Mutagenic and carcinogenic potential of menadione.

Menadione (2-methyl-l,4-naphthoquinone) or vitamin K3 is a lipid-soluble substance and promotes the hepatic biosynthesis of blood clotting factors. Carcinogenic potential of menadione was determined by a DC polarography method in strictly anhydrous N,N-dimethylformamide (DMF) in the presence of alpha-lipoic acid. Superoxide anion formation was measured after incubation of rat lung, liver and kidney microsomes with menadione. The genotoxic potential of menadione was investigated using the unscheduled DNA synthesis (UDS) and alkaline elution assays. The parameter of potential menadione carcinogenicity tg alpha was 0.0025 indicating no carcinogenic activity of menadione. Superoxide anion was generated in a concentration- and time-dependent manner when menadione was incubated with microsomes. In the mammalian cells (A 549) used for alkaline elution and UDS assays, menadione was cytotoxic at concentrations above 20 nmol/ml. The use of S9 mix (metabolic activation) fractions decreased the cytotoxicity of menadione. In the concentration range of above 20 nmol/ml menadione was genotoxic in the UDS test in absence of metabolic activation. In the presence of metabolic activation the menadione-induced DNA damage and repair was greatly reduced. Treatment of A 549 lung cells with 4-nitroquinoline-N-oxide (NQO) caused significant formation of DNA single-strand breaks both in the absence and presence of metabolic activation. Treatment of A 549 lung cells with menadione caused formation of DNA single-strand breaks in the absence of S9 mix. In the presence of metabolic activation menadione caused no significant formation of DNA strand breaks. Menadione-induced DNA repair in A 549 cells was concentration-, time-, and temperature- dependent. Measurement of unscheduled DNA (UDS) synthesis (repair) following treatment with NQO and menadione yielded strong UDS responses in the absence of S9 mix. Taken together the results of these studies suggest the mutagenic potential of NQO and menadione. These results indicate that menadione undergoes redox cycling with formation of reactive oxygen species which cause DNA damage and repair without having a carcinogenic potential.

Electrochemical behavior and determination of tumor inhibiting or promoting activities of flavonoids.

This paper deals with determination of the tumor inhibiting or promoting activities of 11 flavonoids performed by DC polarography. Flavonoids were tested in the presence of polyaromatic carcinogen 7,12-dimethylbenz(a)anthracene (DMBA) or in combination with DMBA and 12-O-tetradecanoylphorbol-13-acetate (TPA), which is known as a specific tumor promoter for epidermal carcinogenesis. We found that in this experimental system the promotory or inhibitory activities of studied flavonoids depend on the number and position of hydroxyl groups in their chemical structures and are related to the polarographic behavior of these compounds. Flavonoids, which are hydroxylated at the ring B, are reduced in anhydrous DMF on a mercury dropping electrode in two one-electron steps. Absence of the hydroxyl groups in these positions caused their reduction in three one-electron steps. Similarly, flavonoids with hydroxyl groups at the ring B have been shown to inhibit the activities of DMBA (2.7-45.9%) and of DMBA + TPA (47.2-78.2%). Missing hydroxyl groups caused weaker inhibitory activity against DMBA + TPA (19.01-38.74%) and the enhancement of DMBA activity (31.08-66.21%). Presented data demonstrated that the electrochemical method--DC polarography is very sensitive, simple technique for determination of the tumor inhibiting or promoting activities of the studied compounds.

Improved synthesis, antibacterial activity and potential carcinogenicity of 5-amino-1 ,2,4-thiadiazol-3(2H)-one.

This paper reports on the preparation of 5-amino-1,2,4-thiadiazol-3(2H)-one, a sulfur-containing analogue of cytosine with the -CH=CH- group between the positions 5 and 6 of the pyrimidine ring replaced by the divalent sulfur (-S-). Improved procedures for the preparation of thiobiuret, some of its methyl derivatives and 5-amino-1,2,4-thiadiazol-3(2H)-one are documented. Thiobiuret and its N-methyl derivatives were obtained by addition of hydrogen sulfide to the respective 1-cyanoureas. Subsequent oxidation of thiobiuret with hydrogen peroxide in alkaline medium produced 5-amino-1,2,4-thiadiazol-3(2H)-one. This substance was traced back converted to the starting thiobiuret by reaction with cysteine hydrochloride. Alkaline degradation of thiadiazol led to the formation of 1-cyanourea isolated as its silver salt. An investigation of the thiadiazol biological activities has shown that it inhibits the growth of E. coil by 10% at 8.5 microM concentrations, but exhibited no cytostatic activity in L1210, HeLa S3 and HL-60 cell lines. Potential carcinogenicity of the prepared compounds was determined by a DC polarographic method. While the values of the parameter of carcinogenicity for all intermediates indicate only marginal carcinogenic potential, the value of the parameter of carcinogenicity for the thiadiazole indicates possible carcinogenicity of this compound.

Pentacyclic triterpenoic acids: new chemoprotective compounds. Minireview.

Ursolic acid and oleanolic acid are pentacyclic triterpenoic acids having a similar chemical structure and are the major components of some oriental and traditional medicine herbs wildly distributed all over the world. There is a growing interest in the elucidation of the biological roles of both these triterpenoid compounds. This review summarizes the biological activities of presented triterpenoid acids (anti-inflammatory, hepatoprotective, gastroprotective, anti-ulcer, anti-HIV, cardiovascular, hypolipidemic, antiatherosclerotic and immunoregulatory effects). Our interest has been focussed especially on their anti-tumor and chemopreventive activity. Both compounds have been shown to act at various stages of tumor development, including inhibition of tumorigenesis, inhibition of tumor promotion, and induction of tumor cell differentiation. They effectively inhibit angiogenesis, invasion of tumor cells and metastasis. However, the mechanisms by which they act are poorly understood. Ursolic acid and oleanolic acid are relatively non-toxic and could be use as chemopreventive/chemoprotective agents in clinical praxis.

Determination of free radical scavenging activity of quercetin, rutin, luteolin and apigenin in H2O2-treated human ML cells K562.

We investigated protective effects of four flavonoids against H2O2- induced DNA damage in human myelogenous leukemia cells (K562) using the comet assay. The structural difference of studied flavonoids -- quercetin, rutin, luteolin and apigenin -- are characterized by the number of hydroxyl groups on the B ring. The presence of an o-dihydroxy structure on the B-ring confers a higher degree of stability to the flavonoid phenoxyl radicals by participating in electron delocalization and is, therefore, an important determinant for antioxidative potential. The results correlate with earlier published data obtained in murine leukemia cell line L1210. Hydrogen peroxide induced in human K562 cells a concentration-dependent increase of single cell DNA strand breaks. The strongest inhibition against H2O2-induced DNA damage (44%, 42%) was found in a range of luteolin and quercetin concentrations of 20-100 micromol/l. Protective effect of rutin (100 and 1000 micromol/l) was only marginal (8-10%). Apigenin had no protective effect on DNA single strand breaks induced by H2O2. Luteolin and quercetin are therefore effective in the protection of human single cell DNA from oxidative attack.

Taraxasterol and beta-sitosterol: new naturally compounds with chemoprotective/chemopreventive effects.

Substantial attention has been given to primary cancer prevention in daily life. Dietary factors are through to contribute to as much as one-third of the factors influencing the development of cancer. Ones of the components of a plant-based diet are beta-sitosterol and taraxasterol, compounds attracting our specific attention. This review summarizes the biological activities of presented phytosterols (anti-inflammatory, cholesterol-lowering, anti-microbial, anti-bacterial, anti-fungal effects). Our interest has been focussed especially on their anti-tumor and chemopreventive activity. They have been shown experimentally to inhibit colon and breast cancer development. They act at various stages of tumor development, including inhibition of tumorigenesis, inhibition of tumor promotion, and induction of cell differentiation. They effectively inhibit invasion of tumor cells and metastasis. With regard to toxicity, no obvious side effects of phytosterols have been observed in studies to date, with the exception of individuals with phytosterolemia. The exact mechanism by which dietary phytosterols act is not fully understood. However, some mechanisms have been offered. Therefore, they have a bright future in clinical application. Further investigation to explore their potential in tumor treatment may prove to be worthwhile.

The free radical scavenging activity of four flavonoids determined by the comet assay.

Flavonoids are reported to exhibit a wide variety of biological effects, including antioxidant and free radical scavenging activities. The aim of our study was to determine the cytotoxicity of flavonoids quercetin, rutin, apigenin and luteolin and their ability to protect DNA molecules against H2O2-induced damage. Cytotoxicity of studied flavonoids was tested in murine leukemia L1210 cells by the trypan blue exclusion technique. DNA strand breaks were determined using the alkaline single-cell gel electrophoresis (comet assay). Quercetin was found to possess the highest protective effect among the flavonoids studied (45%). The protective activity determined was lower for luteolin (40%). Protective effect of apigenin (600 microM/L) was only marginal (2%). However, at the higher concentration of apigenin (1200 microM/L), this flavonoid induced DNA single strand breaks. This indicates the ability of apigenin to serve as a pro-oxidant. Rutin had no protective effect on DNA single strand breaks induced by H2O2.

Physico-chemical properties and spectrophotometric determination of biologically active 1-alkyl-2-(2-pyridyl)pyridinium bromides.

Physico-chemical properties of compounds prepared from 2,2'-bipyridine, 1-alkyl-2-(2-pyridyl)pyridinium bromides, were investigated by DC polarography and by GC-MS. Their ionization potentials were calculated. Additionally, the formation of associates with bromothymol blue and methyl orange during the spectrophotometric determination was measured. It was determined that 1-alkyl-2-(2-pyridyl)pyridinium ions are reduced by 2 one-electron steps in a DC polarography system. The reduction potentials are not related to the ionization potential values calculated for the substances investigated. The carcinogenic potential (tg alpha) of the parent compound 2,2'-bipyridine and of a series of 1-alkyl derivatives was very low indicating that the compounds are not carcinogenic. The MS fragmentation patterns indicate the low stability of the 1-alkyl substituents. It was shown that 2,2'-bipyridine is either fragmented to two pyridine ions or the--N=CH--fragments are removed. Additionally, spectrophotometric determinations of colored associates of 1-alkyl-2-(2-pyridyl)pyridinium bromides with bromothymol blue and methyl orange were investigated and the optimal condiditions for these determinations are reported.

Comparison of the carcinogenic potential of streptozotocin by polarography and alkaline elution.

The carcinogenic potential of streptozotocin (STZ) was evaluated by the polarographic determination of its reduction potential in the presence of alpha-lipoic acid and detection of DNA single-strand brakes by alkaline elution. After STZ electrochemical reduction in an anhydrous solvent, the half-wave potential (E1/2 ) was determined to be -1.340 V. The parameter of the carcinogenic potential (tg alpha) for STZ was 0.400. This is in good agreement with WHO data regarding STZ carcinogenicity. Additionally, it is in the good agreement with the tg alpha value determined for the positive control used, N-nitroso-N-methylurea (NMU), which was found to be 0.459. The 3 hours exposure of A549 human lung tumor cells to 250, 500, and 1000 nmol/ml STZ was followed by DNA single-strand brakes detection using the alkaline elution method. NMU, the positive control, was tested under identical experimental conditions at the same concentrations. Without metabolic activation, NMU induced a significant formation of DNA single-strand brakes only at 1000 nmol/ml. In the presence of the metabolic activation, NMU caused a significant, concentration-dependent formation of DNA single-strand brakes. In the absence of metabolic activation, STZ induced no significant formation of DNA single-strand brakes at any concentration used. In the presence of metabolic activation, STZ caused a significant, concentration-dependent formation of DNA single-strand brakes. The results of this study underline the crucial role of using a metabolic activation system when carcinogenic potential of drugs and chemicals is investigated in vitro studies. Results of polarographic experiments and alkaline elution correlate well with each other and they indicate that these methods are useful to early predict the carcinogenic potential of STZ and other xenobiotics.

Rapid HPLC analysis of melphalan applied to hyperthermic isolation limb perfusion.

Hyperthermic isolated limb perfusion (HILP) with melphalan (MH) as a standard cytotoxic drug has been performed in 28 patients suffering from malignant melanoma. MH has been administered by HILP via extracorporeal circulation system. The drug given locoregionally reduces subsequent toxicity of organs. For all that residues can leak into the systemic circulation during HILP. Because of known carcinogenic potential and secondary cancer formation, the main interest of this work is to determine MH concentration profile in the patient plasma during and after HILP and evaluation of its potential toxicity in patients. Reversed-phase HPLC assay, which uses isocratic elution and fluorimetric detection has been shown to be sensitive, reliable and suitable for routine analyses. The assay was validated for the concentration range of 50-2500 ng.ml-1 with the limit of detection (LOD) 6.881 ng.ml-1. The samples were treated by methanol precipitation with the recovery more than 80%. The stability of standard solutions and methanolic extracts of MH were also followed. The concentration profile of MH in patient samples has been pursued in three time points during and after chemoperfusion (45 min after application of MH in extracorporeal circulation, 10 min after the joining the extremity to systemic circulation and one hour after the great vessels reconstruction). The concentrations of MH ranged 100-1500 ng.ml-1 and varied from patient-to-patient. Some complications were observed after HILP in 11 patients and are correlated with the higher con- centrations of MH (over 150 ng x ml-1) found in plasma.

Ursolic acid: an anti-tumorigenic and chemopreventive activity. Minireview.

Ursolic acid, UA, as a pentacyclic triterpene is of interest to scientists in the area of oncology because of its cytotoxicity, induction of differentiation, anti-mutagenic, antiviral and anti-invasive activities. UA is capable of inducing apoptosis in tumor cells on one side and to prevent malignant transformation of normal cells on the other side. It also interferes with numerous enzymes, including the ones serving directly to DNA synthesis. The aim of this review is to summarize reports on UA biological properties and to show its main anti-tumor effects and chemopreventive properties in normal cells.

Study of antioxidant effect of apigenin, luteolin and quercetin by DNA protective method.

A DNA protective capacity of three flavonoids, apigenin (AP), luteolin (LU) and quercetin (QU) against free radicals generated by H202, resp. Fe2+ is reported. This effect corresponding with scavenging of free radicals or with chelating of iron was assayed at two concentrations of flavonoids studied (1 microM and 10 microM). The quantitative analysis has shown that LU possesses the highest DNA protective effect of flavonoids investigated in the presence of H2O2. On the other hand, in the presence of 10 microM Fe2+, AP exhibited the highest DNA protective effect at the concentration of 1 microM and the following order was reached at the stoichiometric concentrations (10 microM) of Fe2+. It is believed that this discrepancy is caused by the ability of LU and QU iron-complex formation as it was separately investigated using UV-VIS spectrometry.

Flavonoids as chemoprotective agents in civilization diseases.

Flavonoids, a class of polyphenolic compounds widely distributed in the plant kingdom, are capable of protecting against several chronic and degenerative diseases, among them cancer, cardiovascular diseases, stroke, cataracts and brain and immune dysfunctional states. These substances are common dietary components. They exhibit many biological properties through various mechanism of activity. Early studies of flavonoids investigated their significant antioxidant, antitumor, anti-inflammatory, antiallergenic and hepatoprotective effect. Additionally, their ability to modulate the activity of various enzymes affects normal as well as malignant systems. This review summarizes data on the beneficial effects of flavonoids in humans.

Potential carcinogenic and inhibitory activity of compounds isolated from Lilium candidum L.

This paper deals with determination of potential carcinogenic and inhibitory activity of 10 compounds isolated from the ethanolic extract of Lilium candidum L. perfonned by DC polarography. The series of investigated compounds consisted of two spirostanol saponins, two pyroline derivatives, jatropham and its glucoside, flavonol kaempferol, 2-fenylethyl-alpha-L-arabinopyranosyl-(1-->6)-beta-D-glucopyranoside, 2-phenylethylpalmitate and methylsuccinic acid. Carcinogenic, resp. mutagenic activity data for these compounds, with the exception of kaempferol, are not available in scientific literature. All tested compounds were reduced in N,N-dimethylformamide in one or two well defined steps. They also fonned a reversible complex with alpha-lipoic acid, a compound serving as an indicator of carcinogenic activity. The marginal diffuse current values of these complexes served as a criterion of a very low potential carcinogenicity. The inhibitory activity of isolates were studied in the presence of 12-O-tetradecanoylphorbol-13-acetate, a specific tumor promoter for an epidermal carcinogenesis, and in the presence of a polyaromatic substance 7,12-dimetylbenz(a)anthracene known as a carcinogen. The spirostanol saponins possessed the highest inhibitory activity (> 70%) and jatropham (66%). The inhibitory activity of jatropham glucoside was significantly lower (49%). Practically zero inhibitory activity was measured for the 2-phenylethylpalmitate and methylsuccinic acid.

Tamoxifen in cancer therapy: minireview.

Tamoxifen belongs among relatively new drugs. As it has already been shown, it undoubtedly brings a benefit to oncology patients. However, there are still questions regarding its broader use in therapy or cancer prevention. This review puts together some data available at present time with the aim of elucidating the most important aspects of its use in medical oncology.

Determination of apigenin in rat plasma by high-performance liquid chromatography.

Apigenin (4',5,7-trihydroxyflavone, AP) belongs to a less-toxic and non-mutagenic flavone subclass of flavonoids, the biotransformation and metabolism of which have been little studied until now. Therefore, this study is focussed on the determination of AP in free form. AP was administered to rats via the i.p. route (25 mg kg(-1)) and then the blood was collected at 10, 15, 30 and 45 min after injection. Methanol was used for rat plasma deproteinization. The HPLC assay (mobile phase, 2% formic acid-acetonitrile-methanol, 40:35:25, v/v; flow-rate, 1 ml min(-1); UV detection at 349 nm) for AP determination was validated and used for the quantification of AP in rat plasma. The unknown concentration was calculated from the equation obtained by the least-squares regression analysis (y = 0.521x + 1.130, r2 = 0.998). The highest concentration of AP in plasma was found to be 30 min after injection. The concentration profile of AP obtained here may contribute to until known results about AP metabolism. They could be applied to other studies of AP or related flavonoids because of favourable effects on human health.

Potential carcinogenicity of the synthetic 1,3,6-triazine (6-azapyrimidine) nucleic acid analogues determined by DC polarography. II. Nucleosides of 6-azauracil.

The polarographic reduction of six synthetic 1,3,6-triazine (6-aza) nucleosides with 6-azauracil as the nucleoside base in the strictly anhydrous solutions was studied in the absence and presence of alpha-lipoic acid. The values of the half-wave potentials E1/2 and the parameter of potential carcinogenicity tg alpha were compared for six nucleosides of 6-azauracil and two nucleosides of 4-thio-6-azauracil. The current value of the first diffuse polarographic wave or a new diffuse polarographic wave belonging to the nucleoside-alpha-lipoic acid complex increased with the increase of the alpha-lipoic acid concentration for the all compounds only marginally. Although this diffuse current increase was linear and dependent on the alpha-lipoic acid concentration in anhydrous solutions, the determined index tg alpha values ranged between 0.027 and 0.114. This is an indication of a very low potential carcinogenicity of the all nucleoside analogues investigated.

Separation of structurally related flavonoids by GC/MS technique and determination of their polarographic parameters and potential carcinogenicity.

The present study deals with the investigation of the naturally occurring derivatives of the benzo[b]pyran-4-one - flavonoids - chrysin, tectochrysin and galangin, and with the effect of minor changes in their chemical structure on their separation using GC/MS. In the relation to their close chemical structure, their basic polarographic parameters were also investigated. Their potential carcinogenicity index tg alpha was determined by DC polarography experiments in the presence of alpha-lipoic acid. The tg alpha values for chrysin, tectochrysin and galangin were all under 0.180. This indicates a very minor carcinogenic potential that does not prevent the use of the investigated flavonoids in human.

Kojic acid--a new leading molecule for a preparation of compounds with an anti-neoplastic potential.

Kojic acid as a molecule of natural origin may serve as template for the synthesis of new biologically active compounds. The synthetic KA (pyranone) derivatives possess various kinds of biological activities which are related by their similarity to flavonoids. The most important property is the antifungal and antineoplastic activity and capability of chelating metals. It is shown that the antineoplastic activity of kojic acid derivatives is based on various mechanisms of action on different levels of cellular metabolism and functions what makes this compound interesting for future investigation as cytotoxic agent.

Effect of thioredoxin on the sensitivity of bacteria to chemical damage.

The ability of thioredoxin (Trx) to protect cells from chemical damage was determined by comparing the growth of a control strain of Escherichia coli JM101 and isogenic strain transformed with the plasmid pKKTS1 containing the Streptomyces aureofaciens thioredoxin gene, in the presence of the nucleoside analogs arabinosylcytosine, 5-fluorouridine, ftorafur and carcinogen beta-naftylamine. Arabinosylcytosine showed no effect on the growth of either of the two strains. 5-fluorouridine, ftorafur [1-((R,S)-tetrahydrofuran-2-yl)-5-fluorouracil] and beta-naftylamine demonstrated lower inhibitory effects on the growth of the thioredoxin overproducing strain than on the growth of the control strain. These results suggested that Trx could protect the cells from chemical damage under certain metabolic conditions.