Robert W. Teel

Ellagic acid binding to DNA as a possible mechanism for its antimutagenic and anticarcinogenic action

Ellagic acid (EA), a plant phenol, is reported to possess antimutagenic and anticarcinogenic activity. In the present study, explants of esophagus, trachea, colon, forestomach and bladder from young male Sprague-Dawley rats were incubated in medium containing [3H]EA (4.5 mu Ci/ml) for 24 h at 37 degrees C. DNA from these explants was extracted, purified and quantitated to determine [3H]EA binding to the DNA. Significant covalent binding of [3H]EA to DNA occurred in all the explants. Calf thymus DNA incubated in 0.05 M sodium phosphate buffer containing [3H]EA covalently bound [3H]EA in a concentration dependent manner. Furthermore covalent binding of [3H]EA to calf thymus DNA was inhibited by the addition of unlabeled EA that was concentration dependent over a range of 50-150 microM and by the addition of unlabeled adenosine, cytidine, guanosine or thymidine at a concentration of 1.0 mM. These results suggest that one of the mechanisms by which EA inhibits mutagenesis and carcinogenesis is by forming adducts with DNA, thus masking binding sites to be occupied by the mutagen or carcinogen.

Modulation by phytochemicals of cytochrome P450-linked enzyme activity

Compounds derived from plant sources with putative anticancer properties were studied for their effects on alkoxyresorufin O-dealkylase activity, a measure of cytochrome P450 activity. The phytochemicals investigated included benzyl isothiocyanate, caffeic acid, chlorogenic acid, diosmin, ferulic acid, indole-3-carbinol, phenethyl isothiocyanate and resveratrol. Each phytochemical at concentrations of 0.25 and 0.5 microM was incubated with 0.2 mg hamster liver microsomal protein and 0.5 microM concentrations of benzyloxyresorufin, ethoxyresorufin and methoxyresorufin. Three of the phytochemicals tested, namely benzyl isothiocyanate, phenethyl isothiocyanate and resveratrol, exhibited potent inhibition of alkoxyresorufin O-dealkylase activity. Benzyl isothiocyanate inhibited benzyloxyresorufin O-dealkylase (BROD) activity, ethoxyresorufin O-deethylase (EROD) activity and methoxyresorufin O-demethylase (MROD) activity by 90% at both the 0.25 and 0.5 microM concentrations. Phenethyl isothiocyanate inhibited BROD activity by 69%, EROD activity by 90% and MROD activity by 94% at both concentrations tested. Resveratrol inhibited BROD activity by 69% at the 0.25 microM concentration and by 78% at the 0.5 microM concentration. It inhibited EROD activity by 60% at the 0.25 microM concentration and by 80% at the 0.5 microM concentration. Resveratrol exhibited the greatest inhibitory action toward MROD, i.e. 76% and 84% at the two concentrations tested. Chlorogenic acid significantly affected BROD, EROD and MROD activity only at the 0.5 microM concentration inhibiting by 51%, 47% and 54%, respectively. Caffeic acid affected BROD and MROD activity at 0.5 microM only inhibiting BROD activity by 46% and MROD activity by 40%. Diosmin inhibited EROD activity by 11% at the 0.25 microM concentration and by 61% at 0.5 microM. It inhibited MROD by 47% and 54% at the two concentrations tested but did not significantly alter BROD activity. Ferulic acid significantly inhibited EROD and MROD activity at the 0.5 microM concentration by 28% and 32%, respectively. Indole-3-carbinol significantly inhibited BROD activity by 26% at 0.25 microM and by 42% at 0.5 microM. It inhibited EROD and MROD activity by 28% and 29% at 0.5 microM, respectively. The alkoxyresorufin O-dealkylase reactions are selective for various isoforms of cytochrome P450. Our results suggest that the phytochemicals we tested have varied effects on the enzymatic activity of isoforms of cytochrome P450 that dealkylate benzyloxyresorufin, methoxyresorufin and ethoxyresorufin and therefore may have varied effects on the metabolism of substrates for these isoforms.

Disposition of the plant phenol ellagic acid in the mouse following oral administration by gavage

1. The absorption, distribution and elimination of 3H-ellagic acid, a putative antimutagen and anticarcinogen, was studied in male Swiss-Webster mice following oral administration. 2. Levels of 3H-ellagic acid were highest in blood 30 min after administration, in urine and bile 120 min post-administration, and in liver, lung and kidney 15 min after administration [corrected]. 3. Free ellagic acid and its conjugates were present in urine, bile and blood. H.p.l.c. analysis of the organic solvent extracts of urine, bile and blood indicated the presence of four metabolites in urine, two in blood and one in bile. 4. Sulphate ester, glucuronide and glutathione conjugates of ellagic acid were present in urine, bile and blood. H.p.l.c. analysis of organic solvent extracts after aryl sulphatase or beta-glucuronidase treatment showed that ellagic acid was the major component present. 5. Absorption of 3H-ellagic acid occurred mostly within two hours after oral administration. Levels in blood, bile and tissues were low and almost all of the absorbed dose was excreted in urine. 6. More than 53% of the orally administered 3H-ellagic acid remained in the gastrointestinal tract at 24 h. Approximately 19% was excreted in faeces and 22% in urine at 24 h. 7. Of the 24 h faecal radioactivity 93% was extractable into organic solvents and more than 80% of this fraction was free ellagic acid. Only one metabolite was found in faeces.

Organosulfur compounds of garlic modulate mutagenesis, metabolism, and DNA binding of aflatoxin B1

The effects of two organosulfur compounds of garlic (ajoene and diallyl sulfide) and a crude garlic extract on aflatoxin B1 (AFB1)-induced mutagenesis were determined using rat liver 9,000 g supernatant (S-9) as the activation system and Salmonella typhimurium TA-100 as the tester strain. The effects of these compounds on AFB1 binding to calf thymus DNA were also measured. Metabolites of AFB1 were isolated and analyzed by reverse-phase high-performance liquid chromatography. All these compounds inhibited S-9-dependent mutagenesis induced by AFB1. They also inhibited AFB1 binding to DNA. A significant decrease in organo-soluble metabolites of AFB1 was observed with ajoene and garlic extract. An increase of glucuronide and glutathione conjugates was obtained with garlic extract. The results indicate that garlic compounds tested in this study are antimutagenic and, potentially, anticarcinogenic.

Effects of capsaicin on rat liver S9-mediated metabolism and DNA binding of aflatoxin.

At concentrations of 25, 50, and 100 microM, capsaicin, which is the major component in various aspects of Capsicum hot peppers, decreased the binding of aflatoxin (AFB1) to calf thymus DNA by 19%, 44%, and 71%, respectively, in incubations with rat liver S9. At concentrations of 50 and 100 microM, capsaicin decreased the formation of AFB-DNA adducts (AFB1-N7-Gua) by 53% and 75% as determined by high-pressure liquid chromatography (HPLC). HPLC analysis of organo-soluble fractions showed that these effects correlated with a concentration-dependent decrease in S9-mediated metabolism of AFB1 by capsaicin. Capsaicin also altered the formation of water-soluble conjugates of AFB1. This was indicated by a decrease in radioactivity in water-soluble fractions and in glutathione conjugates of AFB1 analyzed by HPLC. These results suggest that capsaicin inhibited the biotransformation of AFB1 by modifying Phase I hepatic enzyme activity.

Effect of allixin, a phytoalexin produced by garlic, on mutagenesis, DNA-binding and metabolism of aflatoxin B1

Allixin, a phytoalexin isolated from garlic, was examined for its effects on aflatoxin B1(AFB1)-induced mutagenesis using Salmonella typhimurium TA100 as the bacterial tester strain and rat liver S9 fraction as the metabolic activation system. The effects of allixin on the binding of [3H]AFB1 to calf thymus DNA and on the formation of metabolites of [3H]AFB1 were also determined. Allixin showed a dose-related inhibition of Histidine+ revertants induced by AFB1. Allixin at 75 micrograms/ml inhibited [3H]AFB1 binding to calf thymus DNA and reduced formation of AFB1-DNA adducts. In addition, allixin exhibited a concentration-dependent inhibition of the formation of organosoluble metabolites and the glutathione conjugates of [3H]AFB1. The data indicate that the effect of allixin on AFB1-induced mutagenesis and binding of metabolites to DNA may be mediated through an inhibition of microsomal P-450 enzymes. Allixin may thus be useful in the chemoprevention of cancer.

Effects of capsaicin on liver microsomal metabolism of the tobacco-specific nitrosamine NNK

Chemically-induced mutagenesis and carcinogenesis is modulated by various plant products, some of which are present in the human diet. 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a potent carcinogen in tobacco and tobacco smoke, is activated by microsomal enzymes. In this study, we investigated the effects of capsaicin on the in vitro metabolism of NNK. Capsaicin is the principal component of Capsicum fruits used widely by humans as a food additive. Liver microsomes from saline-injected, phenobarbital-induced and beta-naphthoflavone-induced hamsters were used. Microsomes from phenobarbital and beta-naphthoflavone-induced animals expressed decreased NNK reduction and enhanced pyridine-N-oxidation, but did not significantly alter alpha-carbon hydroxylation of NNK. Capsaicin (0.5 mM) inhibited the formation of all metabolites of NNK by all microsomal fractions and inhibited alpha-hydroxylation by phenobarbital-induced microsomes more than by either of the other two treatments. Our results suggest that capsaicin, as a naturally occurring dietary constituent, possesses antimutagenic and anticarcinogenic properties through the inhibition of xenobiotic metabolizing enzymes.

Ellagic acid metabolism and binding to DNA in organ explant cultures of the rat.

Ellagic acid (EA) is a plant phenolic compound with postulated antimutagenic and anticarcinogenic activity. In this study, explants of esophagus, forestomach, colon, bladder, trachea, lung and liver from male Sprague-Dawley rats (130-140 g) were incubated in culture medium containing [3H]EA (20 microM, 4.5 microCi/ml) for 24 h at 37 degrees C. After extraction, purification and quantitation of explant DNA significant differences in the binding of EA to the DNA was observed. The most binding occurred in esophagus and the least in lung. Analysis of the organsoluble fraction of the culture medium by high performance liquid chromatography yielded 3 metabolites of EA. None of the metabolites were identified. Elution of water-soluble metabolites from an alumina column showed that there were sulfate ester, glucuronide and glutathione conjugates of EA in the explant culture medium from all the organs. The profile of water-soluble conjugates was very similar between colon and forestomach and between trachea and lung. These results indicate that EA binds to DNA in different tissues and that tissues metabolize EA to both organosoluble and water-soluble products.

Chinese medicinal herbs modulate mutagenesis, DNA binding and metabolism of aflatoxin B1

Oldenlandia diffusa (OD) and Scutellaria barbata (SB) have been used in traditional Chinese medicine for treating liver, lung and rectal tumors while Astragalus membranaceus (AM) and Ligustrum lucidum (LL) are often used as an adjunct in cancer therapy. In this study, we determined the effects of aqueous extracts of these four herbs on aflatoxin B1 (AFB1)-induced mutagenesis using Salmonella typhimurium TA100 as the bacterial tester strain and rat liver 9000 x g supernatant as the activation system. The effects of these herbs on [3H]AFB1 binding to calf-thymus DNA were assessed. Organosoluble and water-soluble metabolites of AFB1 were extracted and analyzed by high-performance liquid chromatography (HPLC). Mutagenesis assays revealed that all of these herbs produced a concentration-dependent inhibition of histidine-independent revertant (His+) colonies induced by AFB1. At a concentration of 1.5 mg/plate, SB and OD in combination exhibited an additive effect. The trend of inhibition of these four herbs on AFB1-induced mutagenesis was: SB greater than LL greater than AM. LL, OD and SB significantly inhibited AFB1 binding to DNA, reduced AFB1-DNA adduct formation, and also significantly decreased the formation of organosoluble metabolites of AFB1. Our data suggest that these Chinese medicinal herbs possess cancer chemopreventive properties.

Modulation of cytochrome P-450IA1-mediated mutagenicity, DNA binding and metabolism of benzo[a]pyrene by Chinese medicinal herbs

Oldenlandia diffusa (OD) and Scutellaria barbata (SB) have been used in traditional Chinese medicine for treating liver, lung and rectal tumors. We previously showed that they inhibited mutagenesis, DNA binding and metabolism of benzo[a]pyrene (BaP) and aflatoxin B1 (AFB1) bioactivated by Aroclor 1254-induced rat hepatic S9. The purpose of this study was to investigate the effects of OD and SB on the cytochrome P-450IA1-mediated mutagenicity of BaP in Salmonella typhimurium TA100 using beta-naphthoflavone (beta NF)-induced rat hepatic S9. We also determined the effects of OD and SB on cytochrome P-450IA1-linked ethoxyresorufin O-deethylase (EROD) activity in beta NF-induced hepatic microsomes. In addition, we studied the effects of these two herbs on BaP metabolite binding to calf thymus DNA and using high performance liquid chromatography (HPLC) we investigated the effects of OD and SB on the metabolism of BaP by beta NF-induced S9. Our experimental results showed that OD and SB inhibited the mutagenicity of BaP in the presence of either non-induced or beta NF-induced S9. SB significantly inhibited BaP binding to DNA. These effects correlated with the inhibition of cytochrome P-450IA1-linked EROD activity in beta NF-induced microsomes and with an inhibition of beta NF-induced S9 mediated metabolism of [3H]BaP as determined by HPLC. These results suggest that OD and SB may possess antimutagenic activity by inhibiting P-450IA-mediated metabolism of BaP.