Pro/Antigenotoxic Activity of Usnic Acid Enantiomers In Vitro.

The effect of usnic acid enantiomers on the genotoxic effects of dioxidine and methyl methanesulfonate was studied in vitro in human peripheral blood lymphocytes by the DNA comet method. We found that usnic acid enantiomers in a concentration range of 0.01-1.00 µM demonstrated pronounced antigenotoxic activity and reduced DNA damage induced by genotoxicants by 37-70%. In the same concentration range, the test enantiomers reduced the level of atypical DNA comets (hedgehogs) induced by genotoxicants by 23-61%. The test compounds did not modulate the effects of genotoxicants in a concentration of 10 µM and potentiated them in a concentration of 100 µM. The modifying activity of usnic acid did not depend on spatial configuration and on the used model genotoxicant.

Effect of green juice and their bioactive compounds on genotoxicity induced by alkylating agents in mice.

Kale juice (Brassica oleracea L. var. acephala D.C.) is a reliable source of dietary carotenoids and typically contains the highest concentrations of lutein (LT) and beta-carotene (BC) among green leafy vegetables. As a result of their antioxidant properties, dietary carotenoids are postulated to decrease the risk of disease occurrence, particularly certain cancers. The present study aimed to (1) examine the genotoxic and antigenotoxic activity of natural and commercially available juices derived from Brassica oleracea and (2) assess influence of LT or BC against DNA damage induced by alkylating agents such as methyl methanesulfonate (MS) or cyclophosphamide (CP) in vivo in mice. Male Swiss mice were divided into groups of 6 animals, which were treated with water, natural, or commercial Brassica oleraceae juices (kale), LT, BC, MMS, or CP. After treatment, DNA damage was determined in peripheral blood lymphocytes using the comet assay. Results demonstrated that none of the Brassica oleraceae juices or carotenoids produced genotoxic effects. In all examined cell types, kale juices or carotenoids inhibited DNA damage induced by MMS or CP administered either pre- or posttreatment by 50 and 20%, respectively. Under our experimental conditions, kale leaf juices alone exerted no marked genotoxic or clastogenic effects. However, a significant decrease in DNA damage induced by MMS or CP was noted. This effect was most pronounced in groups that received juices, rather than carotenoids, suggesting that the synergy among constituents present in the food matrix may be more beneficial than the action of single compounds. Data suggest that the antigenotoxic properties of kale juices may be of therapeutic importance.

Protective effect of capsaicin against methyl methanesulphonate induced toxicity in the third instar larvae of transgenic Drosophila melanogaster (hsp70-lacZ)Bg9.

Capsaicin (trans-8-methyl-N-vanillyl-6-nonenamide) is the main component in hot peppers, including red chili peppers, jalapenos, and habanero, belonging to the genus Capsicum. Capsaicin is a potent antioxidant that interferes with free radical activities. In the present study, the possible protective effect of capsaicin was studied against methyl methanesulphonate (MMS) induced toxicity in third instar larvae of transgenic Drosophila melanogaster (hsp70-lacZ)Bg9. The third instar was allowed to feed on the diet having different doses of capsaicin and MMS separately and in combination. The results suggested that the exposure of third instar larvae to the diet having MMS alone showed significant hsp70 expression as well as tissue DNA and oxidative damage, whereas the larvae feed on the diet having MMS and capsaicin showed a decrease in the toxic effects for 48-h of exposure. In conclusion, capsaicin showed a dose-dependent decrease in the toxic effects induced by MMS in the third instar larvae of transgenic Drosophila melanogaster.

Evaluation of the antimutagenic activity and mode of action of the fructooligosaccharide inulin in the meristematic cells of Allium cepa culture.

This study evaluated the mutagenicity and antimutagenicity of inulin in a chromosomal aberration assay in cultures of the meristematic cells of Allium cepa. The treatments evaluated were as follows: negative control--seed germination in distilled water; positive control--aqueous solution of methyl methanesulfonate (10 µg/mL MMS); mutagenicity--aqueous solutions of inulin (0.015, 0.15, and 1.50 µg/mL); and antimutagenicity--associations between MMS and the different inulin concentrations. The antimutagenicity protocols established were pre-treatment, simultaneous simple, simultaneous with pre-incubation, and post-treatment. The damage reduction percentage (DR%) was 43.56, 27.77, and 55.92% for the pre-treatment; -31.11, 18.51, and 7.03% for the simultaneous simple; 30.43, 19.12, and 21.11% for the simultaneous with pre-incubation; and 64.07, 42.96, and 53.70% for the post-treatment. The results indicated that the most effective treatment for inhibiting damages caused by MMS was the post-treatment, which was followed by the pre-treatment, suggesting activity by bioantimutagenesis and desmutagenesis. The Allium cepa assay was demonstrated to be a good screening test for this type of activity because it is easy to perform, has a low cost, and shows DR% that is comparable to that reported studies that evaluated the prevention of DNA damage in mammals by inulin.

In vivo protective activity of Styrax camporum hydroalcoholic extract against genotoxicity induced by doxorubicin and methyl methanesulfonate in the micronucleus and comet assays.

Styrax camporum Pohl is a tall shrub or a tree with small white flowers, which grows in the states of São Paulo and Minas Gerais and is popularly used for the treatment of gastroduodenal diseases. Considering this last fact, the aim of this study was to evaluate the genotoxic potential of S. camporum hydroalcoholic extract and its influence on genotoxicity induced by doxorubicin and methyl methanesulfonate in Swiss mice using the micronucleus and comet assays, respectively. The animals were treated by gavage with different doses of the extract (250, 500, and 1000 mg/kg body weight). For antigenotoxicity assessment, different doses of the S. camporum extract were administered simultaneously with doxorubicin (micronucleus test; 15 mg/kg) and methanesulfonate (comet assay; 40 mg/kg). The results showed that the S. camporum extract itself was not genotoxic in the mouse micronucleus or comet assay. The number of micronucleated polychromatic erythrocytes was significantly lower in animals treated with the S. camporum extract and doxorubicin when compared to animals treated only with doxorubicin. In the comet assay, the S. camporum extract, at the doses tested, significantly reduced the extent of DNA damage in liver cells induced by methanesulfonate. The putative activity of the active compounds of S. camporum extract may explain the effect of this plant on genotoxicity induced by doxorubicin and methanesulfonate.

Diverse small molecule inhibitors of human apurinic/apyrimidinic endonuclease APE1 identified from a screen of a large public collection.

The major human apurinic/apyrimidinic endonuclease APE1 plays a pivotal role in the repair of base damage via participation in the DNA base excision repair (BER) pathway. Increased activity of APE1, often observed in tumor cells, is thought to contribute to resistance to various anticancer drugs, whereas down-regulation of APE1 sensitizes cells to DNA damaging agents. Thus, inhibiting APE1 repair endonuclease function in cancer cells is considered a promising strategy to overcome therapeutic agent resistance. Despite ongoing efforts, inhibitors of APE1 with adequate drug-like properties have yet to be discovered. Using a kinetic fluorescence assay, we conducted a fully-automated high-throughput screen (HTS) of the NIH Molecular Libraries Small Molecule Repository (MLSMR), as well as additional public collections, with each compound tested as a 7-concentration series in a 4 µL reaction volume. Actives identified from the screen were subjected to a panel of confirmatory and counterscreen tests. Several active molecules were identified that inhibited APE1 in two independent assay formats and exhibited potentiation of the genotoxic effect of methyl methanesulfonate with a concomitant increase in AP sites, a hallmark of intracellular APE1 inhibition; a number of these chemotypes could be good starting points for further medicinal chemistry optimization. To our knowledge, this represents the largest-scale HTS to identify inhibitors of APE1, and provides a key first step in the development of novel agents targeting BER for cancer treatment.

Phenylbutyrate inhibits homologous recombination induced by camptothecin and methyl methanesulfonate.

Homologous recombination is accompanied by extensive changes to chromatin organization at the site of DNA damage. Some of these changes are mediated through acetylation/deacetylation of histones. Here, we show that recombinational repair of DNA damage induced by the anti-cancer drug camptothecin (CPT) and the alkylating agent methyl methanesulfonate (MMS) is blocked by sodium phenylbutyrate (PBA) in the budding yeast Saccharomyces cerevisiae. In particular, PBA suppresses CPT- and MMS-induced genetic recombination as well as DNA double-strand break repair during mating-type interconversion. Treatment with PBA is accompanied by a dramatic reduction in histone H4 lysine 8 acetylation. Live cell imaging of homologous recombination proteins indicates that repair of CPT-induced DNA damage is redirected to a non-recombinogenic pathway in the presence of PBA without loss in cell viability. In contrast, the suppression of MMS-induced recombination by PBA is accompanied by a dramatic loss in cell viability. Taken together, our results demonstrate that PBA inhibits DNA damage-induced homologous recombination likely by mediating changes in chromatin acetylation. Moreover, the combination of PBA with genotoxic agents can lead to different cell fates depending on the type of DNA damage inflicted.

Anti-clastogenic effect of beta-glucan extracted from barley towards chemically induced DNA damage in rodent cells.

beta-Glucan (BG) was tested in vitro to determine its potential clastogenic and/or anti-clastogenic activity, and attempts were made to elucidate its possible mechanism of action by using combinations with an inhibitor of DNA polymerase. The study was carried out on cells deficient (CHO-k1) and cells proficient (HTC) in phases I and II enzymes, and the DNA damage was assessed by the chromosomal aberration assay. BG did not show a clastogenic effect, but was anti-clastogenic in both cell lines used, and at all concentrations tested (2.5, 5 and 10 microg/mL) in combination with damage inducing agents (methylmethane sulfonate in cell line CHO-k1, and methylmethane sulfonate or 2-aminoanthracene in cell line HTC). BG also showed a protective effect in the presence of a DNA polymerase beta inhibitor (cytosine arabinoside-3-phosphate, Ara-C), demonstrating that BG does not act through an anti-mutagenic mechanism of action involving DNA polymerase beta.

Antimutagenic activity of berry extracts.

Plants are proven sources of useful anti-tumor and chemopreventative compounds. Hence, identification of phytochemicals useful in dietary prevention and intervention of cancer is of paramount importance. The initial step in the formation of cancer is damage to the genome of a somatic cell producing a mutation in an oncogene or a tumor-suppressor gene. Fresh juices and organic solvent extracts from the fruits of strawberry, blueberry, and raspberry were evaluated for their ability to inhibit the production of mutations by the direct-acting mutagen methyl methanesulfonate and the metabolically activated carcinogen benzo[a]pyrene. Juice from strawberry, blueberry, and raspberry fruit significantly inhibited mutagenesis caused by both carcinogens. Ethanol extracts from freeze-dried fruits of strawberry cultivars (Sweet Charlie and Carlsbad) and blueberry cultivars (Tifblue and Premier) were also tested. Of these, the hydrolyzable tannin-containing fraction from Sweet Charlie strawberries was most effective at inhibiting mutations.

Inhibitory effect of chamomile essential oil on the sister chromatid exchanges induced by daunorubicin and methyl methanesulfonate in mouse bone marrow.

Different preparations of chamomile (Matricaria chamomilla) are used to treat various diseases, including inflammation and cancer; however, no studies on the plant's antigenotoxic capacity have been made. The aim of the present work was to determine the inhibitory effect of the chamomile essential oil (CO), on the sister chromatid exchanges (SCEs) produced by daunorubicin and methyl methanesulfonate (MMS) in mouse bone marrow cells. CO was analyzed and was found to contain 13 compounds, mainly bisabolol and its oxides, chamazulene, farnesene, germacrene and other sesquiterpenes. Initially, a toxic and a genotoxic analysis of CO were made; both showed negative results. To determine whether CO can inhibit the mutagenic effects induced by daunorubicin, one group of mice was administered corn oil, another group was treated with the mutagen (10 mg/kg), a third group was treated with 500 mg/kg of CO; three other groups were treated first with CO (5, 50 and 500 mg/kg) and then with 10 mg/kg of daunorubicin. In the case of MMS, the experimental groups consisted of the following: the negative control group which was administered corn oil, a group treated with 25 mg/kg of MMS, a group treated with 1000 mg/kg of CO, and three groups treated first with CO (250, 500 and 1000 mg/kg) and then with MMS (25 mg/kg). The results indicated a dose-dependent inhibitory effect on the SCEs formed by both mutagens. In the case of daunorubicin, a statistically significant result was observed in the three tested doses: from the lowest to the highest dose, the inhibitory values corresponded to 25.7, 63.1 and 75.5%. No alterations were found with respect to the cellular proliferation kinetics, but a reduction in the mitotic index was detected. As regards MMS, the inhibitory values were 24.8, 45.8 and 60.6%; no alterations were found in either the cellular proliferation kinetics or in the mitotic indices. Our results suggest that CO may be an effective antimutagen that could be considered for further study.

Chemoprotection profiles of sodium thiosulfate on methyl methanesulfonate-induced mutagenesis of bacteriophage T4.

BACKGROUND: The alkylation of nucleic acids is primarily responsible for chemical carcinogenesis. Even during disease treatment, several alkylating drugs interact with nucleic acids and cause severe toxic effects. Thus good chemoprotectants are necessary. For our study we chose a simple model organism, bacteriophage T4 (a nucleoprotenic particle), and alkylating agent methyl methanesulfonate (MMS) to study its lethal effects. Sodium thiosulfate (STS), used as a chemoprotectant, has been tested against alkylating drugs. MATERIAL/METHODS: Bacteriophage T4D(o) were exposed to different molarities of MMS for several pre-termination incubations. Alkylation reactions were stopped with different concentrations of STS at given pre-termination incubation periods and further incubated up to 24 hours. The viability (survival frequency) of phage T4 was studied at various post-termination intervals by plaque count assay. RESULTS: Our results show that the survival frequency is strongly influenced by MMS dosage and exposure time. However, the antidotal effect of STS on MMS-induced lethality directly corresponds to STS dosage. Survival frequencies with 1% quench solution were lower than with 5% quench solution at all molarities of MMS and at different pre- and post-termination periods. CONCLUSIONS: Our studies confirmed the role of STS in the cytoprotection of bacteriophage T4. In the presence of 1% STS, a moderate inhibition in cytotoxicity was observed, while 5% STS exhibited a significant inhibition against the cytotoxic activity of MMS, presumably due to a rapid covalent binding of the methyl group (carbocation - an electrophile) of MMS with the nucleophilic sulfur atom of STS.

Investigation of genotoxic and antigenotoxic activities of chlorophylls and chlorophyllin in cultured V79 cells.

Chlorophyll and its derivatives are examples of plant compounds (purified and/or extracted) which appear to protect DNA from damage caused by chemical or physical agents, although some studies have identified clastogenic activity of these compounds. This study was carried out to assess the genotoxic activity of chlorophyll-a (Chl-a), -b (Chl-b) and chlorophyllin (Chl) and their antigenotoxic activity against the DNA damage induced by methyl methanesulphonate (MMS) under conditions of simultaneous, pre-, post-treatment, and simultaneous treatment after pre-incubation of the chemical with MMS. The micronucleus (MN) test was used in binucleated cells (induced by cytochalasin-B) of a mammalian cell line (V79). The three concentrations of Chl-a, Chl-b or Chl (0.1375, 0.275, 0.55microM) were not genotoxic and the genotoxic action of MMS (400microM) decreased (74-117%) under all treatment conditions. The results showed that there was no significant difference among the treatment types, the concentration or the nature of chlorophyll used. The data obtained suggest that Chl-a, Chl-b and Chl when associated with the DNA damaging agent, MMS, may protect the DNA by desgenotoxic action and/or by bio-antigenotoxic mechanisms, with the similar efficiency.

Antimutagenic activity of fluphenazine in short-term tests.

Fluphenazine, an antipsychotic drug that belongs to the phenothiazine family, reduced the genotoxicity of direct- and indirect-acting mutagens in the Ames test, both in the presence and in the absence of promutagen-activating S9 fraction. In short-term tests on human lymphocytes, the inhibitory effect of fluphenazine on the genotoxicity of standard mutagens was strongest in the cytokinesis-blocked micronucleus assay and in the thioguanine resistance test, and weakest in the sister chromatid exchange test. Fluphenazine also considerably reduced the level of free radicals estimated in in vitro samples of human granulocytes. The results suggest that, in the range of the tested concentrations, fluphenazine could be considered for use to prevent the genotoxicity of daunorubicin, methyl methanesulfonate, benzo[a]pyrene, and mitomycin C. Reduction in the level of free radicals appears to be an important mechanism of the antimutagenic action of fluphenazine.

Derivatization of the interface cysteine of triosephosphate isomerase from Trypanosoma brucei and Trypanosoma cruzi as probe of the interrelationship between the catalytic sites and the dimer interface.

In the interface of homodimeric triosephosphate isomerase from Trypanosoma brucei (TbTIM) and Trypanosoma cruzi (TcTIM), one cysteine of each monomer forms part of the intersubunit contacts. The relatively slow derivatization of these cysteines by sulfhydryl reagents induces progressive structural alterations and abolition of catalysis [Garza-Ramos et al. (1998) Eur. J. Biochem. 253, 684-691]. Derivatization of the interface cysteine by 5, 5-dithiobis(2-nitrobenzoate) (DTNB) and methylmethane thiosulfonate (MMTS) was used to probe if events at the catalytic site are transmitted to the dimer interface. It was found that enzymes in the active catalytic state are significantly less sensitive to the thiol reagents than in the resting state. Maximal protection against derivatization of the interface cysteine by thiol reagents was obtained at near-saturating substrate concentrations. Continuous recording of derivatization by DTNB showed that catalysis hinders the reaction of sulfhydryl reagents with the interface cysteine. Therefore, in addition to intrinsic structural barriers, catalysis imposes additional impediments to the action of thiol reagents on the interface cysteine. In TcTIM, the substrate analogue phosphoglycolate protected strongly against DTNB action, and to a lesser extent against MMTS action; in TbTIM, phosphoglycolate protected against the effect of DTNB, but not against the action of MMTS. This indicates that barriers of different magnitude to the reaction of thiol reagents with the interface cysteine are induced by the events at the catalytic site. Studies with a Cys14Ser mutant of TbTIM confirmed that all the described effects of sulfhydryl reagents on the trypanosomal enzymes are a consequence of derivatization of the interface cysteine.

Cassia senna inhibits mutagenic activities of benzo[a]-pyrene, aflatoxin B1, shamma and methyl methanesulfonate.

Ethanol extract of Senokot tablets (Cassia senna concentrate used as vegetable laxative), was found to be non-mutagenic while it inhibited the mutagenicity of benzo[a]pyrene, shamma, aflatoxin B1 and methyl methanesulfonate in the Ames histidine reversion assay using the Salmonella typhimurium tester strain TA98. While the Senokot extract completely inhibited the mutagenicity of promutagens (i.e. metabolic activation dependent) like benzo[a]pyrene and shamma, it reduced the mutagenic activity of the direct acting mutagen methyl methanesulfonate by only 58%. The mutagen aflatoxin B1 showed a 25-fold increase in the number of histidine revertants per plate at low concentrations (1.0-4.0 micrograms/plate) in the presence of metabolic activation system while at high concentrations (10.0-30.0 micrograms/plate) it proved to be weakly mutagenic (with a 5-fold increase in the number of histidine revertants/plate) without metabolic activation. The Senokot extract completely inhibited the mutagenic effect of low concentrations of aflatoxin B1 in the presence of metabolic activation but not that resulting from higher concentrations without metabolic activation. The results obtained with benzo[a]pyrene, shamma and aflatoxin B1 indicated that the antimutagenic effects of Senokot extract could be largely due to an interaction with the metabolic process involved in the activation of procarcinogens. However, the results obtained with methyl methanesulfonate suggested that factors in Senokot may also interact with direct mutagens to produce some antimutagenic effects. An ethanol extract of crude senna leaves found to be weakly mutagenic also inhibited (though less than Senokot) the mutagenic effect of benzo[a]pyrene suggesting that the antimutagenic principle is present in the complex plant material itself.

Todralazine influence upon the mutagenicity of some direct- and indirect-acting mutagens.

Todralazine decreased the mutagenic activity of tested direct- and indirect-acting mutagens. Despite the marked differences between efficient todralazine doses (ED50) it was observed that, in the case of tested indirect mutagens as well as in some of the direct mutagens, the decrease of mutagenicity by todralazine was very strong, exceeding 80% in some cases.

Effect of beta-carotene on clastogenic effects of mitomycin C, methyl methanesulphonate and bleomycin in Chinese hamster ovary cells.

The effect of beta-carotene on the frequencies of micronuclei (MN) induced in cytochalasin blocked binucleated Chinese hamster ovary cells (CHO) by a bifunctional alkylating agent mitomycin C (MMC), a monofunctional alkylating agent methyl methanesulphonate (MMS) and a radio-mimetic agent bleomycin (BLEO) was investigated. Four different modes of application of the combination of clastogens and beta-carotene were examined (pre-treatment, simultaneous treatment, pre- +simultaneous treatment and post-treatment). The results obtained showed no effect of beta-carotene on the frequencies of MN induced by MMS, a slight but not statistically significant reduction of MMC-induced MN only when beta-carotene was used in low concentrations (0.25 and 0.5 microM) and a potentiation of the clastogenicity of bleomycin by beta-carotene in three of the treatment regimes utilized, post-treatment being ineffective. On the basis of these results it can be concluded that the effect of beta-carotene on clastogenesis induced by chemicals depends on the type and mechanism of action of the clastogen used as well as the treatment protocol employed.

Inhibition of methyl methanesulfonate-induced unscheduled DNA synthesis in spermatozoa of mice by Poly I-C.

Methyl methanesulfonate (MMS 75 mg.kg-1)-induced unscheduled DNA synthesis (UDS) was inhibited significantly in the spermatozoa of mice injected with Poly I-C (0.05, 0.50, and 5.00 mg.kg-1) at 4 h prior to ip MMS. Radioactivity was reduced from 53 +/- 3 to 50, 45 +/- 5, and 34 +/- 6 dpm/million sperms respectively. The effects of Poly I-C were dose-dependent (r = 0.9498, P less than 0.05) in inhibition of MMS-induced UDS. The effect of serum collected from the mice injected with Poly I-C (0.50 mg.kg-1) had similar effects as that of Poly I-C. Our findings suggest that both Poly I-C alone and mouse serum induced with Poly I-C may prevent the DNA damage produced by MMS.

Mechanisms of chemical mediated cytotoxicity and chemoprotection in isolated rat hepatocytes.

Although N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and methylmethanesulfonate (MMS) cause injury and malondialdehyde formation in rat hepatocytes, MNNG toxicity is much more sensitive to inhibition by antioxidants. In order to quantify the relationship between toxicity and antioxidation potential, we compared 14 antioxidants that protected against MNNG and MMS toxicity. Chemoprotection was quantified as the concentration that delayed by 1 h the decline in trypan blue exclusion to less than or equal to 50%. While chemoprotection against MNNG and antioxidant efficacy were directly related (R = 0.86), chemoprotection against MMS and antioxidant efficacy were unrelated (R = 0.37). Since we hypothesized that protection against MMS involved stabilization of membranes, the capacity of the 14 compounds to stabilize membranes in an unrelated system (i.e. prevention of erythrocyte osmotic rupture) was assayed. Chemoprotection against both MNNG and MMS correlated with reduced RBC fragility (R = 0.97 and 0.70, respectively). One of the better protecting compounds, 4b,5,9b,10-tetrahydroindeno[1,2-b]indole, was also protective against hepatocellular toxicity mediated by acetaminophen, carbon tetrachloride and tert-butyl hydroperoxide, suggesting a fundamental basis in the mechanism of chemoprotection. We propose that methylating agents and perhaps other chemical toxicants destabilize cellular membranes resulting in hepatocellular injury. For MNNG, radical mediated events may result in membrane destabilization; for MMS, membranes are destabilized without concurrent radical events. The current studies provide a basis for future work to determine structure-activity relationships of chemoprotective agents, examine protection mechanisms, and develop better protective compounds.