Thermodynamical model of mixed aggregation of ligands with caffeine in aqueous solution. Part II.

A statistical-thermodynamical model of mixed association in which one component's self-association is unlimited while the second component does not self-aggregate is described. The model was tested with 4',6-diamidino-2-phenyl-indole-dihydrochloride (DAPI) and ethidium bromide (EB) using light absorption spectroscopy and calorimetry. The system is controlled by two parameters, which represent self-aggregation 'neighborhood' association constant KCC and mixed 'neighborhood' association constant KAC. Calculated, using this model, KAC = 58.2 +/- 1 M-1, KAC = 64.6 +/- 2 M-1 for DAPI and EB, respectively, are in good agreement with known values of stacking interactions. The titration microcalorimetric measurement of DAPI-CAF interaction delta H = -11.1 +/- 0.4 kcal/mol is also consistent with this type of reaction. The structures of the stacking complexes were also confirmed by semi-empirical molecular modeling in the presence of water. The data indicate that CAF forms stacking complexes with DAPI and EB, thus effectively lowering the concentration of the free ligands in the solution, and therefore, CAF can be used to modulate aromatic compound activity.

DAPI: a DNA-specific fluorescent probe.

DAPI (4',6-diamidino-2-phenylindole) is a DNA-specific probe which forms a fluorescent complex by attaching in the minor grove of A-T rich sequences of DNA. It also forms nonfluorescent intercalative complexes with double-stranded nucleic acids. The physicochemical properties of the dye and its complexes with nucleic acids and history of the development of this dye as a biological stain are described. The application of DAPI as a DNA-specific probe for flow cytometry, chromosome staining, DNA visualization and quantitation in histochemistry and biochemistry is reviewed. The mechanisms of DAPI-nucleic acid complex formation including minor groove binding, intercalation and condensation are discussed.

Diagnostic value of 169Yb-cis-dichlorodimethionine platinum scintigraphy in patients with malignant lymphomas.

Ytterbium-169 (169Yb) cis-dichlorodimethionine platinum (169Yb-PtCl2Meth2) is a new agent of low toxicity with an affinity for neoplastic tumours. The aim of this study was to assess the diagnostic value of 169Yb-PtCl2Meth2 scintigraphy in patients with malignant lymphomas. 169Yb-PtCl2Meth2 scintigraphy was performed in 67 patients with histologically proven malignant lymphoma. Liver, spleen, kidney and bone were among the normal tissues that showed uptake of the radiopharmaceutical. The compound is excreted in the main through the urinary tract. There was no significant activity in the bowels. For evaluation of the images, uptake of activity was scored according to a 5-point scale. In total, 888 sites were evaluated. The sensitivity, specificity and accuracy of lymphoma detection for the body as a whole was 80, 89 and 87%, respectively. The best results were obtained for the head (sensitivity 91%, specificity 89%), neck (sensitivity 84%, specificity 90%) and chest (sensitivity 82%, specificity 84%). In the sub-diaphragmatic region, sensitivity and specificity were 76 and 84%, respectively. Scintigraphy with 169Yb-PtCl2Meth2 appears to be a sensitive, non-invasive procedure for the staging of malignant lymphomas. The results suggest that it is possible to monitor the therapy of malignant lymphoma by means of 169Yb-PtCl2Meth2 scanning.

Experimental toxic liver damage and hepatic plasma clearance of 99mTc-mebrofenin (iminodiacetate derivative). III. Chronic CCl4-induced liver damage with eventual cirrhosis in rabbits.

Chronic damage to liver parenchyma was induced in rabbits by the long-term administration of carbon tetrachloride. The animals were serially sacrificed 3, 6 and 9 months after the start of intoxication, and examined histopathologically. The biological response was qualitatively assessed from results of histological studies, and measured utilizing series of typical biochemical indices of liver damage, 99mTc-mebrofenin (an-IDA-derivative) plasma clearance by the liver, and quantified indices of uptake and organ transfer of the compound. It was found that the plasma clearance and transfer parameters show association with chronic liver damage. The reduction of plasma 99mTc-mebrofenin clearance in intoxicated rabbits was also associated with changes in the biochemical indices of liver function and damage.

Caffeine prevents apoptosis and cell cycle effects induced by camptothecin or topotecan in HL-60 cells.

Caffeine (3,7-dihydro-1,3,7,-trimethyl-1H-purine-6,6-dione; CAF) is known to potentiate the cytotoxic effects of DNA damaging agents such as ionizing radiation and alkylating agents. In contrast, however, the cytotoxic and cytostatic activity of aromatic, DNA-intercalating, DNA topoisomerase II inhibitors such as Adriamycin, ellipticine, or mitoxantrone are diminished in the presence of CAF. To resolve whether the protective effect of CAF is associated with a particular mechanism of drug interaction (e.g., intercalation into DNA, inhibition of DNA topoisomerase II), or the aromatic nature of the drug structure, per se, we have presently studied the effects of CAF on the cytostatic and cytotoxic action of camptothecin (CAM) and its less toxic but more water soluble derivative topotecan (TPT) on HL-60 human myelogenous leukemia cells: both drugs have aromatic structures but are nonintercalating inhibitors of DNA topoisomerase I. By using spectroscopy and titration microcalorimetry, we have also studied the direct interaction between CAF and TPT in solution. Low (20 nM) concentrations of CAM or TPT perturbed progression of HL-60 cells through S-phase, whereas higher concentrations (0.15 microM) of these drugs induced apoptosis; both effects were easily demonstrable after 4 h of treatment. When added simultaneously with CAM or TPT, CAF prevented both effects. The protective effect of CAF was concentration dependent and evident within the concentration range of 1-5 mM; nearly total protection was seen at a CAF concentration of 5 mM. The bathochromic and hypochromic shift in the absorption spectrum of the water soluble compound TPT upon addition of CAF indicated that CAF and TPT interact (stack) in a fashion similar to that previously observed for CAF and DNA intercalators. Microcalorimetric measurements of TPT titration with CAF indicate an exothermic reaction between these compounds (the enthalpy change was delta H degree = -4.2 kcal/mol), which is consistent with a stacking model of CAF-TPT interaction. Thus, the ability of CAF to protect HL-60 cells against the cell kinetic effects of CAM or TPT, as in the case of DNA intercalating topoisomerase II inhibitors, is most likely due to formation of complexes between CAF and these aromatic molecules, which result in reducing the effective concentration of the free form of these drugs available to the cells.

Thermodynamical model of mixed aggregation of intercalators with caffeine in aqueous solution.

Recently we presented evidence that some intercalating antitumor agents can form complexes with caffeine and that this process may be responsible for the modifying effect of caffeine on the pharmacological activity of these drugs (F. Traganos et al., Cancer Res. 51 (1991) 3682). Here we describe a statistical-thermodynamical model of mixed associations in which one component's self-association is limited to dimer formation while the second component has the ability of unlimited stacking. The system is controlled by three parameters which represent self-aggregation "neighborhood" association constants KCC and KAA and a mixed "neighborhood" association constant KAC. The model was tested using acridine orange and light absorption spectroscopy as an analytical method for detection of complex formation. The experiments performed at two NaCl concentrations (0.01 and 0.15 M) indicate interesting properties of the three-parameter system in which the first parameter (KCC) is practically independent of ionic strength, the second (KAA) is positively and the third parameter (KAC) is adversely affected by ionic strength.

Experimental toxic liver damage and hepatic plasma clearance of 99mTc-mebrofenin (iminodiacetate derivative). I. Early, acute CCl4-induced liver damage in rabbits.

Liver damage was induced in rabbits by graded doses of carbon tetrachloride. The damage was assessed by means of quantitative morphometry using necrosis and steatosis of the parenchyma as end points. Biological response was measured utilizing a series of typical biochemical indices of liver damage as well as plasma clearance by the liver 99mTc-mebrofenin (an IDA derivative) and quantified indices of uptake and organ transfer of the compound. No correlation was found between the CCl4 dose and its effect (i.e. degree of necrosis and steatosis of liver). However, a very strong negative correlation was found between the degree of necrosis in individual animals and value of the clearance. The extent of the damage was positively correlated with liver transfer rate of 99mTc-MBF. Plasma activity of ALAT, TG, GGTP and AspAT were higher in the poisoned animals, however, only increments of activity of GGTP, ALAT and LDH showed statistically significant correlation with the individually assessed damage (necrosis).

Experimental toxic liver damage and hepatic plasma clearance of 99mTc-mebrofenin (iminodiacetate derivative). II. Recovery from the acute, CCl4-induced liver damage.

Liver parenchyma damage was induced in rabbits by the administration of carbon tetrachloride. The animals were serially sacrificed 3, 10, 17 and 31 days post intoxication and examined morphometrically for the extent of necrosis, steatosis and balloon degeneration of hepatocytes. Biochemical indices of the liver damage were studied as well as hepatic clearance of blood plasma from Tc-99m complex of an IDA derivative Tc99m complex (99mTc-mebrofenin--99mTc-MBF) and its uptake and liver transfer characterizing parameters. It was found that toxic effects of CCl4 were conspicuous up to 10 days after administration of carbon tetrachloride. In that period elevated activity of AspAT, ALAT, GGTP and elevated cholesterol and triglycerides were found in the plasma. As in the first paper of this series of works there has been a highly significant statistical association between the Tc-MBF plasma clearance, the uptake and liver transfer of the compound and parenchyma damage in the organ.

Caffeine modulates the effects of DNA-intercalating drugs in vitro: a flow cytometric and spectrophotometric analysis of caffeine interaction with novantrone, doxorubicin, ellipticine, and the doxorubicin analogue AD198.

Exposure of L1210 cells to DNA-intercalating antitumor drugs Novantrone (mitoxantrone; 20 ng/ml), doxorubicin (0.5 micrograms/ml), ellipticine (5 micrograms/ml), or the doxorubicin analogue AD198 (0.4 micrograms/ml), for 1 h, results in inhibition of cell proliferation, arrest of cells in the G2 phase of the cell cycle, and an increase in the number of cells entering higher DNA ploidy. These effects are significantly reduced when 5 mM concentrations of the methylxanthines caffeine or pentoxifylline are present either simultaneous with, or, in some cases, when added for 1 h immediately following pulse exposure to the drug. Both caffeine and pentoxifylline alone (5 mM) have little effect on cell growth or cell cycle progression. The possible mechanism of cell protection against intercalating drugs provided by caffeine was studied spectrophotometrically by measuring the interaction between Novantrone and the caffeine chromophore and in a model system using permeabilized L1210 cells and measuring the effect of caffeine in reducing binding of the intercalating dye acridine orange to cellular DNA and RNA. The data indicate that the observed protection of cells against intercalating drugs by caffeine or pentoxifylline is most likely a consequence of the direct interaction between the methylxanthines and the planar aromatic molecules of the intercalating drugs: formation of caffeine-drug complexes in solution effectively lowers the concentration of the free drug and thereby reduces its pharmacological activity. The principle of selective entrapment of the intercalator by compounds like caffeine may be considered in designing strategies to modulate the activity of intercalating drugs in vivo, e.g., in lowering drug toxicity when inadvertently applied at too high doses.

Interactions of nucleic acids with fluorescent dyes: spectral properties of condensed complexes.

Interaction of cations with nucleic acids (NA) often results in condensation of the product. The driving force of aromatic cation-induced condensation is the cooperative interaction between ligand and single-stranded (ss) NA. This type of reaction is highly specific with regard to the primary and secondary structure of NA, and results in destabilization of the latter. The spectral properties of fluorescent intercalating and non-intercalating ligands [acridine orange, pyronin Y(G), DAPI, Hoechst 33258, and Hoechst 33342]-NA complexes were studied in both the relaxed and condensed form. The changes in absorption, excitation, and fluorescence emission spectra and fluorescence yield that followed the condensation were examined. Although some of these effects can be explained by changes in solvation of the fluorophore and its interaction with NA bases and the solvent, the overall effect of condensation on spectral properties of the complex is unpredictable. In particular, no correlation was found between these effects and the ds DNA binding mode of these ligands. Nevertheless, the spectral data associated with polymer condensation can yield information about the composition and structure of NA and can explain some nonspecific interactions of these probes.

Effect of derivatives of chrysophanol, a new type of potential antitumor agents of anthraquinone family, on growth and cell cycle of L1210 leukemic cells.

The new C-methyl modified derivatives of the anthraquinones chrysophanol and emodin, recently synthesized by us, are potentially bifunctional agents having the ability to intercalate to nucleic acids and also having alkylating properties. Two of these compounds, namely 3-(N,N-bis(2-chloroethyl)-amino)methyl-1,8-dihydroxy-9,10-anthraquinone (Compound 31.662) and its 1,8-di-O-methylated analog (Compound 31.655) have been presently tested on murine leukemic L1210 cells in vitro with respect to their cell cycle specificity. During the initial 24 h of treatment the cytostatic effects of the drugs predominated, manifesting as suppression of cell progression through S (especially through the early portion of S phase) and G2. After 24 h, the cytotoxic effects became apparent, and there was also the appearance of cells with doubled DNA content suggestive of either endoreduplication or impairment of cytokinesis; these cells at higher ploidy level were progressing through S and G2. The observed effects were time- and dose-dependent, occurring at 0.1-0.4 micrograms/ml concentration of 31.662 and 2.0-10.0 micrograms/ml of its methylated analog, either during continuous- or after a 4-h pulse-treatment. Modulation of the cell cycle by the studied drugs is similar to that generally caused by intercalators as well as alkylating agents. However, because no positive evidence of intercalation of the studied drugs to nucleic acids was found, it is possible that alkylation of DNA or other cell constituents may be the primary lesion(s) leading to perturbation of the cell cycle.

Intercalating agents with covalent bond forming capability. A novel type of potential anticancer agents. 2. Derivatives of chrysophanol and emodin.

Fifty-one new C-methyl-modified derivatives of the anthraquinones chrysophanol and emodin or their various methyl ethers were prepared for structure-activity relationship studies of anticancer activity against mouse leukemia L1210 and human leukemia HL-60 cells. Representative compounds were spectrophotometrically studied for their capacity to interact with natural and denatured DNA. In general, those anthraquinones bearing an amino function interact with DNA. 1,8-Dimethoxyanthraquinones are incapable of intercalating into DNA. 1- or 8-Monohydroxymono-methoxyanthraquinones, however, interact with DNA to some extent. No straightforward correlation is apparent between the DNA-affinity data of the compounds studied spectrophotometrically and their cytotoxic effects. Cytotoxic potencies of these compounds on cell growth inhibition during a 72-h period are inversely correlated to their potencies when inhibiting [3H]TdR incorporation into DNA during the initial 30 min of exposure. Surprisingly, some compounds that showed more cytotoxicity did not inhibit initial TdR incorporation (0-30 min), while some others that strongly inhibited TdR incorporation initially did not exhibit cytotoxicity in 72 h. The results suggest that the cytotoxicity produced by these compounds is time dependent and is not a direct result of initial inhibition of DNA replication.

The antitumor intercalating drug ditercalinium binds preferentially to RNA in Friend erythroleukemia cells.

Ditercalinium (DIT; NSC 335153), a 7H-pyridocarbazole dimer, was reported to be capable of binding with high affinity to DNA by bisintercalation. Both the cytostatic and cytotoxic effects of this drug have been attributed to its binding to DNA. DIT inhibits the growth and is cytotoxic to Friend erythroleukemia (FL) cells. When FL cells were treated with 0.5-2.5 microM DIT and then stained with acridine orange (AO), which differentially stains DNA and RNA, the green, orthochromatic fluorescence representing AO binding to DNA was unchanged, while the metachromatic red luminescence characteristic of AO binding to RNA was reduced by as much as 40% in 4 hr; the effect was DIT-concentration dependent. The reduction in RNA stainability by DIT in the absence of any significant decrease in RNA content, was also observed with another RNA-specific fluorochrome, pyronin Y (PY). These results indicate that in live cells DIT preferentially binds to RNA rather than DNA, preventing stainability of the former by the monointercalating dyes AO and PY. When FL cells were exposed to 10 microM DIT after being first permeabilized by ethanol, the subsequent stainability of DNA in these cells was reduced by up to 67% and RNA by up to 44%, indicating that under these conditions DIT binds to both DNA and RNA. This observation was confirmed by competition experiments between AO and DIT bound to DNA or RNA in permeabilized cells mixed with equivalent numbers of RNA-containing (DNase-treated) or DNA-containing (RNase-treated) cells, respectively. The mechanisms that protect DNA against binding by DIT in live cells are unknown but are lost in fixed cells and may be related to maintenance of cellular and/or nuclear membrane integrity. If the propensity for other intercalating drugs to bind to RNA in live cells is correlated with their antitumor activity as is DIT, the rationale for designing new drugs based solely on their affinity for DNA should be reevaluated.

Selective displacement of nuclear proteins by antitumor drugs having affinity for nucleic acids.

The nuclear chromatin binding sites of the antitumor drugs mitoxantrone, ametantrone, doxorubicin, mithramycin, and actinomycin D and the intercalating ligand ethidium were studied by polyacrylamide gel electrophoresis of the proteins released from rat liver nuclei in the presence and absence of these drugs in buffer of low ionic strength (10 mM NaCl). At 25-50 microM free ligand concentration, each drug produced a specific and reproducible pattern of extractable proteins of different molecular weight by (i) releasing new proteins, (ii) altering the quantity of particular extracted proteins, and/or (iii) selectively entrapping other proteins in the nuclei. Ethidium, up to 100 microM, did not affect release of proteins from the nuclei. These results indicate that each ligand either has different binding site(s) in chromatin or modulates chromatin structure in a specific way by changing the affinity of different sets of proteins for their respective binding sites, resulting in their selective extraction or entrapment. The lack of effect of ethidium indicates that intercalation of the ligand to DNA, per se, does not alter the release of nuclear proteins. If patterns of nuclear proteins selectively released or retained by antitumor drugs are found to correlate with biological activity, this type of analysis may be helpful in new drug design and screening.

Interactions of antitumor drug ditercalinium with nucleic acids in solutions and in situ.

Ditercalinium (7-H-pyridocarbazole dimer), a prospective antitumor drug, is a bis-intercalator characterized by a very high affinity towards nucleic acids. Specificity of binding of this drug has been studied using spectroscopy and flow cytometry. The competition experiments between the drug and 4',6-diamidino-2-phenylindole.2HCl (DAPI) or acridine orange indicate that in solution the drug had higher affinity to DNA whereas in situ it binds preferentially to RNA. The results suggest that despite their high affinity towards DNA, the intracellular target of some intercalating drugs may be RNA.

Condensation of DNA in situ in metaphase chromosomes induced by intercalating ligands and its relationship to chromosome banding.

Interactions of certain intercalating cationic ligands with nucleic acids result in the formation of products that undergo condensation and agglomeration; this transition in solution can be monitored by light-scatter measurements. In the present study, using such intercalators as the antitumor drug mitoxantrone or fluorochromes acridine orange and quinacrine, we induced condensation of DNA in situ in Chinese hamster chromosomes. The in situ products scattered light and could be detected by darkfield- or phase-contrast microscopy. In the darkfield the complexes had a characteristic granular appearance and often generated a banding pattern on the chromosomes. In contrast, condensation of DNA in situ by the nonintercalating polyvalent cations (Co3+, spermine4+), while enhancing the chromosome's image contrast, did not produce the granular products or the banding. The condensation of free DNA, single or double stranded, natural or synthetic, the latter of various base composition and configuration, was also measured in solution. The condensation in solution and in situ was observed at similar concentrations of the respective ligands. The intercalating dye ethidium bromide, which did not condense DNA in solutions of moderate and high ionic strength, also did not generate the granular products or banding on chromosomes. The data also show that both base composition and configuration are important factors in determining the sensitivity of DNA to condensation by particular intercalating ligands. The studies suggest that the phenomenon of DNA condensation by intercalating dyes, which shows a high degree of specificity with respect to primary and secondary structures of DNA, may be associated with mechanisms of chromosome banding induced by the intercalating thiazine dyes in Giemsa staining or by quinacrine. Observation of chromosome banding based on light-scatter detection in darkfield microscopy allows the study of interactions between DNA and the ligands that neither fluoresce nor generate colored products. This principle of chromosome "counter-staining" can be explored by flow cytometry.

Interactions of acridine orange with double stranded nucleic acids. Spectral and affinity studies.

Spectral properties of acridine orange (AO) alone or in complexes with natural and synthetic nucleic acids of various base composition have been studied in aqueous solutions by absorption and fluorescence spectroscopy. The dimerization constant and absorption spectra of the dye in monomeric and dimeric form were established; dimerization of AO resulted in quenching of its fluorescence. Complexes of the dye with synthetic nucleic acids differed in the degree of enhancement of fluorescence quantum yield, varying between 1.42 to 2.38 fold as compared to AO monomer; these differences, however, were not base-dependent. Affinity of the dye to natural and synthetic polymers was studied and analyzed using McGhee-von Hippel model of polymer-ligand interactions. Because the sterical requirement for intercalative binding assumes interaction of dye monomer, the correction for AO dimerization was made in all calculations. All studied DNAs (natural and synthetic ones, the latter being homopolymer pairs or alternating copolymers of A,T or G,C or I,C base composition) had similar intrinsic association constants (KI = 5 X 10(4) - 1 X 10(5), M-1) and binding site size (n = 2.0-2.4 b.p.). The exception was poly(dA).poly(dT), having KI = 1.2 X 10(4) and n = 19.3 b.p. The results of KI measurement for calf thymus DNA and AO in different sodium ion concentration were in good agreement with predictions of the counterion condensation theory. The intercalation of AO into DNA is discussed in view of recent theoretical models of DNA-ligand interactions.

Interactions of pyronin Y(G) with nucleic acids.

Spectral properties of pyronin Y(PY) alone or in complexes with natural and synthetic nucleic acids of various base compositions have been studied in aqueous solution containing 10 or 150 mM NaCl and 5 mM Hepes at pH 7.0. The dimerization constant (KD = 6.27 X 10(3), M-1) and the absorption spectra of the dye in monomeric and dimeric form were established. The complexes of PY with single-stranded (ss) nucleic acids show a hypsochromic shift in absorption, and their fluorescence is quenched by over 90% compared to free dye. In contrast, complexes with double-stranded (ds) RNA or DNA (binding by intercalation) exhibit a bathochromic shift in their absorption (excitation) spectrum, and their fluorescence is correlated with the base composition of the binding site. Namely, guanine quenches fluorescence of PY by up to 90%, whereas A, C, I, T, and U bases exert a rather minor effect on the fluorescence quantum yield of the dye. The intrinsic association constant of the dye to ds RNA (Ki = 6.96 X 10(4), M-1) and to ds DNA (Ki = 1.74 X 10(4), M-1) was measured in 150 mM NaCl; the binding site size was 2-3 base pair for both polymers. Implications of these findings for qualitative and quantitative cytochemistry of nucleic acids are discussed.