The role of nick formation in delayed quinacrine mustard fluorescence in the C-heterochromatin of Apodemus argenteus.

Chromosomes stained with fluorochromes, including quinacrine mustard (QM), emit the brightest fluorescence immediately after exposure to excitation light, and the fluorescence gradually fades with an increase in exposure time. However, in the QM-stained chromosomes of the small Japanese field mouse Apodemus argenteus, most C-heterochromatic regions emit weak fluorescence immediately after exposure to blue light, and they become brightly fluorescent by prolonged exposure (delayed QM fluorescence). We proposed recently that the delayed QM fluorescence is somehow related to nicks produced in C-heterochromatic DNA by blue light irradiation. To test this possibility, we examined the chromosomal distribution of nicks by in-situ nick translation and changes, if any, in the QM fluorescence pattern after methylene blue (MB) -mediated photooxidation, which is considered to induce nicks in chromosomal DNA. It was found that C-heterochromatic regions fluoresced brightly without any delay after exposure to blue light, and that nicks increased considerably in the same regions after the MB-mediated photooxidation. It seems, therefore, that photooxidation and strand breaks in DNA (including nicks) are responsible for the induction of delayed QM fluorescence. Trypsin digestion, on the other hand, abolished delayed QM fluorescence. Thus, not only DNA but also chromosomal protein(s) are involved in the unusual sequence of QM fluorescence patterns in A. argenteus.

The delayed quinacrine mustard fluorescence from the C-blocks of Apodemus argenteus is due to the introduction of nicks into the DNA.

"Delayed QM-fluorescence" refers to the unusual kinetics of fluorescence from most of the C-heterochromatic regions of the chromosomes of the small Japanese field mouse Apodemus argenteus. When stained with quinacrine mustard (QM-stained), these C-heterochromatic regions emit weak fluorescence immediately after exposure to blue light (BL); they emit bright fluorescence within a few minutes; and the intensity of the fluorescence gradually decreases after maximum fluorescence has been recorded. To elucidate the mechanism of this phenomenon, we used acridine orange staining (AO-staining) and a modified version of the in situ nick-translation method. Focusing on the large C-heterochromatic region (C-block) of the X chromosome, we noted that AO-stained C-blocks emitted greenish fluorescence, while QM-stained and BL-exposed (QM-BL-processed) C-blocks emitted reddish fluorescence upon AO-staining after removal of QM. These findings suggested that the C-block DNA of A. argenteus might undergo a structural change, such as strand breaks, during QM-BL processing. Application of the modified in situ nick-translation method revealed the generation of an appreciable number of nicks in the C-block DNA by QM-BL processing. No such nick formation was observed in the C-blocks of three other mammalian species: Apodemus peninsulae, Microtus montebelli, and Urotrichus talpoides. Our findings support the hypothesis that nick formation due to exposure to BL might play a primary role in inducing delayed QM-fluorescence in the C-blocks of A. argenteus. On the basis of the present and earlier findings, we propose a probable mechanism for delayed QM-fluorescence in A. argenteus chromosomes.

Defective DNA cross-link removal in Chinese hamster cell mutants hypersensitive to bifunctional alkylating agents.

DNA repair-deficient mutants from five genetic complementation groups isolated previously from Chinese hamster cells were assayed for survival after exposure to the bifunctional alkylating agents mitomycin C or diepoxybutane. Groups 1, 3, and 5 exhibited 1.6- to 3-fold hypersensitivity compared to the wild-type cells, whereas Groups 2 and 4 exhibited extraordinary hypersensitivity (30- to 90-fold). Mutants from Groups 1 and 2 were exposed to 22 other bifunctional alkylating agents in a rapid assay that compared cytotoxicity of the mutants to the wild-type parental strain, AA8. With all but two of the compounds, the Group 2 mutant (UV4) was 15- to 60-fold more sensitive than AA8 or the Group 1 mutant (UV5). UV4 showed only 6-fold hypersensitivity to quinacrine mustard. Alkaline elution measurements showed that this compound produced few DNA interstrand cross-links but numerous strand breaks that were revealed by proteinase treatment. Therefore, the extreme hypersensitivity of mutants from Groups 2 and 4 appeared specific for compounds the main cytotoxic lesions of which were DNA cross-links. Mutant UV5 was only 1- to 4-fold hypersensitive to all the compounds. Repair kinetics of DNA interstrand cross-link production and removal was measured by alkaline elution for AA8 and mutants UV4 and UV5 after exposure to diepoxybutane. Although the initial number of cross-links was similar for the three cell lines, during 24-h incubation, the efficiency of removal of cross-links was lowest in UV4 and intermediate in UV5. These results suggest that the different levels of sensitivity of the five complementation groups to bifunctional alkylation damage are specifically related to different efficiencies of DNA cross-link removal. The phenotype of hypersensitivity to both UV radiation and cross-link damage exhibited by the mutants in Groups 2 and 4 appears to differ from those of the known human DNA repair syndromes.

Immunochemistry of conjugates prepared from serum albumins and acridine nitrogen mustards (ICR mutagens).

Antibodies elicited in rabbits by immunization with conjugates prepared from serum albumins and nitrogen mustard derivatives of quinacrine (atebrin) were found to have strong binding sites complementary to the quinacrine hapten. The characteristic absorption spectrum of quinacrine made possible accurate determinations of the antigen-antibody composition of the serological precipitates. Conclusive evidence that such antibodies, in addition to reacting with the quinacrine component of heterologous protein test conjugates, bind quinacrine itself, as well as closely related acridine haptens, was provided by quantitative inhibition studies. Atebrin and the hydroxy precursors of several heterocyclic nitrogen mustards caused more than a 50% inhibition of the antigen-antibody reactions. The antibodies elicited by the quinacrine-protein conjugates in ascites tumor-bearing mice substantially neutralized the antitumor effectiveness of the low dosages (0.5 to 2.0 mumol/kg) of the acridine nitrogen mustards that were required for a demonstration of chemotherapeutic activity. In contrast, nitrogen mustard, which has no quinacrine moiety, was not affected. Immunization with unaltered serum albumin had no influence on the activity of the acridine nitrogen mustards. Quantitative in vitro inhibition studies allowed satisfactory predictions in vivo immunological reactivity.

Acetylcholine receptor. Binding properties and ion permeability response after covalent attachment of the local anaesthetic quinacrine.

Membrane vesicles rich in nicotinic acetylcholine receptor prepared from Torpedo californica electric tissue have been irreversibly modified with quinacrine mustard, an alkylating derivative of the local anaesthetic quinacrine. The reaction blocked the ion channel regulated by the acetylcholine receptor. Acetylcholine still bound to the modified membrane vesicles with KD approx. 10(-8). The number of binding sites was reduced by up to 50%. Stopped-flow experiments showed that in contrast to what had been found with the reversibly binding quinacrine no fluorescence changes caused by energy transfer from the irradiated protein to the fluorescent local anaesthetic occurred after addition of agonist. This indicates that the conformational changes associated with the activation of the ion channel are blocked by the covalent reaction with quinacrine mustard. Analysis of the membrane vesicles by SDS-polyacrylamide gel electrophoresis showed that all polypeptide chains assumed to be part of the receptor complex had reacted with the mustard. Even small components, probably lipids, migrating with the dye front, showed fluorescence.

Inhibition of the bovine-heart mitochondrial F1-ATPase by cationic dyes and amphipathic peptides.

The bovine heart mitochondrial F1-ATPase is inhibited by a number of amphiphilic cations. The order of effectiveness of non-peptidyl inhibitors examined as assessed by the concentration estimated to produce 50% inhibition (I0.5) of the enzyme at pH 8.0 is: dequalinium (8 microM), rhodamine 6G (10 microM), malachite green (14 microM), rosaniline (15 microM) greater than acridine orange (180 microM) greater than rhodamine 123 (270 microM) greater than rhodamine B (475 microM), coriphosphine (480 microM) greater than safranin O (1140 microM) greater than pyronin Y (1650 microM) greater than Nile blue A (greater than 2000 microM). The ATPase activity was also inhibited by the following cationic, amphiphilic peptides: the bee venom peptide, melittin; a synthetic peptide corresponding to the presence of yeast cytochrome oxidase subunit IV (WT), and amphiphilic, synthetic peptides which have been shown (Roise, D., Franziska, T., Horvath, S.J., Tomich, J.M., Richards, J.H., Allison, D.S. and Schatz, G. (1988) EMBO J. 7, 649-653) to function in mitochondrial import when attached to dihydrofolate reductase (delta 11.12, Syn-A2, and Syn-C). The order of effectiveness of the peptide inhibitors as assessed by I0.5 values is: Syn-A2 (40 nM), Syn-C (54 nM) greater than melittin (5 microM) greater than WT (16 microM) greater than delta 11,12 (29 microM). Rhodamines B and 123, dequalinium, melittin, and Syn-A2 showed noncompetitive inhibition, whereas each of the other inhibitors examined (rhodamine 6G, rosaniline, malachite green, coriphosphine, acridine orange, and-Syn-C) showed mixed inhibition. Replots of slopes and intercepts from Lineweaver-Burk plots obtained for dequalinium were hyperbolic indicating partial inhibition. With the exception of Syn-C, for which the slope replot was hyperbolic and the intercept replot was parabolic, steady-state kinetic analyses indicated that inhibition by the other inhibitors was complete. The inhibition constants obtained by steady-state kinetic analyses were in agreement with the I0.5 values estimated for each inhibitor examined. Rhodamine 6G, rosaniline, dequalinium, melittin, Syn-A2, and Syn-C were observed to protect F1 against inactivation by the aziridinium of quinacrine mustard in accord with their experimentally determined I0.5 values.(ABSTRACT TRUNCATED AT 400 WORDS)

Reconstitution of active acetylcholine receptor by hybridisation of binding site-blocked with ion channel-blocked acetylcholine receptor protein.

The nicotinic acetylcholine receptor regulates the ion permeability of the postsynaptic membrane. This report presents evidence that the transmitter binding site and the ion channel may be located on distinct subunits. By hybridisation of receptor complexes, in which the transmitter binding site was blocked with complexes in which the ion channel was irreversibly inhibited, we reconstituted active acetylcholine receptor complexes. The reconstituted system was similar to the native receptor in its ability to regulate the ion permeability of lipid vesicles in response to nicotinic cholinergic effectors.

Quinacrine mustard and lipophilic cations inhibitory to both vacuolar H(+)-ATPase and F0F1-ATP synthase.

Various lipophilic cations, such as quinacrine mustard and dequalinium, which are known to inhibit mitochondrial F1-ATPase, strongly inhibited vacuolar H(+)-ATPase purified from bovine adrenal chromaffin granules. Quinacrine mustard bound irreversibly to vacuolar H(+)-ATPase subunit A, and the 115 kDa accessory polypeptide and dithiothreitol had no effect. The binding was competitively inhibited by chlorpromazine and quinacrine, and these compounds specifically reduced the amount of labeling of subunit A. Quinacrine mustard also prevented the binding of [alpha-32P]ATP to subunit A but had no effect on the binding of [3H]N-ethylmaleimide to either subunit A or the 115 kDa accessory polypeptide. These results suggest that the binding site of quinacrine mustard in subunit A is not related to the N-ethylmaleimide-binding site(s), which is important for activity.

The modulation of the DNA-damaging effect of polycyclic aromatic agents by xanthines. Part I. Reduction of cytostatic effects of quinacrine mustard by caffeine.

Recently, accumulated statistical data indicate the protective effect of caffeine consumption against several types of cancer diseases. There are also reports about protective effect of caffeine and other xanthines against tumors induced by polycyclic aromatic hydrocarbons. One of the explanations is based on biological activation of such carcinogens by cytochromes that are also known for metabolism of caffeine. However, there is also numerous data indicating reverse effect on cytotoxicity of anticancer drugs that inhibit the action of topoisomerase I (e.g. Camptothecin or Topotecan) and topoisomerase II inhibitors (e.g. Doxorubicin, Mitoxantrone or mAMSA). In this work we tested the hypothesis that the caffeine protective effect is the result of sequestering of aromatic mutagens by formation of stacking (pi-pi) complexes. As the models for the study we have chosen two well-known mutagens, that do not require metabolical activation: quinacrine mustard(QM, aromatic, heterocyclic nitrogen mustard) and mechlorethamine (NM2, aliphatic nitrogen mustard). The flow cytometry study of these agents' action on the cell cycle of HL-60 cells indicated that caffeine prevents the cytotoxic action of QM, but not that of NM2. The formations of stacking complexes of QM with caffeine were confirmed by light absorption, calorimetric measurements and by molecular modeling calculation. Using the statistical thermodynamics calculations we calculated the "neighborhood" association constant (K(AC)=59+/-2M(-1)) and enthalpy change (DeltaH(0')=-116cal mol(-1)); the favorable entropy change of complex formation (DeltaS(0')=7.72cal mol(-1)K(-1), due to release of several water molecules, associated with components in the process of complex formation). The Gibbs' free energy change of QM-CAF formation is DeltaG(0')=-2.41kcal mol(-1). We were unable to detect any interaction between NM2 and caffeine either by spectroscopic or calorimetric measurement. In order to establish, whether the intercalation of QM plays any role in cytotoxic effect we tested, as a control, non-alkylatiatig, but also intercalating QM derivative-quinacrine (Q). The later had no cytostatic effect on HL-60 cell even at there order of higher concentration than QM or NM2 but, similar to QM forms (which we demonstrated) stacking complexes with caffeine (K(AC)=75+/-3M(-1)). These results strongly indicate, that the attenuating effect of caffeine on cytotoxic or mutagenic effects of some mutagens, is not the results of metabolic processes in the cells, but simply the physicochemical process of sequestering of aromatic molecules (potential carcinogens or mutagens) by formation of stacking complexes with them. The caffeine may then act as the "interceptor" of potential carcinogens (especially in the upper part of digesting track where its concentration can reach the concentration of mM level). There is, however, no indication either in the literature or in our experiments that xanthines can reverse the damage to nucleic acids when the damage to DNA has already occurred.

Chlorophyllin protects cells from the cytostatic and cytotoxic effects of quinacrine mustard but not of nitrogen mustard.

Chlorophyllin (CHL), the sodium and copper salt of chlorophyll, is capable of inhibiting the mutagenic activity of many chemical compounds. Several mechanisms have been advanced to explain the antimutagenic activity of CHL, including its antioxidant properties and its ability to form complexes with mutagens. The present study was designed to reveal whether the heterocyclic aromatic nature of a potential mutagen is essential to its sensitivity to CHL. Toward this end, the inhibitory effect of CHL on two compounds of similar chemical reactivity (mustards), that either embodied an aromatic structure (quinacrine mustard; QM) or did not (nitrogen mustard; NM), were compared. Human leukemic HL-60 and breast carcinoma MCF-7 cells were treated with QM or NM in the absence or presence of various concentrations of CHL. Both QM and NM when administered for 1-2 h at micromolar concentrations exerted similar effects; both arrested cells in G2 phase of the cell cycle, induced apoptosis and reduced the clonogenicity of MCF-7 cells. The simultaneous addition of 0.22 M CHL to cultures receiving QM virtually abolished the QM-induced inhibition of cell growth and clonogenicity. In contrast, CHL had no effect on reducing the cytostatic or cytotoxic activity of NM. CHL alone, at a concentration of 0.22 M, had minimal effect on growth of HL-60 cells slightly perturbing their progression through G2. The results are consistent with the model that explains the inhibition of the activity of mutagens or antitumor drugs with aromatic structures by CHL as mediated by its ability to sequester these molecules within heterologous mutagen:CHL complexes that are maintained by stacking interactions. Therefore, excess of chlorophyll in the diet, by sequestering aromatic mutagens (or antitumor drugs with a heterocyclic structure, if taken orally), may inhibit their accessibility to cells, thereby reducing their activity.

Simultaneous detection of SCE and Q-bands on human chromosomes by a double-staining technique.

Human lymphocytes were cultured for two cell cycles in the presence of bromodeoxyuridine (BrdU), and the resulting metaphase chromosomes were first stained with quinacrine mustard (QM) and then, immediately afterwards, with Hoechst 33258, without any intermediate destaining. Both Q-banding patterns and sister chromatid differential staining were photographed subsequently on the same metaphase using two different filter blocks of the fluorescence microscope.

Karyotypic findings in a colonic villous adenoma.

Chromosomes from a patient with a villous adenoma of the colon were examined in detail with fluorescent banding. Of the cells analyzed, 73% had a modal chromosomal number of 48-52. The karyotypic findings show clonal evolution, an additional chromosome #8 being the most frequent abnormality, suggesting that this may be the primary chromosomal change, but other changes such as loss of a Y chromosome and the presence of marker chromosome #1 with duplication of the long arm also were observed. Previous cytogenetic data on large-bowel tumors is summarized. We hypothesize that this chromosomal evidence suggests that tubular adenoma, villous adenoma, and adenocarcinoma may be interrelated steps of a growth disorder.

Separation of human X- and Y-bearing sperm using percoll density gradient centrifugation.

Human X- and Y-bearing sperm, which were detected by the quinacrine mustard fluorescent staining method, were separated in density gradient centrifugation using Percoll (Pharmacia Fine Chemicals, Uppsala, Sweden). The apparent percentages of Y-bearing sperm in normo- and oligospermia were 46.7% +/- 3.4% (n = 13) and 46.6% +/- 1.2% (n = 10), respectively. The separation was observed to some extent in Ficoll Paque (Pharmacia Fine Chemicals); then it was further achieved using Percoll. After centrifugation, sperm were distributed widely on various interfaces of the discontinuous Percoll density gradient (1.06 to 1.11 gm/ml). The content of Y-bearing sperm in the fraction lighter than 1.06 gm/ml was 73.1% +/- 3.3% and was reduced gradually along the density increment. Finally, it was found to be 27.4% +/- 3.4% in the sediment (more than 1.11 gm/ml).

Interactions of chlorophyllin with acridine orange, quinacrine mustard and doxorubicin analyzed by light absorption and fluorescence spectroscopy.

The present study was designed to estimate the ability of chlorophyllin (CHL) to interact with two acridine mutagens, quinacrine mustard (QM) and acridine orange (AO), and with the antitumor anthracycline doxorubicin (Dox). To this end, aqueous solutions of QM, AO or Dox during titration with CHL were subjected to spectrophotometry and spectrofluorimetry to detect possible interactions between these reagents. The data indicate that CHL forms complexes with AO, QM or Dox in these solutions. The presence of the complexes was manifested by a bathochromic shift of the absorption spectra, as well as by strong quenching of the fluorescence of each of these mutagens in the presence of CHL. CHL, thus, may serve as an interceptor of these mutagenic acridines in different in vivo or in vitro applications. Its ability to interact with Dox may potentially be utilized to detoxify patients overdosed with this or similar drugs.

Facile preparation of 6-chloro-9-amino-2-hydroxyacridine, a urinary metabolite of quinacrine and quinacrine mustard.

6-Chloro-9-amino-2-hydroxyacridine was found to be a metabolite of both quinacrine and the antimalarial alkylating agent quinacrine mustard. Its structure was confirmed by a one-step reaction of quinacrine with 48 percent hydrobromic acid. The presence of this compound as a metabolite of quinacrine mustard suggests a possible in vivo activation mechanism for its antitumor activity and a pharmacological basis for its toxicity to the liver. In vitro experiments showed that this new compound does react with chromosomes and, therefore, can be both a useful chromosome stain and an intercalating agent.

Sister chromatid exchanges--a sensitive assay of agents damaging human chromosomes.

Sister chromatid exchange frequencies in human lymphocyte chromosomes are greatly increased by alkylating agents, but ionizing radiation has little if any such effect. Scoring these exchanges may provide a useful technique for exploring the mechanisms of chromosome breakage and repair.

Adaptive enhancement and kinetics of nucleotide excision repair in humans.

An adaptive response, low doses of a mutagen rendering cells more able to subsequently cope with higher doses of that or a related challenging mutagen, enhances nucleotide excision repair in human fibroblasts. After fibroblasts were flashed with 20 J/m2 of UVC, the cyclopyrimidine dimer frequency at any single dinucleotide position remained unchanged for several hours before abruptly displaying first order kinetics of repair. These kinetics were determined by ligation-mediated PCR along exon 9 of the human p53 gene. When a chronic dose of quinacrine mustard (QM) preceded the UVC challenge, the duration of the cyclobutane pyrimidine dimer (CPD) repair lags were reduced by a factor of three and the kinetic half-lives for CPD repair were reduced by a factor of three. The observed repair kinetics are consistent with the following model. The UVC dose required (K(m)) to generate a substrate concentration which half-saturates the cell's repair capacity is 3 J/m2 for the high affinity (6-4) photoproducts and greater than 100 J/m2 for the low affinity cyclobutane dimers. After 20 J/m2 of UVC, the repair enzyme is saturated with (6-4) photoproducts; these competitively inhibit CPD repair by binding all available repair enzyme. After the (6-4)s are repaired, the CPD concentration is less than K(m)(CPD) and so CPD repair kinetics initiate with first order kinetics. QM-induced enhancement, by increasing the concentration, Vmax, of repair enzyme, shortens the duration of (6-4) saturation and increases the rate constant for cyclobutane dimer repair. The data exactly fit the expectations from Michaelis kinetics. Transcription coupled repair is less amenable to Michaelis interpretations and enhanced global repair was almost as rapid as the slightly enhanced transcription coupled repair. We infer that repair enhancement is unable to proportionally increase the number of matrix attachment sites necessary for transcription coupled repair. Understanding competitive inhibition between adduct classes and adaptive enhancement of Vmax is important to understanding the effects of high doses of mutagen mixtures.

Alleviation of mutagenic effects of polycyclic aromatic agents (quinacrine mustard, ICR-191 and ICR-170) by caffeine and pentoxifylline.

Previous studies performed by others indicated that apart from its other biological effects, caffeine (CAF) may have a role in protection of organisms against cancer. However, biological mechanism of this phenomenon remained unknown. Recent studies suggested that caffeine can form stacking (pi-pi) complexes with polycyclic aromatic chemicals. Therefore, one might speculate that effective concentrations of polycyclic aromatic mutagens could be reduced in the presence of caffeine. Here we demonstrate that caffeine and another xanthine, pentoxifylline (PTX), effectively alleviate mutagenic action of polycyclic aromatic agents (exemplified by quinacrine mustard (QM), 2-methoxy-6-chloro-9-(3-(2-chloroethyl)aminopropylamino)acridine.2HCl (ICR-191) and 1,3,7-propanediamine-N-(2-chloroethyl)-N'-(6-chloro-2-methoxy-9-acridinyl)-N-ethyl.2HCl (ICR-170)), but not of aliphatic mutagens (exemplified by mechlorethamine), in the recently developed mutagenicity test based on bacterium Vibrio harveyi. Biophysical studies indicated that caffeine and pentoxifylline can form stacking complexes with the aromatic agents mentioned above. Molecular modeling also confirmed a possibility of stacking interactions between examined molecules.

Comparative mutagenicity of 4 DNA-intercalating agents in L5178Y mouse lymphoma cells.

The mutagenicity of 4 known intercalating agents acridine orange (AO), quinacrine mustard (QM), proflavin (PF) and ethidium bromide (EB) has been investigated in L5178Y mouse lymphoma cells. Methyl methanesulfonate (MMS) was used as a positive control in these studies. AO, QM and PF induced mutation in the excess thymidine- and thioguanine-selective systems. These 3 compounds were negative in the ouabain- and cytosine-arabinoside-selective systems while EB was positive only in the cytosine arabinoside system. It would appear that the EB-induced mutagenesis is different from that of AO, QM and PF though all are intercalating agents. Since the molecular origin of cytosine arabinoside mutants is unknown, further interpretation of the EB results is not possible.

Induction of sister-chromatid exchanges and exchange aberrations by UV light and quinacrine mustard in relation to chiasma formation in a standard line and two oligochiasmatic mutants of Vicia faba L.

The frequencies of sister-chromatid exchanges (SCE) and exchange aberrations (EA) in root tip cells were compared to chiasma formation in pollen mother cells of a standard line and two oligochiasmatic lines of Vicia faba L. SCE and EA were induced by UV light and quinacrine mustard. Between the lines SCE frequencies were not different. The background level of SCE was doubled after UV irradiation and 4 times higher after exposure to quinacrine mustard in all Vicia lines analysed. However, the induced frequencies of EA were found to be different under the same treatment conditions for the standard line and the oligochiasmatic mutants. Between the frequencies of induced EA and the frequencies of chiasmata a correlation could be shown. The relationship between the formation of SCE and EA due to the reduced ability of meiotic recombination in the mutants of Vicia faba is discussed.