In vitro and in vivo evaluation of a standardized Curcuma longa Linn formulation in cervical cancer.

BACKGROUND: The anti-cancer activity of phytomolecules present in turmeric or haridra (Curcuma longa Linn) extracts against cancer has been described in various 'in vitro and in vivo' studies. OBJECTIVE: In the present study, in vitro and in vivo anti-cancer and chemo-preventive activity of a new standardized Supercritical Turmeric Oil Extract (SCTOE) NBFR-03 was evaluated in cervical cancer models. METHODS AND MATERIALS: In vitro cytotoxicity of this formulation was assessed at 10, 20, 40, and 80 µg/ml concentrations, in three cervical cancer cell lines (HeLa, SiHa, ME180) using Sulforhodamine B assay. The in vivo anti-cancer activity was evaluated in two groups of female nude mice; the first one was with tumor xenograft implants and at the same time treatment was started with 96 µl/kg/day p.o. and 192 µl/kg/day p.o. NBFR-03 for three months. The second group was kept as chemoprevention group where mice were pre-treated with the formulation (96 µl/kg/day p.o.) for two weeks and injected with cancer cell suspension with continued treatment for three months. RESULTS: No cytotoxicity was seen in any cell line with the extract when compared to positive control (Adriamycin 10 µg/ml). In mice the first treatment group with tumor xenograft implants did not show any significant anti-tumor activity but showed a trend where higher dose group had smaller tumor volumes as compared to lower dose group and controls (p = 0.37 and p = 0.34 respectively). The chemopreventive group with pre-treated mice also showed smaller tumor size as compared to controls (p = 0.163). CONCLUSION: NBFR-03 turmeric oil extract showed a promising trend in mice pre-treated with NBFR-03. There is a scope for further studying the potential of this extract as complementary therapy and as a chemopreventive.

Chemopreventive effect of 4'-demethyl epipodophyllotoxin on DMBA/TPA-induced mouse skin carcinogenesis.

The chemopreventive effect of topical application of 4'-demethyl epipodophyllotoxin (DMEP), an antimitotic agent, on a two-stage skin carcinogenesis model in Swiss Albino mice induced by 9, 10-dimethylbenz[a]anthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA) was investigated. Two topical applications with 0.24% DMBA over 1 week, followed later by 5 nmol of TPA twice weekly produced 100% incidence of tumors in these animals by 18 weeks. Treatment of animals with DMEP (until the end of the experiment), 30 min before TPA treatment, significantly reduced the tumor incidence, tumor volume and the conversion efficiency of papillomas to squamous cell carcinomas. The tumor formation and growth was also delayed by DMEP pre-treatment. Application of DMEP protected against the losses provoked in levels of glutathione and activity of catalase and superoxide dismutase in skin and liver of animals by the application of DMBA/TPA. Thus, DMEP might possibly be exerting its chemopreventive activity by acting as an antioxidant.

Chemosensitivity of advanced larynx carcinoma cells in vitro and significance of multidrug resistance markers in these tumors.

Thirty cases of previously untreated advanced larynx carcinoma were checked for in vitro chemosensitivity and presence of the resistance markers viz. P-glycoprotein (P-gp) glutathione-S-transferase-pi (GST-pi) and protein kinase C (PKC) overexpression. The cytotoxicity testing was done using MTT assay and the resistance markers were checked by immunohistochemical methods using monoclonal antibodies. The drug combinations employed in MIT assay were 5FU* + MTX*, 5FU + cisPt*, 5FU + Mito*, cisPt + Mito and MTX + Mito (*5FU = 5Fluorouracil, MTX-methotrexate, cisPt-cisplatin and Mito = mitomycin C). No statistically significant correlation was observed between resistance to the above drug combinations and presence of the resistance markers under consideration. A statistically significant correlation was observed between node positivity and expression of resistance markers which indicates that presence of one or more of these markers in these tumors may be considered as a negative prognosis marker. CisPt-Mito was found to be the most effective drug combination in vitro, in the cases studied.

Chemopreventive efficacy of a betel leaf extract against benzo[a]pyrene-induced forestomach tumors in mice.

The effect of betel leaf extract and some of its constituents, eugenol, hydroxychavicol, beta-carotene and alpha-tocopherol, on benzo[a]pyrene-induced forestomach neoplasia in male Swiss mice was examined. Betel leaf and its constituents decreased the number of papillomas per animal with the maximum protection, considering molar dosage, exhibited by beta-carotene and alpha-tocopherol. Except for beta-carotene, eugenol, hydroxychavicol and alpha-tocopherol increased the levels of reduced glutathione in the liver while glutathione S-transferase activity was enhanced by all except eugenol. Of seven sources, Banarasi betel leaves showed the maximum amounts of beta-carotene and alpha-tocopherol.

Mutagenicity and carcinogenicity of tea, Camellia sinensis.

Aqueous, caffeine free and tannin fractions of commercial tea and tannic acid were tested for mutagenicity in Ames test. Tea fractions of tannic acid were non mutagenic in strains TA 100, TA 98, TA 1535 and TA 1538 of Salmonella typhimurium with or without metabolic activation (rat-S9 mix) at different doses tested. In strain TA 98 the above tea fractions and tannic acid inhibited the S9 mix mediated mutagenicity of tobacco in a dose dependent manner. The different tea fractions at 60 degrees C, did not increase the tumor incidence in Swiss mice by gavage feeding. They also failed to produce tumors when injected subcutaneously. Caffeine free tea extract decreased the tobacco induced liver tumors but had no effect on lung tumors. The same fraction was ineffective in hexachlorocyclohexane induced liver tumors in Swiss mice.

Chemopreventive efficacy of betel leaf extract and its constituents on 7,12-dimethylbenz(a)anthracene induced carcinogenesis and their effect on drug detoxification system in mouse skin.

Effects of topically applied betel leaf extract (BLE) and its constituents. beta-carotene, alpha-tocopherol, eugenol and hydroxychavicol on 7,12-dimethylbenz(a)anthracene (DMBA) induced skin tumors were evaluated in two strains of mice. BLE, beta-carotene and alpha-tocopherol, significantly inhibited the tumor formation by 83, 86, 86% in Swiss mice and 92, 94 and 89% in male Swiss bare mice respectively. Hydroxychavicol showed 90% inhibition in Swiss bare mice at 24 weeks of treatment. Eugenol showed minimal protection in both strains of mice. The mean latency period and survivors in BLE, beta-carotene, alpha-tocopherol and hydroxychavicol treated groups were remarkably high as compared to DMBA alone treated group. Intraperitoneal injection of betal leaf constituents showed a significant effect on both glutathione and glutathione S-transferase levels in the Swiss mouse skin.

Effect of long-term treatment of two tobacco-specific N-nitrosamines on the vitamin A status of mice.

The effect of long-term treatment of two important tobacco-specific N-nitrosamines, N'-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), on the depot or circulating levels of vitamin A of Swiss and BALB/c male mice was studied. It was observed that treatment of both NNN and NNK in Swiss and BALB/c mice decreased liver vitamin A levels significantly. NNK treatment also caused a decrease in the levels of vitamin A in plasma.

Activation of N'-nitrosonornicotine by hydrogen peroxide in vitro.

Betel-quid ingredients were found to produce reactive oxygen species, such as superoxide anion and hydrogen peroxide, in vitro. We demonstrated that N'-nitrosonornicotine (NNN) can be converted to its active metabolite, hydrogen peroxide, nonenzymatically in the presence of ferrous ions and ethylenediaminetetracetic acid (EDTA) at pH 7.2. Three ultimate metabolites of NNN--NNN-1-N-oxide, 4-hydroxy-4-(3- pyridyl)butyric acid and 4-oxo-4-(3-pyridyl)butyric acid--and nornicotine were detected by high-performance liquid chromatography. 3H-NNN and 14C-NNN interact with calf thymus DNA in the presence of hydrogen peroxide, ferrous ion and EDTA. The results suggest that formation of reactive oxygen species in the presence of NNN may be a key factor in the initiation of oral tumours in tobacco and betel-quid chewers.

Antimutagenic and anticarcinogenic effects of betel leaf extract against the tobacco-specific nitrosamine 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK).

Earlier studies showed that betel leaf inhibits the mutagenic action of standard mutagens like benzo[a]pyrene and dimethylbenz[a]anthracene. Since tobacco-specific nitrosamines are the major carcinogens present in unburnt forms of tobacco, we studied the effect of an extract of betel leaf on the mutagenic and carcinogenic actions of one of the most potent, 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK). Betel-leaf extract and hydroxychavicol suppressed the mutagenicity of NNK in both the Ames and the micronucleus test. In studies in mice, betel-leaf extract reduced the tumorigenic effects of NNK by 25%. Concurrent treatment with the extract also inhibited the decreases in levels of vitamin A in liver and plasma induced by NNK. Betel leaf thus has protective effects against the mutagenic, carcinogenic and adverse metabolic effects of NNK in mice.

Effect of vitamin A status of rats on metabolizing enzymes after exposure to tobacco extract or N'-nitrosonornicotine.

The effects of N'-nitrosonornicotine (NNN) and tobacco extract on hepatic and pulmonary biotransformation enzymes were studied in rats fed vitamin A-sufficient or -deficient for semisynthetic diets. Basal levels of cytochrome P450, benzo[a]pyrene hydroxylase, benzphetamine demethylase, glutathione S-transferase and glutathione were lower in the group on the deficient diet. Treatment with tobacco extract or NNN significantly increased the levels of these enzymes in the sufficient diet group. However, in the deficient group, phase I enzymes were significantly increased, but glutathione and glutathione S-transferase levels were drastically reduced. Urine from animals on the deficient diet and treated with tobacco extract or NNN were mutagenic in the Ames Salmonella/microsome test. The results suggest that altered metabolism resulting from a vitamin A-deficient diet may be an important factor in susceptibility to carcinogens.

Effect of nutritional status on mutagenicity of urine excreted by rats treated with standard/experimental carcinogens.

Urine samples, collected from Sprague Dawley rats treated with extracts of tobacco/masheri, benzo (a) pyrene, N'-nitrosonornicotine, N'-nitrosodiethylamine and maintained on semi-synthetic diets sufficient or deficient in Vitamin A, B and protein were tested for mutagenicity using Salmonella/microsome assay. The mutagenic activity of urine or various treated groups was in the order deficient diet greater than standard laboratory diet greater than nutritionally sufficient diet. Present results confirmed the earlier observations that nutritionally deficient animals are likely to have more exposure to mutagenic metabolites that are generated by increased phase I enzymes and decreased detoxification system.

Effect of tobacco extract and N'-nitrosonornicotine on the carcinogen metabolising enzymes under different dietary vitamin B status.

Studies were carried out to evaluate the changes in the phase I and II enzymes of xenobiotic metabolism, on treatment with tobacco extract (TE) and a tobacco specific carcinogen, N'-nitrosonornicotine (NNN) in Sprague-Dawley rats maintained on vitamin B complex sufficient and deficient semi-synthetic diets. Both TE and NNN significantly increased the hepatic and pulmonary phase I enzymes in the vitamin B sufficient (SB+) and deficient (SB-) animals. However, the percent increase in enzyme activities was drastically higher in the SB- treated group as compared to those in the SB(+)-treated group. On the other hand, TE and NNN significantly depressed the liver and lung glutathione (GSH) level and glutathione S-transferase (GST) activity in the SB- animals, while the opposite effect was observed in the SB(+)-treated animals. Furthermore, both the treatments depleted the hepatic pool of vitamin A, with a concurrent increase in that of vitamin C in SB+ and SB- groups.

Carcinogenicity studies on the two tobacco-specific N-nitrosamines, N'-nitrosonornicotine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone.

The tobacco-specific N-nitrosamines (TSNA) have been implicated in oral cancer. However, except for one study using rats, no study has shown the ability of TSNA in inducing oral tumours in experimental animals. We have studied the carcinogenic potentials of N'-nitrosonornicotine (NNN) and 4-methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in mice and hamsters, wherein the nitrosamines were administered on the tongues of the mice and the cheek pouches of the hamsters to simulate the exposure conditions of humans. It was observed that in Swiss and BALB/c male mice, both NNN and NNK induced tumours of lung, forestomach and liver. However, no oral tumours were induced in mice. The effect of vitamin A depletion was tested in Swiss male mice. It was found that a low vitamin A status did not alter the percentage incidence of tumours induced by both nitrosamines to a significant extent. In the studies using Syrian golden hamsters, long-term treatment of NNK to hamster cheek pouch induced tumours in the lung, liver, stomach and cheek pouch. Subsequently, the effect of hydrogen peroxide (H2O2) on NNK-induced carcinogenicity in hamsters was studied. It was observed that simultaneous administration of NNK and H2O2 to the animals increased the incidence of cheek pouch tumours. Another pertinent observation was that even when a small initiator dose of NNK was given followed by the application of H2O2, a very significant increase in the tumour incidence was observed. This observation suggests that H2O2 could act as a promoter to NNK-induced carcinogenesis. In conclusion it may be stated that both NNN and NNK do not show any strain or species specificity. They failed to produce tumours at the site of application in mice but in hamsters few cheek pouch tumours were seen or were induced when NNK was applied alone. The cheek pouch tumour incidence increased when H2O2 was given concurrently or when applied for a long period after a low initiator dose of NNK was administered in the cheek pouch.

Mutagenic and cytogenetic studies of N'-nitrosonornicotine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone.

The tobacco specific nitrosamines (TSNA) N'-nitrosonornicotine (NNN) and 4-(Methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK) were tested for mutagenic and clastogenic effects using a battery of short-term test systems. These test systems include the Ames test, micronucleus test (MNT), induction of chromosomal aberrations and sister chromatid exchange (SCEs). NNN and NNK were tested for their potency in inducing mutations in the Ames Salmonella/microsome assay and their clastogenic action were tested by the micronucleus inducing ability in vivo using Swiss mice. Studies on the induction of chromosomal aberrations and SCE exchange were carried out using human peripheral blood lymphocyte cultures. In the Ames test and MNT, NNN was positive but in comparisons with NNK, NNK was a more potent mutagen. Present studies clearly proves the genotoxic potential of both NNN and NNK and between the two NNK is more potent.

Anticarcinogenic effect of betel leaf extract against tobacco carcinogens.

Epidemiological studies have implicated that betel quid offers some protection to tobacco induced carcinogenesis. Earlier studies in our laboratory have shown betel leaf extract (BLE) to be antimutagenic against standard mutagens and tobacco-specific N'-nitrosamines (TSNA), N'-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). In the present study, we have tested the anticarcinogenic effect of BLE using Swiss male mice. Two protocols of study were used to test this effect. In the first protocol, the effect of BLE was tested against the standard carcinogen benzo[a]pyrene (BP) using Wattenberg's stomach tumor model, Cancer Res., 41 (1981) 2820-2823. In this protocol, BLE inhibited the tumorigenicity of BP to a significant extent. In the second protocol, the effect of BLE against the two tobacco-specific nitrosamines, NNN and NNK was studied using long-term studies on Swiss male mice. The nitrosamines were administered on the tongues of the mice, while the BLE was supplied in drinking water. Two doses of NNN (22 mg and 72 mg) and one dose of NNK (22 mg) were used. In this study, it was observed that the number of tumor bearing animals decreased, but the difference was significant only in the group treated with the low dose of NNN in combination with BLE. However, in all the BLE treated animals, irrespective of the dose of nitrosamine, the hepatic vitamin A and C levels were elevated significantly as compared to the corresponding nitrosamine-treated controls. These results indicate that BLE has a promising anticarcinogenic role to play in tobacco induced cancer.(ABSTRACT TRUNCATED AT 250 WORDS)

Hydroxychavicol: a new anti-nitrosating phenolic compound from betel leaf.

Hydroxychavicol and eugenol are the phenolic compounds isolated from betel leaf (piper betel). The modulation of nitrosation of methylurea by sodium nitrite at pH 3.6 and 30 degrees C was studied. The formation of mutagenic N-nitrosomethylurea was monitored by checking the mutagenicity of reaction mixture in Salmonella typhimurium strain TA100 and TA1535 without S9 mix. Hydroxychavicol and eugenol exhibit dose-dependent suppression of nitrosation in vitro without affecting the survival of the bacteria. Pre- or post-treatment of bacterial cells from S. typhimurium strains TA100 and TA1535 with phenolics did not modify the mutagenicity of nitrosomethylurea. The blocking of hydroxy group(s) in the benzene ring by acetylation abolishes the anti-nitrosating activity of the molecule(s). The nitrosation inhibition by hydroxychavicol is through scavenging of nitrite ions in the media, thus making them non-available for the nitrosation of methylurea.

Antimutagenic effects of betel leaf extract against the mutagenicity of two tobacco-specific N-nitrosamines.

Epidemiological studies have implicated chewing tobacco alone to be more hazardous than chewing tobacco with betel quid. Experimental studies have shown that betel leaf is antimutagenic against standard mutagens like benzo[a]pyrene and dimethylbenz[a]anthracene. Since the tobacco-specific N-nitrosamines (TSNA) are the only carcinogens present in unburnt forms of tobacco, including chewing tobacco, we tested the effect of an extract of betel leaf against the mutagenicity of the two important TSNA, viz., N'-nitrosonornicotine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, using the Ames Salmonella/microsome assay with TA100 +S9 and the in vivo micronucleus test. In both the test systems it was observed that betel leaf extract suppressed the mutagenic effects of both the nitrosamines to a significant extent.

Protective effect of hydroxychavicol, a phenolic component of betel leaf, against the tobacco-specific carcinogens.

The phenolic compound, hydroxychavicol (HC), present in betel leaf, was synthesised and tested for its antimutagenic effect against the mutagenicity of the 2 tobacco-specific N-nitrosamines (TSNA), N'-nitrosonornicotine (NNN) and 4-(nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK), in 2 different test systems, viz. the Ames Salmonella/microsome assay and the micronucleus test using Swiss male mice. We are reporting the synthesis of HC of a high degree of purity. We observed that HC suppressed the mutagenic effects of NNN and NNK in both test systems used. These results indicate that HC may have a role to play in reducing the risk of oral cancer in betel quid with tobacco chewers.

Modulations in the biotransformation of tobacco extract and N'-nitrosonornicotine under differential dietary protein status.

The modulation of the phase I and phase II biotransformation enzymes upon treatment with tobacco extract (TE) and N'-nitrosonornicotine (NNN) was investigated using male Sprague-Dawley rats fed differential protein diets. It was observed that the animals fed a low protein diet showed an overall decrease in the basal levels of hepatic and pulmonary phase I and II enzymes. TE and NNN significantly decreased the detoxifying system in the low-protein-fed animals. Animals fed 20% protein, however, showed significant increases in glutathione and glutathione S-transferase upon treatment. Furthermore, TE and NNN treatment brought about a significant depletion in the hepatic pool of vitamin A with a concomitant increase in the vitamin C levels.

Curcumins as inhibitors of nitrosation in vitro.

The effects of turmeric extract and its pure yellow pigments curcumin I, II and III were tested on the nitrosation of methylurea by sodium nitrite at pH 3.6 and 30 degrees C. The nitrosomethylurea formed was monitored by checking the mutagenicity in S. typhimurium strains TA1535 and TA100 without metabolic activation. Turmeric extract as well as curcumins exhibit dose-dependent decreases of nitrosation. Curcumin III was the most effective nitrosation inhibitor among the compounds tested. The simultaneous treatment of inhibitor with nitrosation precursors was essential and pre- or post-treatment of inhibitor had no effect on the mutagenicity of nitrosomethylurea. The binding of nitrite with the inhibitors was studied at pH 3.6 and 30 degrees C. Curcumin I shows a dose-dependent depletion of nitrite ions thus making nitrite non-available for nitrosation. Curcumin I and III when tested also showed a time-dependent depletion of nitrite ions at pH 3.6 and 30 degrees C. Curcumin III has a higher affinity for nitrite ions than curcumin I.