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.     

RNA binding efficacy of theophylline,theobromine and caffeine

The binding of naturally occurring methylxanthines such as theophylline, theobromine and caffeine to nucleic acids are reckoned to be pivotal as they are able to modulate the cellular activities. We explore the interaction of yeast RNA binding efficacy of the above xanthine derivatives by using UV absorption differential spectroscopy and Fourier Transform Infrared (FTIR) spectroscopy. Both the analyses show discrimination in their binding affinity to RNA. The differential UV-spectrum at P/D 3.3 reveals the greater RNA binding activity for theophylline (85 +/- 5%), whereas moderate and comparatively less binding activity for theobromine (45 +/- 5%) and caffeine (30 +/- 5%) and the binding activity was found to depend on concentration of the drugs. In FTIR analysis we observed changes in the amino group (NH) of RNA complexed by drugs, where the NH band is found to become very broad, indicating hydrogen bonding (H-bonding) with theophylline (3343.4 cm(-1)), theobromine (3379.8 cm(-1)) and caffeine (3343 cm(-1)) as compared to the free RNA (3341.6 cm(-1)). Furthermore in RNA-theophylline complex, it is observed that the carbonyl (C=O) vibration frequency (nu(C=O)) of both drug (nu(C=O)=1718, 1666 cm(-1)) as well as RNA (nu(C=O)=1699, 1658 cm(-1)) disappeared and a new vibration band appeared around 1703 cm(-1), indicating that the C=O and NH groups of drug and RNA are effectively involved in H-bonding. Whereas in RNA-theobromine and RNA-caffeine complexes, we found very little changes in C=O frequency and only broadening of the NH band of RNA due to complexation is observed in these groups. The changes in the vibrations of G-C/A-U bands and other bending frequencies are discussed. Thus the discrimination in the binding affinity of methylxanthines with RNA molecule shows that strong RNA binding drugs like theophylline can selectively be delivered to RNA targets of microbial pathogens having the mechanism of RNA catalysis.     

Extraction and quantification of daunomycin and doxorubicin in tissues

The measurement of intracellular concentrations of the anti-cancer drug doxorubicin was performed by the application of a simple cell extraction technique combined with a rapid high-performance liquid chromatographic separation. Quantitation was done by fluorescence detection. The extraction procedure was non-degradative and the mean recovery of drug was 95%. A high drug extraction efficiency was confirmed with radiolabeled [³H]doxorubicin. The method is applicable to normal and neoplastic tissue.     

The enhancement of the mutagenic effect of ultraviolet radiation inEscherichia coli by caffeine and acriflavine

The mutational synergism of caffeine and acriflavine was studied in five types ofEscherichia coli mutants induced by u. v.-radiation. The following types of mutations were compared: streptomycinrresistance (strain B/r), streptomycin-independence (strain Sd-4), and reversions to prototrophy (strains WP-14 pro⁻, WP-2 try⁻, and WP-2 try⁻ hcr⁻). In all hcr⁺ strains tested the presence of caffeine or acriflavine in a post-irradiation plate medium slightly decreases the survival of u.v.-irradiated cells and increases considerably the frequency of induced mutations. The mutational synergism of caffeine and acriflavine in the str-r and str-i mutants is observed only within the range of low doses. The abovementioned dose-dependence of the synergistic effect is discussed from the point of view of qualitative difference between the premutational damage caused by low and high doses. The post-irradiation treatment by caffeine slightly increases the frequency of induced prototrophs also in the WP-2 hcr⁻ strain. This finding is explained by the inhibition of the residual HCR-activity of the strain. The post-irradiation mutational synergism of acriflavine was not found in the WP-2 hcr⁻ strain.     

Caffeine, pentoxifylline and theophylline form stacking complexes with IQ-type heterocyclic aromatic amines

Methylxanthines (MTX), in particular caffeine (CAF), are known as the most widely consumed alkaloids worldwide. Many accumulated statistical data indicate the protective effect of CAF intake against several types of cancer. One of the possible explanations of this phenomenon is direct non-covalent interaction between CAF and aromatic mutagen/carcinogen molecules through stacking (π-π) complexes formation. Here we demonstrate that CAF and other MTX, pentoxifylline (PTX) and theophylline (TH), form stacking complexes with carcinogenic imidazoquinoline-type (IQ-type) food-borne heterocyclic aromatic amines (HCAs). We estimated neighborhood association constants (K_AC of the order of magnitude of 10² M⁻¹) in neutral and acidic environment and enthalpy changes (ΔH values between −15.1 and −39.8 kJ/mol) for these interactions using UV-Vis spectroscopy, calculations based on thermodynamical model of mixed aggregation and titration microcalorimetry. Moreover, using Ames test with Salmonella typhimurium TA98 strain and recently developed mutagenicity assay based on bioluminescence of Vibrio harveyi A16 strain, we demonstrated a statistically significant reduction in HCAs mutagenic activity in the presence of MTX.     

The interaction of lysozyme with caffeine,theophylline and theobromine in solution

The interactions of lysozyme with caffeine (Caf), theophylline (Tph) and theobromine (Tbr) were investigated using UV–Vis absorption, fluorescence, synchronous fluorescence, and three-dimensional fluorescence spectra techniques. The results revealed that Caf (Tph or Tbr) caused the fluorescence quenching of lysozyme by the formation of Caf (Tph or Tbr)–lysozyme complex. The binding constants (K _A) and thermodynamic parameters (ΔG°, ΔH°, ΔS°) at two different temperatures, the binding locality, and the binding power were obtained. The results showed that the process of binding Caf (Tph or Tbr) to lysozyme was a spontaneous molecular interaction procedure and the hydrophobic and electrostatic interactions play a major role in stabilizing the complex; The distance r between donor (lysozyme) and acceptor (Caf, Tph or Tbr) was obtained according to fluorescence resonance energy transfer. The effect of Caf (Tph or Tbr) on the conformation of lysozyme was analyzed using synchronous fluorescence and three-dimensional fluorescence spectra techniques. The results showed that the binding of Caf (Tph or Tbr) to lysozyme induced some micro-environmental and conformational changes in lysozyme and disturbed the environment of the polypeptide of lysozyme.     

Simultaneous microdetermination of theophylline, caffeine and phenobarbital in blood collected on paper

A sensitive and selective gas chromatographic method has been developed for the simultaneous determination of theophylline, caffeine and phenobarbital. Blood collection is performed by dropping 30 μl of blood onto a disc of a special paper. Vinbarbital is used for quantitation by the internal standard method. The chromatographic separation is performed on a 3% OV-17 column, after pentylation of the methylxanthine and internal standard, and the compounds are detected with a nitrogen-sensitive detector.The sensitivity of the method allows the monitoring of theophylline therapy in premature newborns by the differential determination of caffeine and theophylline. The sampling method does not affect the accuracy and precision and is very suitable for the collection of small blood samples.     

Effect of caffeine and of pentoxifylline on the motility and metabolism of human spermatozoa

Human spermatozoa were washed and incubated with 6 mM-caffeine or 0.15-1.2 mM-pentoxifylline. Sperm motility was measured by time-lapse photography, the rate of glycolysis by the release of tritiated water from 1 mM-[3-3H]D-glucose and the rate of mitochondrial respiration by the release of 14CO2 from 1 mM-[U-14C]-L-lactate or 1 mM-[2-14C]pyruvate. Caffeine stimulated the majority of spermatozoa to convert from the 'rolling' to the 'yawing' mode of progression with a concomitant increase in lateral head displacement from 4.1 +/- 0.09 microns (343) to 6.7 +/- 0.25 microns (105) (mean +/- s.e.m. (number of spermatozoa)). There was a 45% decline in the percentage of progressively motile spermatozoa and a very small decrease in their velocity. Pentoxifylline had only a slight effect on lateral head displacement or percentage motility but produced a significant increase in velocity. Both compounds increased the rate of glycolysis by greater than 40% but elevated the rate of 14CO2 production to a smaller extent. The concentrations of ATP and ADP changed very little. We conclude that the glycolytic pathway in human spermatozoa can respond efficiently to changes in energy demand.     

Interaction of caffeine and of theophylline with liver glutamate dehydrogenase

Caffeine and theophylline inhibited the activity of rat liver glutamate dehydrogenase (GDH), but not that of beef liver GDH, in forward and reverse directions of the enzyme reaction. In the forward direction, approximately 16 mM caffeine or 16 mM theophylline inhibited 50 per cent of the rat liver GDH activity (I50); while in the reverse direction, the I50 of caffeine and theophylline was 15 mM and 8 mM, respectively. The inhibition produced by caffeine was cooperative in both directions, while that of theophylline was negatively cooperative in the forward direction and non-cooperative in the reverse. However, ADP reduced the inhibitory effect of caffeine and theophylline to the extent of 40% and 80%, respectively. The Ki values obtained for caffeine and theophylline were different in the presence of various concentrations of substrates and coenzymes. Based upon these data, we presume that certain subtle changes occurring in the conformation of the rat liver GDH (probably at the ADP/NADH site) in comparison with those of the beef liver GDH may be responsible for its inhibition by caffeine and theophylline.     

Effect of cyclic AMP,theophylline and caffeine on the glucose repression of sporulation inSaccharomyces cerevisiae

Summary Repression of the sporulation ability ofSaccharomyces cerevisiae by glucose present in the presporulation medium was studied. Glucose lowered sporulation ability when added to the presporulation medium containing yeast extract but did not do so when added to the presporulation medium without glucose. The glucose-repressed sporulation ability was recovered by the addition of cyclic AMP, and theophylline or caffeine to the presporulation culture. Theophylline promoted the action of cyclic AMP, but caffeine did not. The effect of caffeine to reverse glucose repression was greater than that of cyclic AMP and theophylline.     

Cilostazol and pentoxifylline decrease angiogenesis,inflammation, and fibrosis in sponge-induced intraperitoneal adhesion in mice

AimsAdhesion formation following abdominal intervention is an abnormal peritoneal healing process. Our aim was to investigate the effects of controlling adhesion development by inhibiting its key components (angiogenesis, inflammation and fibrosis) using phosphodiesterase (PDE) inhibitors.Main methodsTwo PDE inhibitors including cilostazol a PDE3 inhibitor (40 and 400 mg/kg), and pentoxifylline (PTX), a PDE 1–5 inhibitor (50 and 500 mg/kg) were used for a period of 7 days to inhibit angiogenesis, inflammation, and fibrosis in a murine model of sponge-induced peritoneal adhesion. Angiogenesis was assessed by hemoglobin content, vascular endothelial growth factor (VEGF) levels, and morphometric analysis. Accumulation of neutrophils and macrophages was determined by measuring myeloperoxidase (MPO) and N-acetylglucosaminidase (NAG) activities, respectively. Levels of TNF-α were also determined. Fibrosis was assessed by determining the amount of collagen in the implant; TGF-β1 levels in the implant were also measured.Key findingsOur results show that the treatments attenuated the main components of the adhesion tissue by reducing the amount of fibrovascular tissue that infiltrated the sponge matrix (wet weight). Hemoglobin content and VEGF levels were also decreased by approximately 40%. Neutrophil accumulation was unaffected by the compounds. However, NAG activity was reduced by pentoxifylline, but not by cilostazol. These compounds also decreased the levels of the pro-inflammatory and pro-fibrogenic cytokines TNF-α and TGF-β1, respectively, and collagen synthesis.SignificanceOur results suggest that cilostazol and PTX decreased the development of peritoneal adhesions in the model, which might be associated with cyclic nucleotide modulation. Therapies to intervene in these pathways may be beneficial for the prevention of these lesions.     

Fluorescence emission properties of 8-aza analogues of caffeine,theophylline, and related N-alkyl xanthines

Fluorescence emission properties of 8-azacaffeine, 8-azatheophylline and other N-alkylated 8-azaxanthines (8-azaXan) have been examined. It is shown that N-methylated 8-azaxanthines, as well as 8-azatheophylline, are highly fluorescent in aqueous medium as the neutral, and, in some instances, also as the monoanionic, forms. 8-Azacaffeine exhibits moderate emission, but its isomer, 1,3,8-trimethyl-8-azaXan, is highly fluorescent. All three 8-azaxanthines monomethylated on the triazole ring, as well as 8-azaxanthosine, exhibit increased acidity in the excited state. Some fluorescent pyrazolo[4,3-d]pyrimidine-5,7-diones, xanthine congeners of pyrazolo[4,3-d]pyrimidines, are also reported. Many of these are good fluorescent probes in enzymatic, receptor binding, and nucleic acid systems, some examples of which are presented. In particular, 8-azaXan is an excellent fluorescent probe for purine nucleoside phosphorylases, as a fluorogenic substrate in the reverse, synthetic pathway.     

Spectral Analysis of Naturally Occurring Methylxanthines (Theophylline,Theobromine and Caffeine) Binding with DNA

Nucleic acids exist in a dynamic equilibrium with a number of molecules that constantly interact with them and regulate the cellular activities. The inherent nature of the structure and conformational integrity of these macromolecules can lead to altered biological activity through proper targeting of nucleic acids binding ligands or drug molecules. We studied the interaction of naturally occurring methylxanthines such as theophylline, theobromine and caffeine with DNA, using UV absorption and Fourier transform infrared (FTIR) spectroscopic methods, and especially monitored their binding affinity in the presence of Mg²⁺ and during helix-coil transitions of DNA by temperature (T_m) or pH melting profiles. The study indicates that all these molecules effectively bind to DNA in a dose dependent manner. The overall binding constants of DNA-theophylline = 3.5×10³ M⁻¹, DNA-theobromine = 1.1×10³ M⁻¹, and DNA-Caffeine = 3.8×10³ M⁻¹. On the other hand T_m/pH melting profiles showed 24–35% of enhanced binding activity of methylxanthines during helix-coil transitions of DNA rather than to its native double helical structure. The FTIR analysis divulged that theophylline, theobromine and caffeine interact with all the base pairs of DNA (A-T; G-C) and phosphate group through hydrogen bond (H-bond) interaction. In the presence of Mg²⁺, methylxanthines altered the structure of DNA from B to A-family. However, the B-family structure of DNA remained unaltered in DNA-methylxanthines complexes or in the absence of Mg²⁺. The spectral analyses indicated the order of binding affinity as “caffeine≥theophylline>theobromine” to the native double helical DNA, and “theophylline≥theobromine>caffeine to the denatured form of DNA and in the presence of divalent metal ions.