Chromatographic analyses of Lavandula angustifolia and Rosmarinus officinalis extracts and their biological effects in mammalian cells and cell-free systems.

Knowledge of biological properties of natural compounds allows to understand their therapeutic value, efficacy and security. We investigated: composition of Lavandula angustifolia (LA) and Rosmarinus officinalis (RO) extracts, their antioxidant capacity, cytotoxicity and genotoxicity, their DNA-protective potential against DNA damage induced in hamster V79 cells by several genotoxins or in plasmid DNA by Fe2+ ions and activity of antioxidant enzymes in cells treated with these extracts. Higher cytotoxicity, observed at higher concentrations of extracts, was accompanied by the increased level of single-strand (ss) DNA breaks as well as formamidopyrimidine DNA glycosylase (Fpg) sensitive sites. LA and RO extracts were able to protect DNA of hamster cells as well as plasmid DNA against ss DNA breaks induced by genotoxins and Fe2+. LA extract mildly increased the activity of superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT), while RO extract decreased the activity of SOD, but increased the activity of CAT and GPx. Cell-free tests confirmed antioxidant activity of both extracts. The biological properties of LA and RO extracts showed that they could have a positive impact on human health.

Influence of different chemical agents (H2O2, t-BHP and MMS) on the activity of antioxidant enzymes in human HepG2 and hamster V79 cells; relationship to cytotoxicity and genotoxicity.

We investigated activities of antioxidant enzymes (AEs), superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT) in human HepG2 and hamster V79 cells treated with a scale of concentrations of hydrogen peroxide (H2O2), tert-butyl hydroperoxide (t-BHP) and methyl methanesulfonate (MMS). Cytotoxicity and genotoxicity of these substances were evaluated simultaneously. We have found out that H2O2, t-BHP and MMS predictably induce significant concentration-dependent increase of DNA lesions in both cell lines. Cytotoxicity detected in V79 cells with help of PE test was in a good conformity with the level of DNA damage. MTT test has proved unsuitable, except for MMS-treated V79 cells. Compared with human cells HepG2, hamster cells V79 manifested approximately similar levels of SOD and CAT but ten times higher activity of GPx. Across all concentrations tested the most significant increase of activity of the enzyme CAT was found in H2O2- and t-BHP-treated HepG2 cells, of the enzyme SOD in t-BHP- and MMS-treated V79 cells, and of the enzyme GPx in H2O2-treated V79 cells. We suggest that stimulation of enzyme activity by the relevant chemical compounds may result from transcriptional or post-transcriptional regulation of the expression of the genes CAT, SOD and GPx. Several authors suggest that moderate levels of toxic reactants can induce increase of AEs activities, while very high levels of reactants can induce their decrease, as a consequence of damage of the molecular machinery required to induce AEs. Based on a great amount of experiments, which were done and described within this paper, we can say that the above mentioned principle does not apply in general. Only the reactions of t-BHP affected HepG2 cells were consistent with this idea.

Carvacrol and rosemary essential oil manifest cytotoxic, DNA-protective and pro-apoptotic effect having no effect on DNA repair.

For several thousand years natural products were successfully used to treat a variety of diseases and to maintain health in humans, but until now it is not fully known what causes these medicinal effects. In our study we assessed the cytotoxic, DNA-protective and pro-apoptotic effect of two frequently occurring natural compounds, carvacrol and rosemary essential oil, on human hepatoma HepG2 cells. In addition we examined the in vitro incision repair activity of liver cell extracts prepared from hepatocytes isolated from Sprague-Dawley (SD) rats fed with water containing carvacrol or rosemary oil. Using conventional and modified single cell gel electrophoresis we proved that incubation of HepG2 cells with selected concentrations of carvacrol and rosemary oil significantly protected cellular DNA against two dangerous oxidative agents, hydrogen peroxide (H(2)O(2)) and 2,3-dimethoxy-1,4-naphthoquinone (DMNQ). It is interesting that despite this DNA protection, the addition of both volatiles to the drinking water of SD rats had no effect on incision repair capacity of hepatocyte extracts. In this paper we also showed that carvacrol and rosemary oil can trigger apoptotic cell death pathways in HepG2 cells, which is probably connected with their cytotoxicity.

Cytotoxic, anti-carcinogenic and antioxidant properties of the most frequent plant volatiles.

KEYWORDS: Flowers, berries, leaves, barks and roots of different plants have been used through the ages as a source of flavor in food and perfume preparations. The volatiles responsible for the flavor of botanicals can be extracted from the plant material as "essential oils" (EOs), called also volatile oils or ethereal oils. The term essential is intended to indicate that the oil is the fragrant essence of the plant from which it is extracted. EOs are constituted by hydrocarbons (monoterpenes and sesquiterpenes) and oxygenated compounds (alcohols, esters, ethers, aldehydes, ketones, lactones, phenols and phenol ethers). Of the numerous groups of naturally occurring compounds examined so far terpenes are known as fragrances and flavoring agents. The data reported in this review including the data obtained in our laboratory show that many of EOs exhibit a range of biological activities inclusive of antioxidative, anti-mutagenic and anti-carcinogenic activities. Most of them belong to phytochemicals with chemopreventive potential. On the other hand some herbal products can cause serious adverse effects. A complex research of toxic, genotoxic, anti-mutagenic and anti-carcinogenic effects of EOs is therefore very important.

Structure of flavonoids influences the degree inhibition of Benzo(a)pyrene - induced DNA damage and micronuclei in HepG2 cells.

Flavonoids are plant derivatives of flavone of which chemical structure is characterized by various degrees of hydroxylation and glycosidic substitution. In the present study we investigated the protective effect of two structurally different groups of flavonoids against-benzo[a]pyrene (B(a)P)-induced genotoxic effects on human hepatocellular carcinoma (HepG2) cells. The first group of flavonoids: fisetin, kaempferol, galangin, quercetin, and luteolin, hydroxylated at the 3´,4´-position on the B ring, 3 - position of C ring and on the A ring was able to inhibit significantly B(a)P-induced genotoxic effects in a greater degree than the second group of flavonoids: chrysin, 7-hydroxyflavone, 7,8-dihydroxyflavone and baicalein (hydroxylated on the A ring) which showed a statistically significant inhibition of genotoxicity mainly at higher concentrations (10 and 25 µM). The tenth flavonoid tested rutin, which contains hydroxyl group at the position 3 of C ring, substituted by the sugar rutinose, was not able to inhibit effectively genotoxic changes induced by B(a)P. Our results, obtained with help of micronucleus test and single cell gel electrophoresis (comet assay) suggest that inhibition of B(a)P-induced DNA lesions and micronuclei correlates with the structural arrangement and organization of the hydroxyl groups in the molecular structure of the flavonoids tested.

Ex vivo assessment of protective effects of carvacrol against DNA lesions induced in primary rat cells by visible light excited methylene blue (VL+MB).

Carvacrol belongs to frequently occurring phenolic components of essential oils (EOs) and it is present in many kinds of plants. Biological effect of this phenol derivative on human beings is however not sufficiently known. The present study was undertaken to evaluate the level of VL+MB-induced oxidative DNA lesions in hepatocytes and testicular cells (freshly isolated from control or carvacrol-watered rats) by the modified single cell gel electrophoresis (SCGE). The results showed that carvacrol significantly reduced the level of VL+MB-induced oxidized bases (EndoIII- and Fpg-sensitive sites) only in hepatocytes but not in testicular cells. Chromosomal aberration assay of primary hepatocytes, isolated from control or carvacrol-watered rats did not testify any genotoxic activity of carvacrol. We suggest that in vivo applied synthetic carvacrol, whose antioxidative activity was confirmed by DPPH assay, exhibits primarily a strong hepatoprotective activity against oxidative damage to DNA.

Effects of borneol on the level of DNA damage induced in primary rat hepatocytes and testicular cells by hydrogen peroxide.

The aim of this paper was to evaluate genotoxic effects of borneol and its ability to change DNA-damaging effects of H2O2 in rat hepatocytes and testicular cells. Both in vitro and ex vivo approaches were used in the case of hepatocytes. Testicular cells were tested only ex vivo, i.e. shortly after isolation from rats supplemented by borneol. Cytotoxicity of borneol increased in in vitro conditions in a concentration-dependent manner and it was associated with DNA-damaging effects at toxic concentrations. While non-toxic concentrations of borneol applied in vitro protected cells against H2O2-induced DNA damage and interfered only partly with rejoining of H2O2-induced DNA strand breaks, cytotoxic concentrations of borneol manifested synergy with H2O2, i.e. enhanced DNA-damaging effects of H2O2. On the other side, borneol given to rats in drinking water decreased the level of DNA damage induced by H2O2 in both hepatocytes and testicular cells. Our results show that though at higher concentrations (2-h treatment with >2 mM borneol >0.3084 mg/ml) borneol acts cytotoxically and genotoxically on primary hepatocytes cultured in vitro, if given to rats during 7 days in a daily concentration of 17.14 or 34.28 mg/kg it reduces genotoxicity of H2O2 in both hepatocytes and testicular cells.

Yeast cell wall polysaccharides as antioxidants and antimutagens: can they fight cancer?

Polysaccharides represent the major part of the yeast cell wall dry weight and build the skeletal carcass defining cell wall stability and cell morphology (beta-D-glucans) or constitute amorphous matrix and cell surface fibrous material (mannans and mannoproteins). It is known that yeast cell wall beta-D-glucans reveal immunomodulating properties, which allows for their application in anti-infective and antitumor therapy. Recent data also suggest that polysaccharides reveal antioxidant activity that can result in their protective function as antioxidants, antimutagens, and antigenotoxic agents. The paper provides a review of our continuing research involving water-soluble derivatives of beta-D-glucan isolated from the baker's yeast Saccharomyces serevisiae and of a glucomannan isolated from the industrial yeast Candida utilis. The results are confronted with the available literature data. The derivatives of beta-D-glucan demonstrated potent inhibitory effect on lipid peroxidation comparable to that of the known antioxidants and exerted DNA protection from oxidative damage. The free radical scavenging activity was confirmed by spin-trap electron paramagnetic resonance. Antimutagenic and antigenotoxic activity of the yeast polysaccharides was demonstrated using yeast, bacterial, and algal models. The derivatives of beta-D-glucan exerted potent enhancement of tumor necrosis factor alpha (TNF-alpha) released from murine macrophages and revealed synergistic effect with cyclophosphamide in the treatment of Lewis lung carcinoma and two types of lymphosarcoma in murine models. The results indicate significant protective antioxidant, antimutagenic, and antigenotoxic activities of the yeast polysaccharides and imply their potential application in anticancer prevention/therapy.

Carvacrol given to rats in drinking water reduces the level of DNA lesions induced in freshly isolated hepatocytes and testicular cells by H(2)O(2).

Carvacrol represents a very frequent constituent of essential oils and occurs in many kinds of plants. Though human beings comequite often into close contact with this phenol derivative, its biological effects are not sufficiently known. In this paper we investigated the influence of carvacrol given to rats in drinking water on resistance of their liver and testicular DNA against the oxidative agent hydrogen peroxide H(2)O(2). Carvacrol was dissolved in tap water and given to rats either in concentrations of 30 and 60 mg/1 kg/day during 7 days or in concentrations of 15 and 30 mg/1 kg/day during 14 days. Control animals were given tap water only. After the given time the rats were sacrificed and hepatocytes and testicular cells were isolated and treated with different concentrations of H(2)O(2) (0-250 microM, 5 min, on ice). Then the level of DNA lesions was detected by single cell gel electrophoresis. The results of both types of application of carvacrol showed that DNA of cells isolated from carvacrol-treated animals was significantly more resistant to damaging effects of hydrogen peroxide than DNA of control animals. We assume that the observed DNA-protective effects of carvacrol, which was given to rats during a short time of their life, could be associated with an increase of antioxidant activity of liver and testicular cells in these animals.

Reduction of genotoxic effects of N-nitrosomorpholine in human hepatoma cells and hamster lung cells by carboxymethyl chitin-glucan.

N-nitrosomorpholine (NMOR) belongs to the group of N-nitrosamines and represents a known hepatocarcinogen. Exposure to this compound is considered to be a potential health hazard to humans. There is, however, considerable evidence that the effect of many carcinogenic agents can be markedly influenced or altered by various natural substances. The objective of this study was to assess the DNA-protective and anticlastogenic effects of the derivative of a natural compound, carboxymethyl chitin-glucan (CM-CG), against genotoxicity of N-nitrosomorpholine (NMOR) in human hepatoma cells HepG2 and hamster lung cells V79 cultured in vitro. The exponentially growing cells were pre-treated during 24 h with three different concentrations of CM-CG (150, 750 and 1500 mg/ml) and then treated with different concentrations of NMOR. DNAprotective effects of CM-CG were evaluated by single-cell gel electrophoresis (SCGE, comet assay) and anticlastogenic effects by chromosomal aberration assay. At the SCGE assay a short-term (30 min) and at the chromosomal aberration assay a continuous treatment with NMOR was used. In both HepG2 and V79 cells pre-treated with CM-CG, a significant decrease of the percentage of DNA lesions induced by NMOR was observed along with a reduction of NMOR-induced chromosomal aberrations. We did not find any substantial differences between the genotoxic effects of NMOR on HepG2 and V79 cells, which have different histopathological origins and different levels of metabolizing enzymes. Three different concentrations of CM-CG exerted a similar protective effect against NMOR-induced DNA lesions and chromosomal aberrations in both HepG2 and V79 cells.

Comparative study of DNA-damaging and DNA-protective effects of selected components of essential plant oils in human leukemic cells K562.

Eucalyptol, carvacrol and thymol represent components of plant essential oils characterized by a wide range of biological effects toward microorganisms, fungi, insects, etc. However, till now only a few investigations have been carried out to study the effects of essential oils and their components on human cells cultured in vitro. The aim of our work was therefore to compare cytotoxic and DNA-damaging effects of eucalyptol, carvacrol and thymol on human leukemic K562 cells cultured in vitro and to investigate their possible protective (antioxidant) effects against hydrogen peroxide-induced DNA damage. Testing of cytotoxic activity was performed by the trypan blue exclusion technique. The amount of DNA lesions in K562 cells treated with the plant volatiles studied or their combinations with hydrogen peroxide (H2O2) was measured by alkaline single cell gel electrophoresis (SCGE; comet assay). We found out that eucalyptol, carvacrol and thymol differed in their cytotoxic and genotoxic effects on K562 cells. As a very important we consider the finding that carvacrol and thymol significantly reduced the level of DNA damage induced in K562 cells by the strong oxidant H2O2. Neither DNA-damaging nor DNA-protective effect was observed using eucalyptol pre-treatment of K562 cells. We assume that DNA-protective effects of carvacrol and thymol can be accompanied by their antioxidant action.

DNA-protective effects of two components of essential plant oils carvacrol and thymol on mammalian cells cultured in vitro.

Many components of essential volatile oils show antioxidant activity and may serve e.g. as a natural replacement of synthetic antioxidant food additives. However, it is important to evaluate such compounds also for their pro-oxidant and toxic properties as their plant origin doesn't secure their safety for living beings, including humans. The aim of this study was therefore to investigate cytotoxic, genotoxic and DNA-protective effects of the long-term (24 h) incubation of mammalian cells with two components of essential plant oils (carvacrol and thymol) in in vitro conditions. Cytotoxicity testing was in all cell lines (human hepatoma cells HepG2, human colonic cells Caco-2 and hamster lung cells V79) performed on the basis of trypan blue exclusion. Plating efficiency was evaluated only in V79 cells which manifest a high colony forming ability. The amount of DNA lesions induced in cells treated with hydrogen peroxide, carvacrol, thymol or combinations of carvacrol or thymol with hydrogen peroxide was measured by standard alkaline single cell gel electrophoresis in human cells HepG2 and Caco-2. Trypan blue exclusion test showed that carvacrol was mildly more cytotoxic than thymol and that Caco-2 cells were mildly more resistant to both carvacrol and thymol than HepG2 and V79 cells. At concentrations = IC20-40, the compounds studied did not induce DNA strand breaks either in human cells HepG2 or in cells Caco-2. Incubation of HepG2 and Caco- 2 cells in the presence of the whole scale of concentrations of carvacrol or thymol led in both cases to a significant protection of the cells studied toward DNA strand breaks induced by a potent oxidant hydrogen peroxide.

Reduction of DNA-damaging effects of anti-HIV drug 3'-azido-3'-dideoxythymidine on human cells by ursolic acid and lignin biopolymer.

In this study we verified our assumption that the genotoxicity of the effective anti-HIV drug 3'-azido-3'-dideoxythymidine (AZT) on human cells could be reduced by non-toxic concentrations of two antioxidants that occur frequently in nature (ursolic acid and lignin biopolymer). Cytotoxicity of these natural compounds, well-known by their antimutagenic effects, was evaluated by the trypan blue exclusion technique. Genotoxic activity of AZT was measured on the basis of AZT-induced single and double strand breaks to DNA in two histopathologically different types of human cells, hepatoma cells HepG2 and colonic cells Caco-2. Induction of DNA strand breaks was measured by the comet assay processed in parallel at pH > or = 13.0 (standard alkaline technique which enables to recognize single strand DNA breaks of different origin) and at pH = 9.0 (neutral technique which enables to recognize double strand DNA breaks). As the level of AZT-induced double strand DNA breaks was rather low, protective effects of the antioxidants tested were evaluated only against AZT-induced single strand DNA breaks by the standard alkaline comet assay. Our findings showed that 1 h pre-incubation of cells with ursolic acid or lignin preceding to 3 h treatment of cells with AZT (3 mg/ml) significantly decreased in both cell types the level of AZT-induced single strand DNA breaks. Pre-incubation of HepG2 or Caco-2 cells with a mixture of both natural antioxidants did not increase the effects of individual treatments. This study confirms that AZT is genotoxic toward both used cell types of human origin and that ursolic acid and biopolymer lignin can protect the cells studied against genotoxic effect of AZT.

Carboxymethyl chitin-glucan enriched diet exhibits protective effects against oxidative DNA damage induced in freshly isolated rat cells.

The connection between dietary intake of carboxymethyl chitin-glucan (CM-CG, approximately 200 mg/kg body weight, during 21 days) and the response of freshly isolated rat cells to genotoxic treatment with a combination of photosensitizer Methylene Blue and visible light (MB+VL) was evaluated in presented study. Blood lymphocytes, testicular cells, and hepatocytes were isolated from rats fed by a standard or CM-CG enriched diet and in ex vivo conditions challenged with oxidative agent. Induced DNA damage was assessed using a modified comet assay. When added to the diet, CM-CG itself did not induce any negative effect on the health condition of animals or on level of DNA breaks in rat cells. Moreover, the cells isolated from CM-CG fed animals were more resistant to oxidative stress induced by visible light-excited Methylene Blue. In conclusion, we have demonstrated that carboxymethyl chitin-glucan represents a natural fungal polysaccharide that is able to exert antimutagenic properties upon application in diet.

Diet containing fungal (1-->3)-beta-D-glucan derivative exhibits protective effects against DNA lesions induced in freshly isolated rat cells.

Dietary effect of water-soluble derivative - carboxymethyl chitin-glucan (CM-CG) on the level of DNA lesions induced by hydrogen peroxide (H2O2) was examined in ex vivo experiments. Lymphocytes, testicular cells, alveolar macrophages and epithelial II cells were isolated from Sprague Dawley rats fed a common or CM-CG enriched diet (200 mg/kg of body weight) during 21 days. Freshly isolated cells were in in vitro conditions exposed to H2O2 and the levels of DNA breaks were evaluated by single cell gel electrophoresis. A dose-dependent increase of DNA breaks was observed after treatment with hydrogen peroxide in all studied cell types. The levels of DNA breaks in cells isolated from CM-CG supplemented animals were lower compared to the levels of DNA breaks in cells isolated from animals fed a common diet. Intake of CM-CG enriched diet did not increase the level of DNA damage in different kinds of freshly isolated rat cells and equipped the cells with resistance to the treatment with hydrogen peroxide.

Repair of oxidative DNA lesions in blood lymphocytes isolated from Sprague-Dawley rats; the influence of dietary intake of lignin.

Living organisms possess a variety of self-protective mechanisms which decrease the free radical attack on DNA and so reduce the risk of cancer. Protection of DNA by endogenous antioxidant systems may be significantly increased by numerous exogenously administered antioxidants. Many of them represent important dietary factors. Biopolymer lignin with its phenolic structure can be included into this group of micronutrients. The aim of the present work was to investigate: 1. the effect of biopolymer lignin, given to Sprague-Dawley (SD) rats in diet, on the level of oxidative DNA lesions induced by oxidative stress in freshly isolated peripheral blood lymphocytes in vitro and 2. the influence of lignin on kinetics of rejoining of DNA strand breaks induced in lymphocytes under these conditions. As model oxidative agents were used H2O2 and visible light in the presence of the photosensitizer Methylene Blue. We found out that dietary intake of lignin caused a significant decrease of H2O2-induced DNA strand breaks and visible light-induced oxidative DNA lesions in freshly isolated rat lymphocytes, but it did not influence the kinetics of rejoining of DNA strand breaks.

Induction of cytotoxicity and ssDNA breaks by 9-bromo-5-morpholino-tetrazolo[1,5-c]quinazoline in tumor cells cultured in vitro.

9-Bromo-5-morpholino-tetrazolo[1,5-c]quinazoline (BMTQ) acted cytotoxically on murine leukemia cell line L1210 and human colon carcinoma cells Caco-2. We found the two highest concentrations of BMTQ (149.2 and 74.6 microM) induced an acute cytotoxic effect, however other tested concentrations (<74.6 microM) manifested a concentration/dependent and time/dependent cytotoxic effect. The sensitivity of murine leukemia cells L1210 and human colon carcinoma cells Caco-2 was expressed in the same order. The cytotoxicity of BMTQ was not accompanied by changes of the cell cycle profile. Following the cytotoxicity-related effects of BMTQ we observed the induction of ssDNA breaks after BMTQ treatment. All the concentrations of BMTQ increased the level of ssDNA breaks 1.3-2.9 times (after 2 h of treatment) and 1.6-2.8 times (after 4 h of treatment) in Caco-2 cells compared to the control. No apoptotic DNA fragmentation induced by BMTQ in Caco-2 cells was recorded.

Chromosomal aberrations, sister chromatid exchanges and micronuclei induced by pentoxifylline in in vitro cultivated Chinese hamster cells (V79) and human blood lymphocytes.

Pentoxifylline (PTX) is a methylxanthine widely used in clinical practice. The mechanism of PTX effects on cellular and molecular level have not been fully explained yet. The present study was carried out to investigate the cytogenetic effect of this drug using cultured Chinese hamster V79 cells and human blood lymphocytes in vitro. The occurrence of chromosomal aberrations (CA), sister chromatid exchanges (SCE) and micronuclei (MN) was observed after the treatment of cells by different concentrations (0.002-2.0mg/ml) of PTX. In exposed V79 cells and lymphocytes as well, the dose-dependent increases of the above mentioned cytogenetic endpoints were found. The statistically significant increase has appeared at lower PTX concentrations in human lymphocytes than in V79 cells in all the investigated parameters. Our results show that, the applied concentrations of PTX has the clastogenic effect on in vitro cultured V79 cells and human lymphocytes. These findings are notable because of the frequent use of this drug and may serve as preliminary data to the further detailed examination of PTX action on molecular level.

Induction of micronuclei by 7H-dibenzo[c,g]carbazole and its tissue specific derivatives in Chinese hamster V79MZh1A1 cells.

The clastogenicity/aneugenicity of N-heterocyclic polycyclic aromatic pollutant 7H-dibenzo[c,g]carbazole (DBC) and its two synthetic derivatives N-methyl DBC (MeDBC) and 5,9-dimethyl DBC (diMeDBC) was evaluated in the genetically engineered Chinese hamster V79 cell line V79MZh1A1 with stable expression of human cytochrome P4501A1 and in the parental V79MZ cell line without any cytochrome P450 activity. While none of the three carbazoles changed significantly the level of micronuclei in the parental V79MZ cells, a variable, but statistically significant rise of micronucleus frequencies was assessed in V79MZh1A1 cells. DBC induced dose-dependent increase in the number of micronuclei at harvest times of 24 and 48h and MeDBC at sampling time of 48h in V79MZh1A1 cells in comparison to untreated cells, however, no significant time-dependent increase in micronucleus frequencies was found. The use of the antikinetochore immunostaining revealed that DBC and MeDBC induced approximately equal levels of both kinetochore positive (C+) and kinetochore negative (C-) micronuclei. DiMeDBC, a strict hepatocarcinogen, did not manifest any effect on micronucleus induction in V79MZh1A1 cells. These studies suggest that genetically engineered Chinese hamster V79 cell lines expressing individual CYP cDNAs are a useful in vitro model for evaluation the role of particular cytochromes P450 in biotransformation of DBC and its tissue and organ specific derivatives.

Contemporary trends in in vivo and in vitro testing of chemical carcinogens.

In the sixties of the last century it was realized that many human cancers are caused by environmental carcinogens and that the best way how to reduce cancer is first to identify in environment chemical carcinogens and second to prevent people from being exposed to such carcinogens. Epidemiological studies are probably the only way to confirm human carcinogenesis, however, this approach is so retrospective that carcinogens can be identified only after many victims have appeared. Carcinogenicity testing in long-term, medium-term, and short-term studies is therefore the only way for the prospective identification of possible human carcinogens. End-points of interest in a carcinogenicity study are primarily preneoplastic and neoplastic changes, but also include degree of malignancy, time to tumor appearance, multiplicity of (pre)neoplasia, and occurence of metastases. Long-term bioassays are designed and conducted to detect all of these end-points. Medium-term bioassays are mainly based on the detection of putative preneoplastic lesions and short-term tests can provide very important information concerning genotoxic effects of studied compounds.