Antimutagenic potential of glucosinolate-rich seed extracts of broccoli (Brassica oleracea L var italica Plenck).

In the current study, isolation of glucosinolate degradation products was done in 4 different incubation solutions with different pHs based on the fact that distinct hydrolytic products are formed at different pHs. All the extracts were tested against direct-acting mutagens (4 nitro-o-phenylenediamine [NPD]), sodium azide, and indirect-acting mutagen (2-aminofluorene [2AF]). It was observed that extracts inhibited mutagenesis induced by the S9-dependent mutagen (2AF) more significantly than direct-acting mutagens. Two different modes of experimentation (pre-incubation and co-incubation) were used, and it was observed that the extracts showed better results in the pre-incubation mode of experimentation. Out of the 4 extracts tested, 0.1 mol/L of HCl extract was found to be the most effective in inhibiting mutagenesis with both TA 98 and TA 100 strains of Salmonella typhimurium. All other extracts also showed pronounced antimutagenic potential. The results of this study indicate the presence of potent antigenotoxic factors in broccoli, which are being explored further for their mechanism of action.

Ellagic acid inhibits growth and arylamine N-acetyltransferase activity and gene expression in Staphylococcus aureus.

It is well documented that N-acetyltransferase (NAT) plays a key role in the N-acetylation of arylamine compounds. Ellagic acid was demonstrated to elicit dose-dependent bacteriostatic activity and inhibition of N-acetylation of 2-aminofluorene (AF). N-acetylation of AF in S. aureus was determined by high preformance liquid chromatography. The apparent values of Km and Vmax of NAT were decreased after co-treatment with 0.5 mM ellagic acid in the cytosol of S. aureus. PCR also indicated that ellagic acid inhibited NAT gene expression (NAT mRNA) in S. aureus.

Mechanisms of arsenite-mediated decreases in benzo[k]fluoranthene-induced human cytochrome P4501A1 levels in HepG2 cells.

Polycyclic aromatic hydrocarbons (PAHs) and heavy metals are often environmental cocontaminants that could interact to alter PAH carcinogenicity. The heavy metal, arsenite, and the PAH, benzo[k]fluoranthene, were used as prototypes to investigate, in human HepG2 cells, mechanisms whereby the bioactivation of benzo[k]fluoranthene by human CYP1A1 could be diminished by arsenite-mediated decreases in CYP1A1 induction by benzo[k]fluoranthene. To determine whether arsenite down-regulates CYP1A1 transcription, quantitative real-time reverse transcriptase-polymerase chain reaction assays and luciferase reporter gene expression assays were used with HepG2 cells treated with benzo[k]fluoranthene and arsenite, separately and as a mixture. Benzo[k]fluoranthene (0.5 microM) and arsenite (5 microM) markedly decreased benzo[k]fluoranthene-mediated induction of CYP1A1 mRNA by 45%. Plasmids containing the CYP1A1 promoter region (pHu-1A1-FL) were induced 7.4-fold over vehicle by benzo[k]fluoranthene (0.5 microM), whereas arsenite (1, 2.5, or 5 microM) decreased reporter gene expression by 46%, 45%, and 61%, respectively. The plasmid, pHu-1A1-Delta100-FL, lacked xenobiotic response element (XRE) sites at -1061 and -981 and showed greater responsiveness relative to pHu-1A1-FL, by 1.7-fold. Benzo[k]fluoranthene (0.5 microM) and arsenite (1, 2.5, or 5 microM) decreased reporter gene expression by 0%, 27%, and 39%, respectively, relative to expression levels produced by benzo[k]fluoranthene alone. Arsenite is stable for at least 48 h in the HepG2 cell medium with respect to its ability to diminish CYP1A1 benzo[k]fluoranthene induction. Arsenite did not affect benzo[k]fluoranthene induction directly through XRE sites, nor did it affect the stability of CYP1A1 mRNA. Thus, arsenite affects the transcriptional regulation of the benzo[k]fluoranthene-mediated induction of CYP1A1 and could diminish PAH carcinogenicity by decreasing bioactivation by CYP1A1.

PCR and flow cytometric analysis of paclitaxel-inhibited arylamine N-acetyltransferase activity and gene expression in human osteogenic sarcoma cells (U-2 OS).

Human epidemiological studies suggest an association between N-acetyltransferase (NAT) activity and the incidence of bladder and colorectal cancers. In this study, paclitaxel was selected to examine the inhibition of arylamine NAT activity, gene expression and 2-aminofluorene-DNA adduct formation in a human osteogenic sarcoma cell line (U-2 OS). The activity of NAT was determined by high performance liquid chromatography (HPLC) assay for the amounts of acetylated 2-aminofluorene (AF) and p-aminobenzoic acid (PABA) and nonacetylated AF and PABA. Human osteogenic sarcoma cell cytosols and intact cells were used to examine the NAT activity, gene expression and AF-DNA adduct formation. The results demonstrated that NAT activity percent of NAT in examined cells, gene expression (NAT1 mRNA) and AF-DNA adduct formation in human osteogenic sarcoma cells were inhibited and decreased by paclitaxel in a dose-dependent manner. The results also demonstrated that paclitaxel decreased the apparent values of Km and Vmax from intact human osteogenic sarcoma cells (U-2 OS). Thus, paclitaxel is an uncompetitive inhibitor of the NAT enzyme.

Paclitaxel (taxol) inhibits the arylamine N-acetyltransferase activity and gene expression (mRNA NAT1) and 2-aminofluorene-DNA adduct formation in human bladder carcinoma cells (T24 and TSGH 8301).

Acetylator polymorphism in man results from differential expression of human liver N-acetyltransferase. N-Acetyltransferase enzyme activity has been demonstrated to be involved in some types of chemical carcinogenesis. Paclitaxel (taxol) had been shown to affect N-acetyltransferase activity of human lung cancer cells. In this study, paclitaxel was chosen to investigate the effects of arylamine N-acetyltransferase activity (N-acetylation of substrate), gene expression and 2-aminofluorene-DNA adduct formation in human bladder carcinoma cell lines (T24 and TSGH 8301). The N-acetyltransferase activity (N-acetylation of substrates) was determined by high performance liquid chromatography assaying for the amounts of acetylated 2-aminofluorene and p-aminobenzoic acid and nonacetylated 2-aminofluorene and p-aminobenzoic acid. Intact human bladder carcinoma T24 and TSGH 8301 cells were used for examining N-acetyltransferase activity, gene expression and 2-aminofluorene-DNA adduct formation. The results demonstrated that the N-acetyltransferase activity, gene expression (NAT1 mRNA) and 2-aminofluorene-DNA adduct formation in intact human bladder carcinoma cells were inhibited and decreased by paclitaxel in a dose-dependent manner. The effects of paclitaxel on the apparent values of Km and Vmax of N-acetyltransferase enzyme from intact human bladder carcinoma cells were also determined in these cell lines. A marked influence of paclitaxel was observed on the decreasing apparent values of Km and Vmax from intact human bladder carcinoma cells (T24 and TSGH 8301). Thus, paclitaxel is an uncompetitive inhibitor to the NAT enzyme.

Luteolin induces N-acetylation and DNA adduct of 2-aminofluorene accompanying N-acetyltransferase activity and gene expression in human bladder cancer T24 cell line.

Arylamine N-acetyltransferase (NAT) plays an important role in the first step of arylamines metabolism. Luteolin has shown antibacterial, antioxidant and antineoplastic activity. To glean insights into the mechanism of action of luteolin, we assessed the effects of luteolin on NAT activity and gene expression and DNA-2-aminofluorene (AF) adduct formation in human bladder cancer T24 cells. By using high performance liquid chromatography, the amounts of N-acetylation of AF and DNA adducts were determined and quantitated. Gene expression was performed by using polymerase chain reaction and gel electrophoresis. The results indicated: 1) luteolin displayed a dose-dependent inhibition of NAT activity and gene expression (NAT1 mRNA) in T24 cells; 2) time-course experiments showed that N-acetylation of AF measured from intact T24 cells was inhibited by luteolin for up to 48 hours; 3) using standard steady-state kinetic analysis, it was demonstrated that luteolin was a possible uncompetitive inhibitor of NAT activity in T24 cells; and 4) the DNA-AF adduct formation in T24 cells was inhibited by luteolin. This report is the first finding which has shown luteolin to affect human T24 cell NAT activity and gene expression (NAT1 mRNA) and DNA-AF adduct formation.

Antimutagenic activities of acetone and methanol fractions of Terminalia arjuna.

The antimutagenic effect of benzene, chloroform, acetone and methanol fractions from Terminalia arjuna, a well-known medicinal plant, was determined against Acid Black dye, 2-aminofluorene (2AF) and 4-nitro-o-phenylenediamine (NPD) in TA98 Frameshift mutagen tester strain of Salmonella typhimurium. Among the different fractions, the antimutagenic effect of acetone and methanol fractions was more than that observed with other fractions. Co-incubation and pre-incubation modes of experimentation did not show much difference in the antimutagenic activity of the extracts. Moreover, these fractions inhibited the S9-dependent mutagens, 2AF and Acid Black dye more effectively than the direct-acting mutagens. Studies are under way to isolate and elucidate the nature of the antimutagenic factor in acetone and methanol fractions.

Detection of drug-induced, superoxide-mediated cell damage and its prevention by antioxidants.

The mode of the cytotoxic activity of three benzo(c)fluorene derivatives was characterized. The observed morphological changes of lysosomes or variations of mitochondrial activity are assumed to be the consequence of cell protection against oxidative damage and/or the part of the damage process. To establish the relationship between the quantity of superoxide (O2*-) generated and the degree of damage resulting from O2*-, a simple system based on measurement of 3-(4-iodophenyl)-2-(4-nitrophenyl)-5-phenyltetrazolium chloride (INT) reductase activity in the presence of superoxide dismutase (SOD) was used. The functionality of the chosen battery of in vitro tests was proved using several known superoxide inducers: cyclosporin A (CsA) and benzo(a)pyrene (BP), as well as noninducers: citrinin (CT) and cycloheximide (CH). From the results followed that the cell growth tests are much better indices of toxicity than the other tests. The model system for the evaluation of the protective capacity of antioxidants against superoxide-induced cytotoxicity included simultaneous exposure of HeLa cells to cytotoxic drugs and to quercetin (Qe), an antioxidant of plant origin. The complete abolishment of the inhibition of cell proliferation and clonogenic survival was concluded to be due to the protective effect of the antioxidant. These observations correlated with the decrease of superoxide content as estimated by the INT-reductase assay in the presence of SOD using the same model system, as well as with the increase of intracellular SOD content and its activity.

Proanthocyanidins from the bark of Hamamelis virginiana exhibit antimutagenic properties against nitroaromatic compounds.

The antimutagenic activity of Hamamelis virginiana bark was examined in the Ames assay. A commercial tincture and a methanolic extract showed dose-dependent inhibitory effects on mutagenicity induced by 2-nitrofluorene. Tannin-free samples did not display any inhibition. Bioassay-guided fractionation resulted in the isolation of two active fractions which were shown to contain oligomeric, proanthocyanidins. They were capable of inhibiting the mutagenicity of selected nitroaromatic compounds. The mechanism of antimutagenic action was also studied. The proanthocyanidins did not act as bioantimutagens, but rather as direct-acting desmutagens. The antimutagenic effect increased with an increasing degree of polymerisation in the proanthocyanidins. The most active fraction consisted of catechin and gallocatechin oligomers with an average polymerisation degree of 9.2.

Modulation of the potency of promutagens and direct acting mutagens in bacteria by inhibitors of the multidrug resistance mechanism.

Multiple drug resistance (MDR) mechanisms are known to limit the effectiveness of some cancer chemotherapies, probably through enhancing P-glycoprotein-mediated drug efflux from mammalian cells. Similar mechanisms appear to act in other organisms, including bacteria, and may affect not only the toxicity but also the mutagenicity of certain chemicals. At least in some experimental situations, MDR can be overcome through concomitant treatment of the cells with various types of inhibitors. Two MDR inhibitors, verapamil, a calcium channel blocker, and trifluoperazine, a calmodulin inhibitor, were assayed for their ability to modulate the potency of nine mutagens with varying mechanisms of action in various Salmonella typhimurium his- strains. Neither verapamil nor trifluoperazine affected the direct mutagenicity of sodium dichromate and 2-methoxy-6-chloro-9[3-(2-chloroethyl)amino-propyl-amino] dihydrochloride (ICR 191) or the S9-mediated mutagenicity of benzo[a]pyrene and 2-amino-3,4-dimethyl-amidazo[4,5-f]quinoline (MeIQ). Both modulators enhanced the direct mutagenicity of doxorubicin. Moreover, trifluoperazine sharply increased the S9-mediated mutagenicity of cyclophosphamide and 2-aminofluorene, while it consistently decreased the mutagenicity of 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2). The contrasting effect towards the aromatic amine 2-aminofluorene and the heterocyclic amine Trp-P-2, representative of important chemical families responsible for the bacterial mutagenicity of cigarette smoke, may explain the observed lack of influence of trifluoperazine on the mutagenicity of a cigarette smoke condensate. These observations extend the known range of chemical types whose mutagenicity can be modulated by inhibitors of MDR and suggest that there may be value in adding MDR inhibitors, especially trifluoperazine, to optimize the detection of mutagenicity by certain types of chemicals in the Salmonella/mammalian microsome mutagenicity test.

[Bioantimutagenic effect of the culture fluid of Streptococcus faecalis on mutagenesis induced by 2-nitrofluorene in Salmonella typhimurium TA 1538 and TA 98]. / Bioantimutagennoe deistvie kul'tural'noi zhidkosti Streptococcus faecalis protiv mutageneza, indutsirovannogo 2-nitrofluorenom u Salmonella typhimurium TA 1538 i TA98.

Streptococcus faecalis growing in rich organic media or synthetic medium containing 0.1% trypton and 0.05% yeast extract was shown to form substances with a bioantimutagenic activity against the mutagenicity of 2-nitrofluorene and 4-nitroquinoline-1-oxide, which induce frameshift and base-pair substitution mutations, respectively. The maximum accumulation of antimutagens in the medium was observed within 16-24 h of growth. The modified Ames test showed that the culture liquid of Str. faecalis possessed a higher antimutagenic activity for Salmonella typhimurium strain TA 1538 than for strain TA 98 with an enhanced SOS response. Fractionation of the culture liquid revealed that the bioantimutagenic activity is localized in the fraction of peptides with molecular masses of less than 12 kDa.

Inhibitory effect of dairy products on the mutagenicities of chemicals and dietary mutagens.

The antimutagenic effects of uninoculated milk and milks cultured with Bifidobacterium or Lactobacillus strains towards the mutagenicity induced by two direct mutagens, 4-nitroquinoline N-oxide and 2-nitrofluorene, and three dietary indirect mutagens, aflatoxin B1, benzo(a)pyrene and quercetin, were investigated using the in vitro Salmonella typhimurium test. Each cultured milk sample and control milk had a significant antimutagenic effect, to an extent varying with the mutagen used. Uninoculated milk had a greater inhibitory effect than cultured milks towards dietary indirect mutagens.

Moderate inhibition of mutagenicity and carcinogenicity of benzo[a]pyrene, 1,6-dinitropyrene and 3,9-dinitrofluoranthene by Chinese medicinal herbs.

The activity of six Chinese medicinal herbs against the environmental mutagens and carcinogens benzo[a]pyrene (B[a]P), 1,6-dinitropyrene (1,6-diNP) and 3,9-dinitrofluoranthene (3,9-diNF) was determined. Samples of Prunella spica, Rheum palmatum, Polygonum multiflorum, Agrimonia pilosa, Ephedra sinica and Teitoutou were tested in an in vitro system. Antimutagenic activity against B[a]P was marked in the presence of extracts (boiled for 2 h in a water bath) whereas that against 1,6-diNP and 3,9-diNF varied from 20 to 86%. The differences in inhibition might be due to inactivation of metabolic enzymes. An extract of P. multiflorum was divided into ether, ethyl acetate and water soluble fractions, which were tested for antimutagenic activity against B[a]P. The antimutagenic action of the ethyl acetate soluble fraction was substantial and dose-dependent. Tannins and related compounds were the major components of the extract, of which epigallocatechin, epigallocatechin gallate, epicatechin gallate and tannic acid strongly inhibited the mutagenicity of B[a]P (2.5 micrograms/plate) in Salmonella typhimurium TA98 with S9 mix. To confirm the results of the in vitro test system, F344/DuCrj male rats were given a subcutaneous injection of B[a]P. Thereafter, they received water extracts of the six Chinese medicinal herbs for 50 weeks and were examined for tumors. The P. multiflorum extract significantly reduced the tumor incidence.

Expression of disodium cromoglygate 'protective' effects observed during V79 cell proliferation.

Disodium cromoglycate (DSCG) in equimolar concentration of 0.56 mumol/l preincubated with an asynchronous cell population decreases the inhibition of proliferation and results in a 100% elimination of inhibition of colony formation induced by benfluron (BF). DSCG protects the cells from unbalanced growth and unbalanced metabolism (e.g., prevention of increase or decrease in protein cell content, glycolytic activity and in amino acid metabolism) which are both the integrating parts of BF cytotoxic reaction. The protective effect of DSCG is manifested also on synchronous cell populations, particularly in G2 phase (26%). DSCG also stabilizes cell membrane by preventing the alterations of its permeability caused by the cytolytic concentrations of benfluron (at a concentration of 1.05 mumol/l and more).

Lipoxygenase: a new pathway for 2-aminofluorene bioactivation.

Soybean lipoxygenase mediated co-oxidation of 2-aminofluorene (2-AF), a putative carcinogen and teratogen, was examined. 2-AF metabolism was followed spectrally as a decrease in absorbance at 286 nm. The enzymatic oxidation displayed a pH optimum at 9.5 with a Km of 0.27 mM and specific activity of 521.7 +/- 46.6 nmol/min per nmol of enzyme. The generation of electrophilic 2-AF intermediate(s) capable of covalent binding to macromolecules was also investigated radiometrically. Significant binding to protein and calf thymus DNA was observed, suggesting clearly bioactivation of 2-AF. Several classical lipoxygenase inhibitors caused a marked inhibition of 2-AF oxidation as well as covalent binding to protein and DNA. These results strongly suggest that lipoxygenase is capable of 2-AF metabolism and this may represent another pathway for bioactivation of arylamines.

Inhibition of 2-aminofluorene mutagenesis in bacteria by inducers of cytochrome P-450d.

Certain chemical inducers of rat liver microsomal cytochrome P-450d are tightly bound to the cytochrome. We investigated the ability of two inducers of cytochrome P-450d, Aroclor 1254 and isosafrole, to inhibit the microsomal activation of 2-aminofluorene to a mutagen as measured in Salmonella typhimurium. Butanol treatment of microsomes from isosafrole-treated rats removed an inhibitory metabolite of isosafrole and increased 2-aminofluorene mutagenesis by approximately 2-fold over controls. Butanol treatment of microsomes from Aroclor 1254-treated rats failed to either remove any of the Aroclor 1254 associated with microsomal cytochrome P-450 or affect 2-aminofluorene-induced mutagenesis. However, addition of Aroclor 1254 to butanol-treated microsomes from isosafrole-treated rats almost completely inhibited 2-aminofluorene mutagenesis. Aroclor 1254 completely inhibited the cytochrome P-450d-dependent estradiol 2-hydroxylase activity of butanol-treated microsomes from isosafrole-treated rats. Thus, we suspect that certain congeners from Aroclor 1254, a widely used mixture for induction of cytochrome P-450 activities, could inhibit cytochrome P-450d and partially mask its ability to metabolize some chemicals to mutagens.

Stimulation of glucosylated lens epithelial Na,K-ATPase by an aldose reductase inhibitor.

In diabetes, glucosylation of the Na,K-ATPase of the lens epithelium makes the pump inefficient. K+ transport and ATP hydrolysis (at near saturating ATP concentrations) are inhibited and the kinetics of ATP hydrolysis become substrate inhibition type. The AR inhibitor (AL1576, Alcon Laboratories) stimulates K+ transport and ATP hydrolysis by glucosylated bovine lens Na,K-ATPase. This inhibitor has a slight stimulatory effect upon the unmodified enzyme function also. The AR inhibitor is not able to prevent glucosylation of the pump in high-glucose-containing medium.

Absence of mutagenic and antimutagenic activities of fluoride in Ames salmonella assays.

The mutagenicity of fluoride (as sodium fluoride, NaF) was investigated with Ames Salmonella/microsome assays in strains of TA97a, TA98, TA100, TA102 and TA1535. The concentrations of NaF tested ranged from 0.44 to 4421 micrograms/plate (0.1 to 1000 ppm F), both with and without microsome activation. In addition, the suggested antimutagenic effect of fluoride was evaluated with known mutagens at various concentrations of NaF (0.44-442.2 micrograms/plate, 0.1-100 ppm F). The data showed that NaF, in amounts from 0.44 to 442.2 micrograms/plate (0.1-100 ppm F), failed to significantly increase the number of the revertants over the number observed in the solvent (distilled deionized water) controls. Increases of NaF to, and beyond, 1100 micrograms/plate (250 ppm F) resulted in a toxic effect and a reduction of the revertants to various degrees among the strains. NaF in the presence of known mutagens did not significantly decrease the number of the revertants. The results of this study indicate that NaF does not have mutagenic or antimutagenic effects in the strains tested with Ames Salmonella assays.

Inhibition of the mutagenicity of aromatic amines by the plant flavonoid (+)-catechin.

Addition of the plant phenolic flavonoid (+)-catechin to rat liver microsomes inhibited the mutagenicity of the aromatic amines 2-aminofluorene and 4-aminobiphenyl in the Ames test. Similarly, (+)-catechin decreased the mutagenicity of N-hydroxy-4-amino-biphenyl, the proximate carcinogen, but, in contrast, had no effect on the mutagenicity of other direct-acting carcinogens such as N-methyl-N'-nitro-N-nitrosoguanidine and 9-aminoacridine. In vitro addition of (+)-catechin gave rise to a dose-dependent inhibition of the cytochrome P-450-dependent benzphetamine N-demethylase and ethoxyresorufin O-deethylase activities. This was achieved by impairment of the electron flow from the reduced pyridine nucleotide to the cytochrome. However, administration of (+)-catechin to rats had no effect on the in vitro mixed-function oxidase activities. It is concluded that the (+)-catechin inhibits the mutagenicity of aromatic amines in the Ames test by interfering with their cytochrome P-450-dependent bioactivation and by direct interaction with the proximate carcinogen, but the former mechanism is unlikely to occur in vivo because the high doses of the flavonoid required are not achieved.