Black tea polyphenols-mediated in vivo cellular responses during carcinogenesis.

Tea (Camellia sinensis), a popular beverage, is consumed worldwide. The biological activities and mechanism(s) of chemopreventive effects of green tea polyphenols (monomeric catechins) have been extensively studied, while similar information regarding newly formed major black tea polyphenols (BTPs-oligomeric, polymeric) is not available. Therefore, this review focuses mainly on compiling the evidence on chemopreventive efficacy of black tea extract (BTE) / BTPs and describing their mechanism(s) of anti-initiating, anti-promoting and anti-progressor action(s) in in vivo experimental systems. Overall, the mechanism(s) implicated in the BTPs-mediated inhibition are diverse and involve effects on multiple molecular pathways and genes.

Effect of polymeric black tea polyphenols on benzo(a)pyrene [B(a)P]-induced cytochrome P4501A1 and 1A2 in mice.

The chemopreventive activity of green tea polyphenols (GTPs) is, in part, due to modulation of cytochrome P450s (CYPs). To investigate the enzyme modulatory properties of major black tea polyphenols, the effect of decaffeinated black tea extract (DBTE) or polymeric black tea polyphenol (PBP) mix was studied on CYP1A1 and CYP1A2 in mouse tissues. Animals receiving 2.5% DBTE or 1% PBP mix or drinking water (15 days) were challenged with single oral benzo(a)pyrene (B(a)P) (1 mg/mouse) treatment on the 14th day. Liver and lung microsomes isolated after 24 h were analysed for CYP1A1 and CYP1A2, using biochemical substrate(s) and Western blot analysis. Treatment with 2.5% DBTE or 1% PBP mix did not significantly alter the basal activity and level of CYP1A1 and CYP1A2, whereas pretreatment with 2.5% DBTE or 1% PBP mix resulted in a significant decrease in both the activity and the level of B(a)P-induced CYP1A1 and CYP1A2 in liver and lungs. The PBP mix possesses enzyme modulatory properties exhibited by monomeric GTPs.

Inhibition of nitrosodiethylamine-induced hepatocarcinogenesis by dietary turmeric in rats.

Turmeric, widely used in food and medicine has been shown to prevent benzo(a)pyrene [B(a)P] or dimethylbenz(a)anthracene (DMBA)-induced forestomach, skin and mammary tumors in mice and/or rats. In this study we examine the modulatory effects of turmeric on nitrosodiethylamine (NDEA)-induced hepatocarcinogenesis in rats. Female Wistar rats were administered NDEA (200 ppm) through drinking water (5 days per week) for 4 weeks. Control and/or NDEA-treated rats received 0, 0.2, 1.0 or 5.0% turmeric diet (w/w) either before (2 weeks), during (4 weeks) and after NDEA exposure (10 weeks) or starting from 24 h after NDEA exposure for 10 weeks. NDEA-treated rats receiving 1 or 5% turmeric before, during and after carcinogen exposure showed significant decrease in number of gamma glutamyl transpeptidase (GGT) positive foci measuring >500 or >1000 microm and decrease in the incidence of NDEA-induced focal dysplasia (FD) and hepatocellularcarcinomas. Decrease in the number of GGT positive foci measuring >1000 microm was also observed in NDEA-treated rats receiving 0.2% turmeric, although no decrease in tumor incidence was noted. On the other hand, similar levels of turmeric treatment (0.2, 1 and 5%) after exposure to NDEA did not show any protective effects. The underlying mechanism(s) of chemoprevention of NDEA-induced hepatocarcinogenesis need to be explored.

Inhibition of cytochrome P450 isozymes by curcumins in vitro and in vivo.

To study the mechanism(s) of turmeric-mediated chemoprevention and to compare the chemopreventive efficacy of turmeric/curcumin(s) against benzo[a]pyrene (B(a)P) and 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK, a tobacco-specific carcinogen), the effects of turmeric/curcumin (C), demethoxycurcumin (dmC), bis-demethoxycurcumin (bdmC) and phenyl and phenethyl-isothiocyanates (PITC and PEITC) on the dealkylation of ethoxyresorufin (ER), methoxyresorufin (MR) and pentoxyresorufin (PR) by rat liver microsomes (in vitro) were studied. These reactions are predominantly mediated by cytochrome P450 (CYP450) isozymes 1A1, 1A2 and 2B1, respectively. In vitro incubation of rat liver microsomes with each of the compounds--C, dmC, bdmC, PITC and PEITC--showed a dose-dependent decrease in carbon monoxide binding to microsomes and also showed a dose-dependent inhibition of CYP 1A1, 1A2 and 2B1 activity, as judged by a decrease in formation of resorufin from respective biochemical probes used. Both the isothiocyanates inhibited activity of CYP 2B1 more readily than that of CYP 1A1/1A2. Significantly lower concentrations of curcumin(s) than isothiocyanates achieved 50% inhibition of activity of CYP 1A1 and 1A2, while concentrations of C (4 microM), bdmC (2.5 microM) required to inhibit CYP 2B1 were slightly higher than that of PEITC (1.3 microM), suggesting curcumin(s) to be effective inhibitors of CYP 2B1 as well. Pretreatment of rats with 1% turmeric through the diet resulted in a significant decrease in induction of B(a)P-induced CYP 1A1 and 1A2 and phenobarbitone (PB)-induced CYP 2B1 in liver, lung and stomach, although the extent of the decrease was different. These results suggest that turmeric/curcumin(s) as in the case of isothiocyanate, PEITC, are likely to inhibit activation of carcinogens metabolized by CYP450 isozymes, namely, CYP 1A1, 1A2 and 2B1.

Effects of turmeric on the activities of benzo(a)pyrene-induced cytochrome P-450 isozymes.

Turmeric and/or its main coloring component, curcumin (diferuloylmethane), have been shown to inhibit benzo(a)pyrene [B(a)P]-induced forestomach papillomas in mice. However, the mechanisms of turmeric-mediated chemoprevention are not well understood. To study the mechanisms of turmeric-mediated chemoprevention, we investigated the effects of turmeric feeding on the activities of isozymes of cytochrome P-450 (CYP450)--namely, ethoxyresorufin O-deethylase (EROD, CYP1A1) and methoxyresorufin O-demethylase (MROD, CYP1A2)--which are predominantly involved in the metabolism of B(a)P. We determined the activities of EROD and MROD by monitoring the formation of resorufin from respective substrates in the presence of microsomal proteins obtained from tissues of control, 1% turmeric, 1 mg B(a)P, and 1% turmeric + 1 mg B(a)P-fed Swiss mice. The results indicate that the administration of turmeric through diet significantly inhibited the activities of both EROD and MROD in forestomach (target organ), liver, and lung. In vitro studies employing curcumin, demethoxycurcumin, and bis-demethoxycurcumin suggest that curcumins are the inhibitors in turmeric. Inhibition of B(a)P metabolizing phase I enzymes (EROD, MROD) may be at least in part one of the possible modes of chemopreventive action of turmeric/curcumin.

Distribution of major and minor alkaloids in tobacco, mainstream and sidestream smoke of popular Indian smoking products.

Various Indian smoking products--cigarette, bidi, chutta and a brand of US cigarette--were analysed by gas chromatography-flame ionization detection (GC-FID) for the levels of nicotine and minor tobacco alkaloids in tobacco, mainstream smoke (MS) and sidestream smoke (SS) employing modified smoking standards, namely two puffs/min. The analysis clearly demonstrated relatively higher levels of nicotine and minor tobacco alkaloids in tobacco from bidi (37.7 mg/g) and chutta (34.5 mg/g) when compared with Indian and US cigarettes (14-16 mg/g) studied. Relatively lower levels (SS/MS) of nicotine in SS from bidi and chutta compared with Indian/US cigarettes, suggest that the contribution of nicotine in SS from a single bidi/chutta to environmental tobacco smoke (ETS) is very much less than that of a single Indian/US cigarette. Reduced levels of nicotine in SS of bidi/chutta result in relatively higher deliveries of nicotine in MS as reflected by higher MS/SS values. The observed differences are likely to be due to difference in tobacco processing, burning rate/temperature and design of the smoking product.

Subchronic oral hepatotoxicity of turmeric in mice--histopathological and ultrastructural studies.

Dietary administration of the whole spice turmeric (0.2%, 1.0%, 5.0%) or ethanolic turmeric extract (ETE, 0.05%, 0.25%) for 14 days, at doses reported to be cancer preventive in model systems, were found to be hepatotoxic in mice. Histopathological evaluation showed coagulative necrosis accompanied by a zone of regenerating parenchymal cells of liver. The ultrastructural changes in liver parenchymal cells were non-specific reaction to injury. Results suggest mouse to be a susceptible species for turmeric induced toxicity.

Subchronic oral toxicity of turmeric and ethanolic turmeric extract in female mice and rats.

Subchronic oral toxicity of turmeric and ethanolic turmeric extract was studied in female Swiss mice and Wistar rats fed turmeric (0, 1 and 5%) and ethanolic turmeric extract (0, 0.05 and 0.25%) through diet for 14 and/or 90 days. The administration of a high dose of turmeric (5%) for longer duration (90 days) showed a significant reduction in body weight gain, alterations in absolute and/or relative liver weights, and hepatotoxicity i.e. focal necrosis or focal necrosis with regeneration both in mice and rats. In mice lower doses of turmeric i.e 0.2 or 1% for 14 days also showed hepatotoxicity and they were found to be more vulnerable to turmeric-induced hepatotoxicity than rats.

Chemopreventive efficacy of curcumin-free aqueous turmeric extract in 7,12-dimethylbenz[a]anthracene-induced rat mammary tumorigenesis.

The modulating effects of turmeric (T), ethanolic turmeric extract (ETE) and curcumin-free aqueous turmeric extract (CFATE) on the initiation or post-initiation phases of DMBA-induced mammary tumorigenesis were investigated in female Sprague-Dawley rats. Dietary administration of 1% T/0.05% ETE 2 weeks before, on the day of DMBA treatment (day 55) and 2 weeks after the single dose (15 mg/animal) of DMBA (during the initiation period) resulted in significant suppression of DMBA-induced mammary tumorigenesis as seen by a reduction in tumor multiplicity, tumor burden and tumor incidence. However, simultaneous administration of 1% T-derived CFATE as the sole source of drinking water during the initiation phase did not suppress DMBA-induced mammary tumorigenesis. Dietary administration of 1% T/0.05% ETE or 1% T-derived CFATE as the sole source of drinking water starting 48 h after DMBA treatment and continuing until the end of the experiment (during the post-initiation period) resulted in significant suppression of DMBA-induced mammary tumorigenesis as seen by reduction in the tumor multiplicity and/or tumor burden although tumor incidence was unaffected. The present data clearly indicate that dietary administration of T/ETE showed strong chemopreventive activity during initiation as well as post-initiation phases of DMBA-induced rat mammary tumorigenesis while CFATE was found to be weakly active only when it was administered during the post-initiation phase.

Inhibitory effects of curcumin-free aqueous turmeric extract on benzo[a]pyrene-induced forestomach papillomas in mice.

The modulating effects of curcumin-free aqueous turmeric extract (CFATE), ethanolic turmeric extract (ETE) and turmeric (T) powder on the benzo(a)pyrene (B(a)P)-induced forestomach tumors were investigated in Swiss female albino mice receiving oral administration of B(a)P at a dose of 1 mg twice weekly for 4 weeks. Administration of 0.2%/1.0%/5.0% turmeric-derived CFATE as sole source of drinking water or 0.01%/0.05%/0.25% ETE in diet or 0.2%/1.0%/5.0% T in diet, 2 weeks before, during and 2 weeks after the last dose of B(a)P (during initiation period) resulted in significant suppression of B(a)P-induced tumorigenesis when compared with the group receiving B(a)P and control diet/drinking water. Among different fractions tested, CFATE appears to be more powerful as not only did it reduce the tumor multiplicity to the lowest levels but it also significantly reduced the tumor incidence. Administration of 5.0% turmeric-derived CFATE as the sole source of drinking water or 0.25% ETE/5.0% T in diet starting from 48 h after the last dose of B(a)P (during the post-initiation period) until the termination of the experiment, also inhibited the formation of multiple gastric tumors by B(a)P, although the suppression of tumor multiplicity was appreciably more in the groups that received 5.0% turmeric-derived CFATE/0.25% ETE treatment during initiation with carcinogen, i.e. 2 weeks before, during and 2 weeks after the last dose of B(a)P. The present data clearly indicate the potential of turmeric-derived CFATE as a powerful chemopreventive fraction and also demonstrate the efficacy of lower, i.e. 1/25th and/or 1/5th of the reported, chemopreventive doses of T/ETE (essentially curcumins) in inhibiting B(a)P-induced forestomach tumors in mice.

Effects of curcumin on the formation of benzo[a]pyrene derived DNA adducts in vitro.

The effects of turmeric (T), curcumins (Cs), aqueous turmeric extract (ATE) and curcumin-free aqueous turmeric extract (CFATE) on the formation of [3H]benzo[a]pyrene ([3H]B(a)P)-derived DNA adducts was studied in vitro employing mouse liver S9. A dose-dependent decrease in binding of [3H]B(a)P metabolites to calf thymus DNA was observed in the presence of T, Cs and ATE but not in the presence of CFATE, suggesting curcumins to be active principles. Further studies employing mouse liver microsomes and individual components of curcumins, i.e. curcumin (C), demethoxycurcumin (dmC) and bisdemethoxycurcumin (bdmC) showed that all the three components brought about dose-dependent; inhibition of [3H]B(a)P-DNA adduct formation and inhibitory activity was in the order C > dmC > bdmC. Investigations on the inhibitory effect of curcumin showed a dose-dependent decrease in cytochrome P450 and aryl hydrocarbon hydroxylase (AHH) activity resulting in relatively larger amounts of unmetabolized B(a)P in the presence of curcumin. Comparison of structures of curcumins with their activity profile suggested the importance of both parahydroxy (p-OH) and methoxy groups (-OCH3) in the structure activity relationship. Experiments to study the mechanism of action of curcumin indicated that the presence of curcumin was essential for the inhibitory effect, as removal of curcumin resulted in restoration of cytochrome P450 activity and the levels of [3H]-B(a)P-DNA adducts to control values. The present studies demonstrate that curcumin is effective in inhibiting [3H]B(a)P derived DNA adducts by interfering with the metabolic enzymes and its physical presence is essential for this effect.

Dose-response effects of malachite green on free radical formation, lipid peroxidation and DNA damage in Syrian hamster embryo cells and their modulation by antioxidants.

Malachite green (MG), consisting of green crystals with a metallic lustre, is very soluble in water and is highly cytotoxic to mammalian cells and also acts as a liver tumour promoter. In view of its industrial importance and possible exposure to human beings, MG poses a potential environmental health hazard. We have earlier reported the possible involvement of reactive free radicals in morphological transformation of Syrian hamster embryo (SHE) cells by MG. In this study we have studied the dose-response effects of MG on free radical formation, lipid peroxidation and DNA damage in SHE cells. Electron spin resonance analysis with 5,5-dimethyl-1-pyrroline-N-oxide as a spin-trapping agent was used to study the production of free radicals from MG. Exposure of SHE cells to MG demonstrated a dose-dependent increase in the generation of free radicals, lipid peroxidation and DNA damage. Treatment of SHE cells with antioxidant enzymes such as catalase (CAT) and glutathione peroxidase (GPx) prior to MG exposure decreased lipid peroxidation and DNA damage, with CAT being more effective than GPx. Since metabolism of MG leads to the generation of free radicals, and CAT and GPx decreased MG-induced lipid peroxidation and DNA damage, the present study confirms the possible relationship between the genotoxicity of SHE cells by MG and the involvement of reactive free radical formation.

Evaluation of frequency of micronucleated oral mucosa cells as a marker for genotoxic damage in chewers of betel quid with or without tobacco.

The frequency of micronucleated cells (MNC) derived from exfoliated human oral mucosal cells has been measured to assess genotoxic damage in chewers of betel quid with tobacco (BQT) and tobacco with lime (T). Significantly elevated frequencies of MNC were observed in the exposed groups (BQT = 4.83 +/- 0.70; T = 5.20 +/- 0.66 per 1000 cells) compared to the control group (C = 2.59 +/- 0.37) although the levels observed were lower than those reported in the literature. No correlation was seen between age, duration and frequency of habits and the frequency of MNC in the 2 habit groups. Clastogenic agents in betel quid possibly involved in micronucleus formation are discussed.

Catechin as an antimutagen: its mode of action.

In the present study, we report that the betel quid ingredient catechu, its extract and pure principle catechin were nonmutagenic in Salmonella typhimurium TA 100, TA 1535, TA 98, and TA 1538 assays with or without metabolic activation. They also exhibited dose-dependent decreases in mutagenicity of benzo(a)pyrene [B(a)P] and dimethylbenz(a)anthracene (DMBA) in strain TA 98 with metabolic activation. We further report that these compounds inhibited activities of cytochrome P-450 and had no effect on glutathione-S-transferase but increased the glutathione content in rat liver tissue. Simultaneous treatment of catechin prevented the mutagenic activity of B(a)P and DMBA metabolites in strain TA 98 in the absence of metabolic activation. Pre- and post-treatment of bacteria with catechin had no effect on the mutagenicity of B(a)P and DMBA metabolites. Catechin also inhibited the in vitro binding of 3H-B(a)P metabolites to calf thymus DNA. Catechu extract and catechin inhibited the nitrosation of methylurea by nitrite at pH 3.6 and 30 degrees C. The formation of nitrosomethylurea in the reaction mixture was monitored by measuring the histidine revertants of strain TA 1535 in the absence of metabolic activation. Pre- and post-treatment of catechu extract or catechin had no effect on the mutagenicity of nitrosomethylurea in TA 1535. The nitrosation inhibition by catechin was through scavenging of nitrite observed at pH 3.6. The above study indicates that catechu in betel quid may act as an antimutagen and may suppress the mutagenic potential of other betel quid mutagens.

Formation and persistence of isoniazid-DNA adducts in mouse tissues.

Further confirmation is provided to support the identity of the product formed by the in vitro reaction of isoniazid (INH) with cytosine as 4-deamino-4-isoniazidocytosine (INH-cytosine). Use of INH-treated mice, in which the tissue DNA is prelabelled by the neonatal administration of [3H]-deoxycytidine, has revealed the presence of two other DNA products in addition to INH-cytosine. Tissue differences in the persistence of these three DNA products suggest the presence of repair reactions for certain adducts. These processes and the effects of hepatotoxicity lead to a selective retention of adducts in the DNA of lung which is the target tissue for INH-carcinogenicity in mice. INH-modified DNA templates are weakly promutagenic during in vitro DNA synthesis. The implications of these observations for the role of INH as an initiating agent and for the species differences in its carcinogenicity are discussed.

Preliminary observations on interaction of 14C-metronidazole with macromolecules in vivo and in vitro.

Preliminary studies on the in vivo and in vitro interactions of 14C-metronidazole with macromolecules showed that the agent or its metabolite(s) can interact with nucleic acids and proteins in vivo. In vitro studies suggest that in absence of DNA synthesis trace amount of metronidazole does bind to DNA/protein and addition of metabolic activation system (from mouse liver) generates more reactive species from metronidazole.

Mutagenicity and carcinogenicity of mono- and diacetyl hydrazine.

Two hydrazine derivatives, monoacetyl hydrazine (MAH) and diacetyl hydrazine (DAH), have been tested for mutagenic response in the Salmonella/mammalian microsome assay and micronucleus test. MAH but not DAH, increased the revertant mutants in TA100 and TA1535 and also increased the frequency of micronuclei in polychromatic erythrocytes. Gavage administration of MAH but not of DAH, resulted in increased incidence of lung tumors. These observations record for the first time the mutagenicity/carcinogenicity of MAH which is one of the metabolites of isoniazid in animals and humans.