends on the nature of leaving ligands X and increases with increasing rin . Platinating activity of the complexes, as determined from the slopes of initial portions of the r(rin ) curves (Fig. 2), increases in the sequence PtII (DAPO)Cbdca, PtII (DAPO)Ox, and PtII (DAPO)(NO3 )2 . Hydrolysis rate

Fig. 2. Degree of DNA platination r vs. starting reagent ratio rin (r ˆ [bound PtII (DAPO)]/[nucleotide], rin ˆ [complex]/[nucleotide]) for di€erent PtII (DAPO)X2 complexes. Platination was carried out in 0.01 M NaHCO3 at 37°C for 24 h. (n) PtII (DAPO)Ox, () PtII (DAPO)(NO3 )2 , (D) PtII (DAPO)Cbdca, (s) cisplatin.

Complex

GPt , %

APt , %

GPt /APt

PtII (DAPO)(NO3 )2 PtII (DAPO)Ox PtII (DAPO)Cbdca

74 ‹ 5 18 ‹ 3 12 ‹ 2

67 ‹ 5 6‹2 <2

1.1 ‹ 0.2 3‹1 >6

of the complexes increases in the same sequence (vide supra). PtII (DAPO)X2 complexes bind to di€erent DNA bases at di€erent rates. Chromatographic examination of acidic hydrolysis products of platinated DNA shows that binding to guanine and adenine predominates. No binding to thymine was observed; binding to cytosine was very slow. For example, at rin ˆ 0.8 the 98‹2% of guanine, 98‹2% of adenine, and 4‹1% of cytosine reacted with PtII (DAPO)Ox at 37°C for 72 h. The degrees of guanine and adenine platination for 1 h at 37°C (Table 2) show that platination rate increases, and the guanine-to-adenine platination degree ratio GPt /APt decreases in the sequence PtII (DAPO)Cbdca, PtII (DAPO)Ox, and PtII (DAPO)(NO3 )2 . 3.3. Properties of platinated DNA 3.3.1. Arrangement of PtII (DAPO) fragments on DNA molecules. ESR spectrum of heavily modi®ed DNA at )196°C suggests magnetic dipole±dipole interaction between nitroxyls (compare spectra c and d in Fig. 1). Distance between nitroxyls L can be determined from parameter d1 /d (see Fig. 1(c)) [27] 0:077 ÿ d
…nm† …5† 1 ÿ d d 0 where subscript `0' indicates the absence of dipole±dipole interaction, (d1 /d)0 value corresponds to d1 /d at r 6 0.01. In our case, (d1 /d)0 ˆ 0.45 (see Table 3). L values can be also determined from the second central moments of ESR lines M2 (mT2 ): L ˆ 0:93 ‡ d1

1:22  …nm†; Lˆp …6† 6 M2 ÿ …M2 †0 where subscript `0' also indicates the absence of dipole± dipole interaction. Eq. (6) is a particular case of a formula given in [28] for a suciently long linear polymer and equal distances between nitroxyls. (M2 )0 value can be measured only at low r. However, in this case, the ESR signal is too weak for accurate measurements. Therefore, (M2 )0 ˆ 3.6 mT2 , which is typical of aqueous solutions of spin-labeled proteins at )196°C, was taken. Distances between nitroxyls were also calculated from r values assuming equal distances between nitroxyls. Let b be the distance from DNA axis to >N±O´ groups. If Z axis coincides with DNA axis and one of nitroxyl groups lies on the X-axis, then the coordinates of two neighbouring nitroxyl groups are (b, 0, 0) and

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A.V. Shugalii et al. / J. Inorg. Biochem. 69 (1998) 67±77

Table 3 Distance between nitroxyls L for modi®ed DNA calculated from degree of modi®cation r and ESR spectrum parameters d1 /d and DM2 a

Complex

r

L

PtII (DAPO)Ox

0.21 0.11 0.08 0.03 0.24 0.17 0.09 0.035 0.03

1.35 2.21 2.59 5.80 1.20 1.62 2.45 5.10 5.80

PtII (DAPO)(NO3 )2

PtII (DAPO)Cbdca a b c

(nm)

b

d1 /d (‹0.01)

L

(nm)

0.76 0.65 0.51 0.48 0.68 0.54 0.47 0.45 0.45

1.17‹0,02 1.31‹0,03 2.31‹0,4 >2,6 1.26 ‹ 0,02 1.33 ‹ 0,03 >3.0 >3.0 >3.0

M2 (mT2 )

L

c

4.8 ) ) ) 4.8 4.55 ) ) )

1.18 ) ) ) 1.18 1.22 ) ) )

(nm)

Calculated from r. Calculated from d1 /d. Calculated from M2 .

(b cos u, b sin u, h), where u ˆ 18/r deg and h ˆ 0.17/r nm. u and h values were calculated for B-DNA. In this case, the distance between neighbouring nucleotide pairs is 0.34 nm, and a single helix turn contains 10 nucleotide pairs. Note that degree of modi®cation r is equal to the number of adducts per one nucleotide (not per a nucleotide pair). L can be calculated from the coordinates of these two points: p L ˆ 2b2 …1 ÿ cos…18=r†† ‡ 0:0289=r2 : …7† Table 3 lists L values calculated from r, d1 /d and DM2 at reasonable assumption b ˆ 0.8 nm. It is seen that the distances determined by the ESR method are similar to those calculated from r. This provides evidence for uniform arrangement of PtII (DAPO) fragments on DNA. An additional information on the arrangement of PtII (DAPO) fragments was obtained from changes in ESR response depending on pH of medium. Acidi®cation of platinated DNA (r > 0.1) solution to [H‡ ] ˆ 0.1 M results in disappearance of the ESR signal for a short period of time (<1 min). Alkalization of this acidic solution to pH 7, results in a rapid recovery of the ESR signal to 75% of its initial amplitude. This can be explained by reversible disproportionation of nitroxyl radicals to diamagnetic products R2 N‡ ˆ O and R2 N-OH in acidic medium [29]:

Incomplete regeneration of the radicals is explained by high reactivity and instability of R2 N‡ ˆ O cations. The rate of acidic disproportionation of >N±O´ for platinated DNA appears to be unusually high. For example, 50% of 2,2,6,6-tetramethylpiperidine-1-oxyl radicals at [>N±O´ ]0 ˆ 10ÿ4 M and [H‡ ] ˆ 0.1 M dispro-

portionate for 135 h [29]. High rate of consumption and regeneration of nitroxyls in PtII (DAPO)±DNA fragments can be explained by intramolecular electron transfer between neighbouring nitroxyl groups. 3.3.2. Integral thermodynamic characteristics of platinated DNA To examine the structure of platinated DNA, we studied dependence of integral thermodynamic characteristics (Tm , DT, and H) and degree of renaturation R on r for DNA modi®ed by various platinum complexes (Fig. 3). Fig. 4 shows the initial portion of Fig. 3 for r ˆ 0±0.03. The obtained data show that the properties of platinated DNA carrying the same PtII (DAPO) fragment depend on the nature of leaving ligands X. Cisplatin and PtII (DAPO)X2 (X2 ˆ (NO3 )2 or Ox) decrease thermal stability of the duplex at low degree of platination (r 6 0.15), but increase it at r > 0.15. At r 6 0.15, cisplatin has the strongest destabilizing e€ect while PtII (DAPO)(NO3 )2 is the weakest destabilizer (Fig. 3(a)). By contrast, platination with PtII (DAPO)Cbdca gives rise to the adducts that stabilize DNA at r 6 0.01 and destabilize it at r > 0.01 (Fig. 4(a)). The feature of PtII (DAPO)Cbdca manifests itself also in the in¯uence on the DT of modi®ed DNA. DT is related to the heterogeneity of DNA nucleotide composition: the higher the heterogeneity, the higher DT is. As distinct to other complexes, PtII (DAPO)Cbdca decreases DT at low r (Figs. 3(b) and 4(b)). H characterizes the ability of a duplex to denaturate upon heating. For starting DNA, H ˆ 37%. All modi®ed DNA exhibit a decrease in H with increasing r, the e€ect of cisplatin being the most pronounced (Fig. 3(c)). Such behaviour can be explained by the formation of locally denaturated regions, which can be revealed by a kinetic formaldehyde method. This method is based on the ability of formaldehyde to react with amino and imino groups of DNA bases in despiralized regions [30]. Formaldehyde causes unwinding of double

A.V. Shugalii et al. / J. Inorg. Biochem. 69 (1998) 67±77

Fig. 3. Tm , DT, H, and R as a function of r for di€erent platinum complexes. (n) PtII (DAPO)Ox, ()PtII (DAPO)(NO3 )2 , (D) PtII (DAPO)Cbdca, (s) cisplatin. Dashed lines show the dependencies of R on r calculated from formula (8) for N ˆ 5000 and p ˆ 0.01 (1) and 0.004 (2).

73

Fig. 4. Tm , DT, and R as a function of r for di€erent platinum complexes (the initial portions of Fig. 3. (n) PtII (DAPO)Ox, ()PtII (DAPO)(NO3 )2 , (D) PtII (DAPO)Cbdca, (s) cisplatin. Dashed line shows the dependencies of R on r calculated from formula (8) for N ˆ 5000 and p ˆ 0.01.

helix at a rate proportional to the content of defects. The rate of unwinding for modi®ed DNA is substantially higher (Fig. 5), which indicates that modi®cation induces additional defects. Content of defects can be determined from the initial portions of kinetic curves in Fig. 5 [30]. For modi®ed DNA, this content is (5 ‹ 3) ´ 10ÿ3 defects/nucleotide pair, which means that the average distance between defects is 125±500 nucleotide pairs. 3.3.3. Interstrand cross-links A number of interstrand cross-links in modi®ed DNA was determined in the experiments on renaturation of melted DNA. The experiments were carried out at low concentration of Na‡ (0.01 M), which makes impossible renaturation of completely separated DNA strands [31]. Random search for a complementary strand and formation of the so-called ``nucleus'' are known to be limiting stages of duplex restoration [32]. Fast restoration of DNA is possible only in the case of incomplete unwinding of strands, each DNA molecule having at least one interstrand cross-link, and a short double helix in the vicinity of this cross-link serving as the nucleus.

Fig. 5. Kinetics of DNA (20 lg/ml) unwinding under the action of formaldehyde (0.8 M) at 55°C. () starting DNA, (n) PtII (DAPO)Ox±modi®ed DNA (r ˆ 0.11).

Comparison of the ®rst and second melting curves (Fig. 6, curves 1 and 10 , respectively) for unmodi®ed DNA shows that cooling of melted DNA does not result in restoration of double helix. A small increase in optical density during the second melting (Fig. 6, curve 10 ) is

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For r > 0.05, measured p values are lower than calculated ones. In this case, a large number of bulky Pt(DAPO) fragments may prevent DNA strands from reassociation.

Fig. 6. Variation of relative optical density for the ®rst (1±3) and the second (10 ±30 ) melting cycles for PtII (DAPO)Ox±modi®ed DNA. (1, 10 ) r ˆ 0, (2, 20 ) r ˆ 0.05, (3, 30 ) r ˆ 0.015.

caused rather by distortion of single-strand short stems then by melting of renaturated DNA regions. Modifying of DNA with PtII (DAPO)Ox to r ˆ 0.05 causes di€erence in melting behaviour: the ®rst and the second melting curves (Fig. 6, curves 2 and 20 ) coincide, thus evidencing complete renaturation of the duplex. Melting curves of the subsequent heating±cooling cycles coincide with curve 2. The di€erence between the ®rst melting curves for unmodi®ed and modi®ed DNA (Fig. 6, curves 1 and 2) indicates the above mentioned partial denaturation of duplex due to modi®cation of DNA. At lower degree of modi®cation (r ˆ 0.015), the duplex is restored only by 30±40%. (compare Fig. 6, curves 3 and 30 ). Two models of partial renaturation can be suggested: (1) a certain part of DNA molecules renaturate completely, while the rest of molecules exhibit no renaturation; (2) all molecules undergo partial renaturation. In the former model, Tm and DT for the ®rst and second meltings are expected to be the same, while for the latter model they must di€er. In our case, the ®rst model is likely realized, because Tm and DT for the ®rst and second melting are virtually identical: Tm ˆ 68.0 and 67.7°C and DT ˆ 13.5 and 14.0°C, respectively. In frame of this model, incomplete renaturation at low r means that only a fraction of DNA molecules contain the cross-links. For example, only 30±40% of DNA molecules contain interstrand cross-links at r ˆ 0.015. The fraction of adducts that form interstrand crosslinks (p) can be estimated as follows. If one DNA molecule contains n adducts, the probability of the absence of cross-links is a ˆ (1 ) p)n . Note that, at the same time, a ˆ 1±0.01R, where R is the degree of renaturation; n ˆ Nr, N is the number of nucleotides in a DNA molecule (in our case N  5000). Therefore, R can be given as

3.3.4. Deplatination of modi®ed DNA by NaCN and thiourea Cyanides and thiourea are able to deplatinate modi®ed DNA [35,36]. These reactions may be useful for di€erentiation of PtII (DAPO)±DNA adducts by their platinum± DNA bond strength. Under the action of NaCN, bifunctional PtII (DAPO) fragments convert to monofunctional ones and then to free complex, tentatively to PtII (DAPO)(CN)2 . Deplatination kinetics was monitored by an increase in the amplitude of three narrow lines related to PtII (DAPO)(CN)2 in ESR spectrum of the reaction mixture (Fig. 7). The amplitude is proportional to the content of free complex. We measured I ˆ h‡ (t)/h‡ (1), where h‡ (t) and h‡ (1) are current and ®nal (24 h after addition of NaCN) amplitudes of the low-®eld ESR line of free complex (see Fig. 1(a)), respectively. Deplatination was carried out in an excess of NaCN (0.1 M). The data points were found to ®t well the equation: I ˆ A1 …1 ÿ exp…ÿk1 t† ‡ …1 ÿ A1 †…1 ÿ exp…ÿk2 t††; …9† in which both terms are the equations of pseudomonomolecular reactions. A1 , k1 and k2 (Table 4) were determined by an Origin MicroCal Software. For di€erent modifying complexes, rate constants k1 and k2 are similar. Note that A1 (the contribution of the slow process, which corresponds to ``strong'' adducts) decreases with decreasing r. Thiourea is less active with respect to PtII (DAPO)± DNA adducts. Noticeable displacement of nitroxyls is observed only at high temperature (87°C) and high thiourea concentration (0.8 M). For DNA samples with r > 0.1, the amplitude of narrow ESR lines is found to increase for 0.5 h since the beginning of the reaction. Subsequent decrease in the amplitude can be explained

Nr

R ˆ ‰1 ÿ …1 ÿ p† Š  100 …%†: …8† Comparison of experimental and calculated R values (Figs. 3(d) and 4(c)) shows that the calculated curve ®ts best to the data points at p ˆ 0.01. Similar values of p were found for DNA adducts with cisplatin [11,33,34].

Fig. 7. Kinetics of PtII (DAPO)±DNA deplatination with 0.1 M NaCN at 20°C for PtII (DAPO)(NO3 )2 ±modi®ed DNA at di€erent r. (1) r ˆ 0.18; (2) r ˆ 0.08; (3) r ˆ 0.04. Solid lines refer to calculated kinetics (Eq. (9)) of deplatination at parameters given in Table 4.

A.V. Shugalii et al. / J. Inorg. Biochem. 69 (1998) 67±77 Table 4 The values of A1 , k1 and k2 for kinetic curves of deplatination of adducts PtII (DAPO)±DNA under the action of 0.1 M NaCN at 20°C Platinating complex

r

A1

k1 (minÿ1 ) k2 (minÿ1 )

PtII (DAPO)(NO3 )2

0.18 0.08 0.04 0.21 0.11 0.08 0.03

0.75 0.42 0.36 0.70 0.49 0.33 0.29

0.006 0.016 0.016 0.010 0.006 0.017 0.018

PtII (DAPO)Ox PtII (DAPO)Cbdca

0.09 0.13 0.15 0.09 0.14 0.17 0.13

by reduction of nitroxyls. For the samples with r < 0.1, reduction prevails over deplatination; therefore, we observed only a decrease in the ESR signal. 4. Discussion The acidic hydrolysis data for modi®ed DNA show that PtII (DAPO)X2 complexes, like other cis-diaminoplatinum ones [10±12], platinate predominantly guanine and adenine. Reactions of DNA with the complexes give rise to formation of a mixture of adducts of general forÿ mula [DNA(PtII (DAPO))n ]2n‡ Xÿ 2n , where X stands for leaving ligands in the starting complexes, bu€er anions, or residues of phosphoric acid of DNA. Platination rates for PtII (DAPO)(NO3 )2 and PtII (DAPO)Ox are close to those for cisplatin (Fig. 2) and PtII (DACH)Cl2 [10]. Thus, four methyl groups at DAPO piperidine ring do not cause substantial steric hindrances for DNA platination as it might be expected from theoretic calculations for structurally similar complexes containing alkyl-substituted 1,2-diaminocyclohexanes as carrying ligands [13]. It is known that cis-diaminoplatinum complexes react with DNA to form ®rst monofunctional adducts, which rapidly and almost completely convert to bifunctional adducts [35]. In case of cisplatin, the fraction of the remaining monoadducts is less than 10% [11]. Our data show that PtII (DAPO)X2 complexes behave similarly. Degree of modi®cation r determined from acidic hydrolysis data is in good agreement with values determined by the ESR and AAS, only if complexes form predominantly bifunctional adducts (see Materials and Methods). The bifunctional PtII (DAPO)±DNA adducts contain no single bonds which allow rotational motion of nitroxyls with respect to DNA molecule. In monofunctional adducts such motion is possible. However, rotation of nitroxyls due to the presence of one single bond accelerates no more than by 2±10 times [37]. Therefore, small di€erence in the mobility of nitroxyls in mono- and bifunctional adducts, as well as a small fraction of monofunctional adducts, make almost impossible di€erentiation of these adducts by ESR spectra. The degree of platination is sensitive to the nature of leaving ligand and increases in the sequence

75

PtII (DAPO)Cbdca < cisplatin  PtII (DAPO)Ox < PtII (DAPO)(NO3 )2 . The rate of hydrolysis of PtII (DAPO)X2 to PtII (DAPO)(OH)2 increases in the same sequence. In water, the most active PtII (DAPO)(NO3 )2 forms aquacomplexes (Scheme 1), in which water molecules can be easily replaced by DNA bases. There is no common opinion in the literature on the in¯uence of leaving ligands X on the mechanism of DNA platination. Kinetic studies [22,23] unambiguously show that, in neutral and weakly acidic media, cisplatin reacts with DNA bases predominantly via [Pt(NH3 )2 Cl(H2 O)]‡ intermediate. Some researchers consider that dicarboxylate complexes (e.g. carboplatin) are actually latent forms of cisplatin and platinate DNA via the same intermediate [38,39]. But as it was shown recently, the rate of hydrolysis of carboplatin [16] and DWR-2114 complex [12], also containing Cbdca as living ligand, does not determine their reactivity and that for this kind of complexes a di€erent mechanism of binding to DNA must occur. The data on the binding of carboplatin to DNA in vitro and in cells also give evidence that carboplatin cannot be considered as just a slow-reacting substitute for cisplatin [40]. Our data show that dicarboxylate complexes, in particular PtII (DAPO)Cbdca, most probably react with DNA bases in the initial form. This suggests that the platination selectivity of complexes with strongly coordinated leaving ligands may di€er from that of easily hydrolyzed complexes. Indeed, for PtII (DAPO)Ox and PtII (DAPO)Cbdca at the low platination degree of G (10±20%, no saturation) the ratio GPt /APt strongly depends on the nature of leaving ligands (Table 2). Substantial di€erence in thermal stability of DNA modi®ed by di€erent PtII (DAPO)X2 complexes (Figs. 3(a) and 4(a)) can also be explained by the in¯uence of leaving ligands on the selectivity of platination. At low r, cisplatin, PtII (DAPO)(NO3 )2 and PtII (DAPO)Ox destabilize the duplex, whereas PtII (DAPO)Cbdca increases Tm and decreases DT (Fig. 4(a) and (b)). The PtII (DAPO) fragment can form inter- or intrastrand cross-links. The experiments on renaturation of melted modi®ed DNA show that the fraction of interstrand cross-links is 1% of the total number of adducts. This value agrees well with estimations for cisplatin [11,33,34]. Interstrand cross-links are scarce and, therefore, they will hardly a€ect the parameters of melting curves. For instance, the stabilizing e€ect of PtII (DAPO)Cbdca for r < 0.05 cannot be explained by the interstrand cross-linking. At p ˆ 0.01 and r < 0.05, the sequence of more than 1000 nucleotide pairs must contain but a single cross-link. The cooperativity length (the number of nucleotides at which the mutual correlation of melting links is lost) is several hundreds of nucleotide pairs [30,41]; therefore, such scarce interstrand cross-linking cannot a€ect melting of DNA fragments containing several thousands of nucleotide pairs. Therefore, thermal stability of modi®ed DNA depends mainly on intrastrand cross-linking.

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For modi®ed DNA, the average distance between nitroxyl groups determined by the ESR method at r P 0.1 (Table 3) is close to that calculated from r under the assumption of equal distances between nitroxyl groups. This implies a uniform arrangement of the adducts along DNA molecules at high degree of platination. But at r 6 0.05, the distance between nitroxyls is too large (L > 3.0 nm) to be accurately measured by the ESR. Thus, this does not exclude selective platination of DNA at low r. At r P 0.1, nitroxyl radicals approach each other to distances enabling the e€ective electron transfer. Therefore, in acidic media (pH 6 1), they undergo unusually rapid and reversible disproportionation to diamagnetic products. Hydrolysis rate and platinating activity of the complexes correlate well with their total toxicity. Toxicity increases in the sequence PtII (DAPO)Cbdca, PtII (DAPO)Ox, and PtII (DAPO)(NO3 )2 : LD50 doses (doses at which 50% of mice died) are 400, 30 and 11 mg/kg, respectively [7]. Platinating activity of cisplatin is intermediate between those for PtII (DAPO)Ox and PtII (DAPO)Cbdca (see Fig. 2). However, toxicity of cisplatin (LD50 ˆ 12 mg/kg [7]) is higher than that for PtII (DAPO)Ox. Antitumor properties of the complexes seem to have no direct correlation with their hydrolytic and platinating activity. One of the most reactive complexes, PtII (DAPO)Ox, appeared to be the most e€ective against mice leukemia L1210, whereas the least reactive and toxic PtII (DAPO)Cbdca appeared to be the most effective against mice leukemia P388 [7]. To conclude, paramagnetic properties of DAPO make possible to obtain by the ESR method new data on interaction of platinum complexes with DNA and on the structure and properties of formed adducts. Nitroxyl radicals were shown to be uniformly arranged along DNA molecule at as high bond Pt-to-nucleotide ratio as 0.1, but probably it is not so at lower degrees of platination. Two types of adducts with di€erent resistance to deplatination under the action of NaCN were revealed by the ESR. It was found that the guanineto-adenine platination rate ratio depends on the nature of leaving ligands. Furthermore, DNA modi®ed by different complexes to equal r exhibits di€erent thermodynamic characteristics. This means that strongly coordinated leaving ligands a€ect platination selectivity. Acknowledgements This work was supported by International Science and Technology Center, project No. 123-94. References [1] N.P. Konovalova, G.N. Bogdanov, V.B. Miller, E.G. Rozantsev, M.B. Neiman, N.M. Emanuel, Dokl. Akad. Nauk SSSR 157 (1964) 707. [2] M. Klimek, Nature 209 (1966) 1256.

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