Tomoe Negishi

Dietary inhibitors of mutagenesis and carcinogenesis

Dietary inhibitors of mutagenesis and carcinogenesis are of particular interest because they may be useful for human cancer prevention. Several mutagenesis inhibitors have been demonstrated to be carcinogenesis inhibitors also, e.g., ellagic acid, palmitoleic acid, and N-acetylcysteine. This means that the search for mutagenesis inhibitors may be useful for discovering anticarcinogenic agents. Many mutagenesis inhibitors have been discovered by the use of short-term assays, particularly the Ames Salmonella test. This simple in vitro system has provided opportunities to elucidate the mechanisms of inhibition. The elucidation of the mechanism may allow us to infer the possible anticarcinogenic activity of the reagent. In this chapter, inhibitors of mutagenesis and carcinogenesis that can arise as components of diet have been reviewed. Most of the inhibitors have been demonstrated to be effective against a specific class of mutagens or carcinogens. Therefore, it may be argued that these inhibitors are antagonistic only to those particular agents. Here again, understanding of the mechanisms of these inhibitions is necessary for the assessment. Dietary inhibitors reviewed in this article include: (1) as inhibitors of mutagenesis: porphyllins, fatty acids, vitamins, polyphenols, and sulfhydryl compounds, (2) as inhibitors of carcinogenesis: vitamins A, E and C, ellagic acid, sulfhydryl compounds, fats, selenium, calcium, and fiber. Further studies in this area of science appear to help establish the recipe of a healthy diet.

Inhibition of N-nitrosation of secondary amines in vitro by tea extracts and catechins

Inhibition of nitrite-mediated N-nitrosation of dimethylamine, morpholine and N-methylaniline by tea extracts and by 6 individual catechins in the extracts was studied. The inhibitions were detected by quantifying the nitrosamines formed. Eight different kinds of teas (5 green teas, a roasted green tea, an oolong tea, and a black tea) were examined for their inhibitory abilities and for their catechin contents, with an attempt to correlate the inhibitory activities to the catechin contents. The results showed that (1) the green tea extracts inhibit strongly the N-nitrosation of the three secondary amines tested, (2) the 6 catechins, notably epigallocatechin, are capable of blocking the N-nitrosations very efficiently, even more efficiently than ascorbic acid, and (3) the inhibition activities of green tea extracts are mostly ascribable to the catechins present in the extracts. These inhibitions occur by rapid reactions between nitrite and the catechins. It was observed that no mutagenicity results from the reaction between the tea extracts and nitrite.

Inhibition of human cytochrome P450 1B1, 1A1 and 1A2 by antigenotoxic compounds, purpurin and alizarin

Recently we have shown that anthraquinone food pigments such as purpurin and alizarin suppress the genotoxic activities of several mutagens including heterocyclic amines and polycyclic aromatic hydrocarbons in the Drosophila DNA repair test and in the Ames test. To investigate the mechanism of this inhibition, we have now examined the effects of these anthraquinone pigments on enzymes that metabolize xenobiotics. The activities of eight human recombinant cytochrome P450 (CYP) isozymes were measured in the presence of purpurin, alizarin or carminic acid. Purpurin and alizarin strongly inhibited the activities of CYP1A1, CYP1A2 and CYP1B1, and weakly suppressed those of CYP2A6 and CYP2E1 in a dose-dependent manner, but did not inhibit those of CYP2C19, CYP3A4 and CYP3A5. Carminic acid did not affect the activities of any CYPs tested. CYP1B1 was the most strongly affected CYP molecule by purpurin and alizarin among CYPs examined in this study. From kinetic analysis, it was shown that the inhibition by purpurin on CYP1B1 was both competitive and non-competitive, and that by alizarin was competitive. The values of slopes obtained from Lineweaver-Burk plots are proportional to the square of purpurin concentration. This observation suggests that two molecules of purpurin are interacting with one molecule of CYP1B1. The K(m) value of CYP1B1 was 11 microM, and the K(i) value of purpurin and alizarin against CYP1B1 was 0.7 microM(2) and 0.5 microM, respectively. We also examined the effects of these pigments on the mutagenicities of MeIQx and B[a]P in the Ames test, using Salmonella typhimurium TA1538 co-expressing each form of human CYP and NADPH-cytochrome P450 reductase (OR). The mutagenicity of MeIQx in TA1538 1A2/OR or 1B1/OR was suppressed by purpurin and alizarin but not by carminic acid. Purpurin also reduced the mutagenicity of B[a]P in TA1538 1A1/OR or 1B1/OR. These results suggest that the antigenotoxic activities of purpurin and alizarin can be explained by inhibition of CYP activities responsible for activating the mutagens.

Porphyrins as potential inhibitors against exposure to carcinogens and mutagens

Studies have shown that there are many substances that can interfere with the actions of carcinogens and mutagens. Porphyrins, which often are constituents of diet, are a class of such inhibitors. Hemin can inhibit selectively the activity of mutagens having polycyclic structures by forming complexes with them. These effects were found with the use of bacterial assays and also by in vitro chemical experiments. A survey of porphyrins for similar effects has been done in our laboratory and it was found that chlorophyll and chlorophyllin act like hemin. These green pigments are antimutagenic in Salmonella and in Drosophila. Work from other laboratories also has supported the antimutagenic character of chlorophyllin. The possibility of modifying human exposure to carcinogens by use of these porphyrins is discussed. A porphyrin-like molecule, copper phthalocyanine trisulfonate, has been shown to have strong affinity to polycyclic compounds. Blue cotton, a cotton preparation bearing this blue pigment as a covalently bound ligand, has been demonstrated to be an adsorbent useful for isolating heterocyclic amines from food and other materials.

Preventive effects of anthraquinone food pigments on the DNA damage induced by carcinogens in Drosophila

We have previously demonstrated the inhibitory effect of chlorophyllin, a green food additive, on the genotoxicities of various carcinogens in Drosophila. Recently, we reported that purpurin, a component of a red food additive produced from madder root (Rubia tinctorium), inhibits the bacterial mutagenicity of heterocyclic amines. In the present study, we examined antigenotoxic activities of various pigments that are either constituents of food or food additives, using Drosophila in vivo DNA repair assay. Third instar larvae of Drosophila were fed a mutagen with or without pigment. The resulting adult flies were monitored for their male (repair deficient)/female (repair proficient) ratios, which reflect the DNA damage. We tested a total of 20 pigments, which are mainly of plant origins, including flavonoids, carotenoids, anthocyanins, anthraquinones and beta-diketone (curcumin)-derivatives, against the genotoxicities of eight carcinogens; IQ, MeIQx, AFB1, NDMA, 2-AAF, DMBA, 4NQO, and MNU. Four anthraquinone pigments (alizarin, purpurin, lac color, and cochineal extract) showed significant antigenotoxic activities. Alizarin and purpurin suppressed the DNA damage induced by IQ, MeIQx, AFB1, NDMA, 2-AAF, DMBA, and MNU. Lac color and cochineal extract showed inhibition against IQ, MeIQx, AFB1, 2-AAF and DMBA. In these inhibitions, suppression of metabolic enzymes may be involved. Since purpurin and alizarin suppressed the activity of MNU, a direct alkylating agent, there may also be a mechanism distinct from enzyme inhibitions in these anthraquinone-mediated suppressions of DNA damage.

The genotoxicities of N-nitrosamines in Drosophila melanogaster in vivo: the correlation of mutagenicity in the wing spot test with the DNA damages detected by the DNA-repair test

The genotoxicities of a series of N-nitrosamines were assayed in the wing spot test and a new short-term test of Drosophila melanogaster. In the spot test, larval flies trans-heterozygous for the somatic cell markers mwh and flr3 were fed the test reagents and the wing hairs in adults were inspected for clones expressing the phenotypes of the markers. In the other test, larval stock consisting of meiotic recombination-deficient (Rec-) double mutant mei-9a and mei-41D5 males and repair-proficient Rec+ females were grown on feed containing the reagents and the DNA damages were detected with the preferential killing of the Rec- larvae as an endpoint. The carcinogenic nitrosamines tested, N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA), N-nitrosodi-n-butylamine (NDBA), N-nitrosomorpholine (NMOR), N-nitro-sopiperidine (NPIP) and N-nitrosopyrrolidine (NPYR), all showed clearly positive activities in both tests. The activities in the wing spot test were ranked in a sequence of NDMA much greater than NMOR greater than NPIP greater than NDEA greater than NPYR greater than NDBA. A similar ranking was obtained in the repair assay. The genotoxicity of N-nitrosodiphenylamine (NDPhA), carcinogenicity studies of which are inconclusive, was marginal in the spot test. The non-carcinogenic N-nitrosoproline (NPRO) and the non-mutagenic N-nitrosothioproline (NTPRO) were negative in the spot test. NDPhA and NPRO were negative in the repair test as well. The DNA-repair test is thus a convenient technique for estimating the mutagenicity of compounds because of its simplicity compared with the wing spot test. These Drosophila tests may be useful in predicting carcinogenic potentials of compounds.

Inhibitory activity of chlorophyllin on the genotoxicity of carcinogens in Drosophila

Antimutagenic activity of copper chlorophyllin against various carcinogenic mutagens was assayed with Drosophila genotoxicity tests, i.e., the wing spot test for detecting somatic cell mutations and the DNA repair test for detecting DNA damage. In these tests, Drosophila larvae were fed carcinogens together with chlorophyllin. Polycyclic aromatic compounds, including heterocyclic amines, polycyclic aromatic hydrocarbons, aromatic amines and aromatic nitro compounds, were subject to inhibition, with a few exceptions. The results support the view that chlorophyllin traps carcinogens by forming complexes, thereby inhibiting the absorption of these compounds from the digestive tract. Consistent with this mechanism, Sepharose-supported chlorophyllin in the feed inhibited the Trp-P-2-induced wing spot formation, while Sepharose itself was ineffective.

The pH-dependent response of Salmonella typhimurium TA100 to mutagenic N-Nitrosamines

The mutagenicity of some N-nitrosodialkylamines, i.e. N-nitrosodimethylamine, N-nitrosodiethylamine, N-nitrosodi-n-butylamine, N-nitrosomorpholine and N-nitrosopyrrolidine, was assayed on Salmonella typhimurium TA100 by the pre-incubation method, and the effect of changing the pH of the pre-incubation mixture was examined. Markedly higher mutagenicities were observed when the pre-incubation of bacteria with nitrosamine and S9 mix was done at pH 5.2, compared with mutagenicities observable after the pre-incubations at conventional pH 7. Pre-incubations at pH 6.2 resulted in responses of intermediate strength. With phenobarbital-induced rat S9, the ratios of mutagenic potency found by the pH 5.2 pre-incubation to that found by the pH 7.2 preincubation were 15-30 for N-nitrosodimethylamine, 5-10 for N-nitrosodiethylamine, 10-20 for N-nitrosodi-n-butylamine, 2-3 for N-nitrosomorpholine and 4-6 for N-nitrosopyrrolidine. The mutagenic potency of each nitrosamine varied with the change of S9 source. The S9 sources examined were PCB-induced rat and mouse livers, and uninduced rat and mouse livers. No exceptions were observed for these S9 preparations regarding the higher mutagenicity at pH 5 than at pH 7. It is speculated that the higher mutagenicity observed by the pH 5 pre-incubation was due to the stability of the active intermediate, alpha-hydroxynitrosodialkylamines, in weakly acidic media.

Mutagenicities of N-nitrosodimethylamine and N-nitrosodiethylamine in Drosophila and their relationship to the levels of O-alkyl adducts in DNA

N-Nitrosodialkylamines are potent carcinogens in experimental animals. Previously, we reported that the mutagenicity of N-nitrosodimethylamine (NDMA) was 10 times higher than that of N-nitrosodiethylamine (NDEA) in the Drosophila wing spot test. To find out how to explain this difference, we have measured the levels of O-alkylated bases in the DNA of exposed Drosophila larvae. Third instar larvae were fed for 3 or 6 h with NDMA or NDEA. Part of the treated larvae were grown to adult flies to score their wings for the presence of mutant spots. From the remaining larvae, DNA was isolated and digested to deoxyribonucleosides, and the digest fractionated by high-performance liquid chromatography (HPLC). The amounts of specific alkyldeoxyribonucleosides present in the fractions were quantified by a radioimmunoassay (RIA) using monoclonal antibodies. Dose-dependent O6-methylguanine, O6-ethylguanine and O4-ethylthymine formations were found to be correlated with the induction frequencies of mutant wing spots. At the same exposure dose, the values of O6-alkylde- oxyguanosine/106 deoxyguanosine were similar for NDMA and NDEA: on feeding 20 micromol/1.5 ml feeding solution, the values for NDMA were 4.0 with 3 h and 18.5 with 6 h of exposure; with 20 micromol NDEA, the corresponding values were 5.4 with 3 h and 14.6 with 6 h of exposure. The wing spot frequencies were very different; however, with NDMA, the total numbers of spots/wing were 3.5 (3 h) and 15 (6 h), and with NDEA 0.8 (3 h) and 0.9 (6 h). Similar discrepancies exist as well between the mutagenicities and the alkylation rates observed for O4-alkylthymidines. These results suggest that the difference between the mutagenic potencies of NDMA and NDEA cannot be explained by the amounts of O-alkyl adducts formed. Different mechanisms are considered by which NDMA and NDEA may produce the genetic effects observed.

Effect of aflatoxin B1 on UDP-glucuronosyltransferase mRNA expression in HepG2 cells

Aflatoxin B1 (AFB1) is a potent mycotoxin that induces hepatocellular carcinoma in many animal species, including humans. In this study, we examined the effects of AFB1 on UDP-glucuronosyltransferase (UGT) mRNA expression in HepG2 cells (human hepatocellular carcinoma cell line). The cells were treated with AFB1 for 48 h at a concentration of 10 μM, and their viability (87%) was not significantly different from that of control cells. Reverse transcription polymerase chain reaction (RT-PCR) analysis demonstrated that the mRNAs of four UGT1As (UGT1A1, UGT1A3, UGT1A4 and UGT1A9) and seven UGT2Bs (UGT2B4, UGT2B7, UGT2B10, UGT2B11, UGT2B15, UGT2B17 and UGT2B28) are expressed in HepG2 cells. The mRNAs of aryl hydrocarbon receptor (AhR), pregnane X receptor (PXR), retinoid X receptor (RXR) and glucocorticoid receptor (GR) as transcriptional regulators were also detected. AFB1 significantly increased mRNA levels of UGT1A3, UGT2B10, UGT2B15 and UGT2B17 in HepG2 cells to 2.5-, 2.0-, 1.9- and 1.5-fold, respectively, whereas the mRNA levels of transcriptional regulators were hardly affected by AFB1. These findings suggest that AFB1 induces UGT2B isoforms rather than UGT1A isoforms in HepG2 cells, and that the change may closely contribute to the toxicity of AFB1.