Effects of monoterpenoids on in vivo DMBA-DNA adduct formation and on phase I hepatic metabolizing enzymes

We have previously demonstrated the anticarcinogenic effects of monocyclic monoterpenes such as limonene when given during the initiation phase of 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary cancer in Wistar-Furth (WF) rats. Here we investigated the possible mechanisms for this chemoprevention activity including limonene's effects on DMBA-DNA adduct formation and hepatic metabolism of DMBA. Twenty-four hours after carcinogen administration, there were approximately 50% of the total DMBA-DNA adducts found in control animals formed in the liver, spleen, kidney and lung of limonene-fed animals. While circulating levels of DMBA and/or its metabolites were not different in control and limonene-fed rats, there was a 2.3-fold increase in DMBA and/or DMBA-derived metabolites in the urine of the limonene-fed animals. Studies of the effects of limonene and sobrerol, a hydroxylated monocyclic monoterpenoid with increased chemoprevention activity, on phase I metabolizing enzymes revealed that these terpenoids modulated cytochrome P450 (CYP) and epoxide hydratase (EH) activity. The 5% limonene diet increased total CYP to the same extent as phenobarbital (PB) treatment when compared to control, while 1% sobrerol (isoeffective in chemoprevention to 5% limonene) did not. However, both 5% limonene and 1% sobrerol diets greatly increased the levels of microsomal EH protein and associated hydrating activities towards benzo[a]pyrene 4,5-oxide when compared to control and PB treatment. These changes also modified the rate and regioselectivity of in vitro microsomal DMBA metabolism when compared to PB treatment or control. Identification of the specific isoforms of CYP induced by these terpenoids was performed using antibodies to CYP isozymes in Western blot analysis and inhibition studies of microsomal DMBA metabolism. Five per cent limonene was more effective than 1% sobrerol at increasing the levels of members of the CYP2B and 2C families but was equally effective at increasing EH. Furthermore, both terpenoid diets caused increased formation of the proximate carcinogen, DMBA 3,4-dihydrodiol. While these terpene-induced changes in hepatic CYP and EH do not explain the anticarcinogenic mechanism of these chemopreventive agents, or the ability of limonene systemically to reduce DMBA-DNA binding, they do reveal novel and selective induction mechanisms of hepatic enzymes.     

Anti-Mutagenic and Anti-Carcinogenic Potential of the Carotenoid Meso-Zeaxanthin

Meso-zeaxanthin was investigated for antimutagenic and anticarcinogenic activity, using the Ames test(Salmonella typhimurium strains TA 98, TA 100, TA 102 and TA 1535) with direct acting mutagens like sodiumazide (NaN3) (5 μg/ plate), nitro-o-phenylendiamin (NPD) (20 μg/ plate), N-methyl- N’-nitro-N-nitrosoguanidine(MNNG) (1μg/ plate) and tobacco extract 50 mg/ plate) and with a mutagen needing microsomal activation,acetamidofluorene (AAF) ( 20 μg/ plate). The carotenoid was found to inhibit the mutagenicity induced byNaN3, NPD and MNNG in a concentration dependent manner, as well as that with AAF and the tobacco extract.Concentrations needed for 50 % inhibiton was found to be 50 μg/ plate for the chemical mutagens and 100 μg/plate for tobacco extract. Using specific resorufin derivatives as substrates in vitro, the concentration of mesozeaxanthinneeded for 50 % inhibition of CYP1A2 (7-methoxyresorufin-O-demethylase) was 5 μg/ml, for CYP2B1/2 (7- pentoxyresorufin-O-depentylase) was 8 μg/ml and for CYP1A1 (7-ethoxyresorufin-O-deethylase) was 12μg/ml, while that of CYP 2E1 (aniline hydroxylase) was 7μg/ml and for CYP 1A, 2A, 2B, 2D and 3A (aminopyrene-N-demethylase) was 10.5 μg/ml. Evaluated using nitroso diethyl amine (NDEA) induced hepatocellular carcinomain rats, treatment with meso-zeaxanthin reduced the tumor incidence when compared to the control group. Theactivity of glutamate oxaloacetate transaminase, glutamate pyruvate transaminase and alkaline phosphatase wasdrastically elevated in both serum and liver tissue of NDEA alone treated control animals and meso-Zeaxanthinpretreated animals showed significant decrease to normal levels, in line with histopathological findings.     

Dehydroepiandrosterone inhibits the expression of carcinogen-activating enzymes in vivo

We investigated the effect of the steroid hormone dehydroepiandrosterone (DHEA) on the hepatic expression and activity of carcinogen-activating enzymes, the cytochromes P450 (CYP) 1A1, 1A2 and 1B1, in Sprague-Dawley rats. In animals fed DHEA at 200 or 400 mg/kg body weight every other day for 2 weeks prior to exposure to the aryl hydrocarbon dimethylbenz[a]anthracene (DMBA, 5 mg/kg), there was a dose-dependent decrease in hepatic CYP activity, as measured by ethoxyresorufin-O (EROD) assay, from 37.1 to 22.9 and 14.7 pmoles/min/10 microg microsomes, respectively. DHEA did not directly inhibit microsomal EROD activity, however, leading us to investigate its effects on enzyme expression. To test this, we examined protein and mRNA levels of the enzymes. Western blot for CYP1A1 and CYP1A2 showed that DHEA inhibited the increase in hepatic CYP1A1 and CYP1A2 enzyme levels that are normally induced by DMBA. DMBA-induced increase in expression of CYP1A1, CYP1A2 and CYP1B1 mRNA was similarly blunted in DHEA-treated animals. DHEA was also able to significantly reduce the basal expression of CYP1A1 and CYP1A2 but not of CYP1B1. These results indicate that DHEA regulates the expression and, hence, the activity of hepatic carcinogen-activating enzymes in vivo, and this may be an important mechanism of its chemopreventive activity.     

Suppression of 7,12-dimethylbenz[a]anthracene-induced mammary carcinogenesis by pre-initiation treatment of rats with beta-naphthoflavone coincides with decreased levels of the carcinogen-derived DNA adducts in the mammary gland

BACKGROUND: Mechanisms underlying prevention by beta-naphthoflavone (beta-NF) of mammary carcinogenesis initiated with 7,12-dimethylbenz[a]anthracene (DMBA) in the rat were elucidated. METHODS AND RESULTS: Treatment of female Sprague-Dawley rats with beta-NF at 40 mg/kg b.wt. for 4 days by oral gavage in corn oil before a single oral dose of DMBA (112 mg/kg b.wt.) suppressed mammary gland carcinogenesis as shown by an increase in the median latent period from 10 to 24 weeks and a 60% decrease in the multiplicity of mammary adenocarcinomas. In contrast, a 20-day treatment with beta-NF starting 3 weeks after DMBA had no significant effects on mammary tumorigenesis. The activities of phase I and phase II enzymes were examined in the liver and mammary gland 24 h after treatment of rats with beta-NF, DMBA, or beta-NF followed by DMBA as in the first bioassay. Treatment with either beta-NF or DMBA increased the hepatic activities of cytochrome P450 (CYP)1A1, 1A2, and 2B1/2, and glutathione S-transferase, and the mammary activity of CYP1A1. The activity of mammary CYP2B1/2 induced by DMBA was decreased by beta-NF. In the liver, the increase of UDP-glucuronosyl transferase (GT) activity in rats treated with beta-NF and DMBA was 2.3-fold greater than in rats treated with DMBA alone. Thus, treatment with beta-NF likely increased the rate of glucuronidation of DMBA dihydrodiols leading to carcinogen detoxification. The levels of the DMBA adducts determined by 32P-postlabeling of the mammary gland DNA were decreased in the beta-NF-pretreated rats. Conclusion: The beta-NF-induced increase in the hepatic UDP-GT activity and decrease in the mammary DNA-DMBA adducts occurred under the same treatment regimen that led to suppression of DMBA-induced mammary carcinogenesis.     

Co-expression of human CYP1A1 and a human analog of cytochrome P450-EF in response to 2,3,7,8-tetrachloro-dibenzo-pdioxin in the human mammary carcinoma-derived MCF-7 cells

Cultured human mammary carcinoma (MCF-7) cells exhibited constitutivecytochrome P450-dependent metabolism of 7,12-dimethylbenz[a]anthracene(DMBA) (45–75 pmol/mg microsomal protein). Exposure ofthe cells to 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD), whichis known to induce CYP1A1, not only resulted in a 30-fold increasein the total microsomal metabolism of DMBA but produced substantialdifferences in the distribution of DMBA metabolites formed.This suggested that different cytochrome P450 (P450) forms predominatedin untreated and induced cells. Comparative studies with TCDD-inducedhuman hepatoblastoma (HepG2) and skin cell carcinoma (SCC-13)cells and also recombinantly expressed human CYP1A1, confirmedthat the DMBA metabolite profile in TCDD-induced MCF-7 cellswas that of human CYP1A1. This distribution, however, differedsubstantially from the regioselectivity of rat CYP1A1 and mouseCypla-1. Rabbit antibodies to rat CYP1A1 completely inhibitedthe DMBA-metabolizing activity of TCDD-induced MCF-7 cells buthad no inhibitory effect on constitutive DMBA metabolism whichwas, however, completely inhibited by chicken antibodies tothe novel P450 in mouse embryo fibroblasts (P450-EF). Anti-P450-EFinhibited only 10% of the DMBA-metabolizing activity in theTCDD-induced MCF-7 cell microsomes. Microsomes from untreatedMCF-7 cells expressed a 52 kDa protein that was immunodetectableby rabbit anti-P450-EF and failed to express immunodetectablelevels of human CYP1A1. DMBA metabolism, therefore, s from P450-EFin uninduced microsomes to CYP1A1 in TCDD-induced microsomes.The mobility of the P450-EF-like protein in MCF-7 cells washigher than that of P450-EF from C3H/10T1/2CL8 (10T1/2) cells(55 kDa). The 52 kDa protein from MCF-7 cells was induced     

Phellinus rimosus (Berk.) pilat attenuates 7,12-dimethylbenz[a] anthracene induced and croton oil promoted skin papilloma formation in mice

The roles of 7,12-dimethylbenz[a]anthracene (DMBA), a polycyclic aromatic hydrocarbon (PAH), and 12-O-tetradecanoylphorbol-13-acetate (TPA) a skin tumor promoter present in croton oil, are clearly implicated in the formation of skin papilloma. The effect of ethyl acetate extract of Phellinus rimosus, a polypore macro fungus, against croton oil-induced skin inflammation, lipid peroxidation and tumor promotion was studied. The antiinflammatory and lipid peroxidation inhibiting activities were determined by topical application of extract of P. rimosus (10 and 20 mg) prior to the application of 0.1 ml of 50% croton oil in acetone. The tumor promotion inhibiting effect of P. rimosus was evaluated against DMBA-initiated, croton oil promoted two-stage carcinogenesis model in mouse skin. The results showed that topical application of the extract (10 and 20 mg) significantly (p < 0.01) and dose dependently attenuate the inflammatory edema as well as lipid peroxidation induced by croton oil. Similarly, topical application of extract (1 and 5 mg) effectively ameliorated the croton oil promoted skin papilloma formation. The results of this study concluded that ethyl acetate extract of P. rimosus showed antitumor activity against DMBA initiated, croton oil promoted skin papilloma formation which can be partially ascribed to the antiperoxidative and anti-inflammatory effects of the extract.     

The role of the human acetylation polymorphism in the metabolic activation of the food carcinogen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ).

The metabolic activation of the heterocyclic food carcinogen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) by two human cytochrome P450 monoxygenases (P4501A1 and P4501A2) and two human N-acetyltransferases (NAT1 and NAT2) was investigated. Various combinations of these enzymes were functionally expressed in COS-1 cells. DNA adducts resulting from the activation of IQ were assayed quantitatively by the 32P-postlabeling procedure. The highest adduct frequency was observed in cells expressing both CYP1A2 and NAT2. CYP1A2 in combination with NAT1 was 3-6 times less active. When expressed alone these enzymes gave rise to low adduct frequencies. Experiments with N-acetyl-IQ as substrate suggest that NAT1 and NAT2 in addition to their known role in N-acetylation display arylhydroxamic acid N, O-acetyltransferase (AHAT) activity. Quantitative differences in adduct formation between IQ and N-acetyl-IQ indicated that metabolic activation of these arylamines preferentially occurs by P4501A2-catalyzed N-hydroxylation followed by O-acetylation mediated through NAT1 and/or NAT2. These data, in combination with the known genetic polymorphism of NAT2, may explain the clinical observation that the acetylation polymorphism constitutes a risk factor in the carcinogenic activation of environmental mutagens.     

Increased CYP1A2 content and capacity to activate Glu-P-1 and Trp-P-2 in liver microsomes of scorbutic ODS rats

Osteogenic Disorder Shionogi (ODS) rats, which cannot synthesizeascorbic acid due to a deficiency of L-gulonolactone oxidase,become scorbutic when not supplied with dietary ascorbic acid.We used the deficient rats to study the effects of ascorbicacid on the amount of cytochrome P450 enzymes in liver microsomes.The total amount of hepatic cytochrome P450 in ODS rats deprivedof ascorbic acid was lower by 40%, whereas ODS rats fed withascorbic acid and the wild strain had the same level of totalhepatic cytochrome P450. Western blot analysis for various formsof cytochrome P450 in liver microsomes indicated that the amountof CYP1A2 was significantly higher in ascorbic acid deficientrats. On the other hand, amounts of CYP2B2 and 3A were lower,and those of CYP2E1 and CYP2C6/11 were unaffected. In accordancewith the higher amount of CYP1A2, Northern blot analysis showedincreased expression of CYP1A2 mRNA. The capacity of microsomesto produce mutagens from 2-amino-6-methyl-dipyrido[1, 2-a:3',2'-d]imidazole acetate (Glu-P-1) and 3-amino-1-methyl-5H-pyrido[4,3-b]indole acetate (Trp-P-2) was higher in scorbutic ODS ratsby the Ames test. These results indicate that the effects ofascorbic acid deficiency on the expression of cytochrome P450in ODS rat livers are form-specific and that the increased CYP1A2is associated with increased metabolic activation of promutagensin the scorbutic state.     

Study on prevention of two-stage skin carcinogenesis by Hibiscus rosa sinensis extract and the role of its chemical constituent, gentisic acid, in the inhibition of tumour promotion response and oxidative stress in mice.

We designed the present study to investigate the role of gentisic acid in the chemopreventive activity of Hibiscus rosa sinensis extract on 7,12-dimethyl benz(a)anthracene (DMBA)/croton oil-mediated carcinogenesis in mouse skin via 12-O-tetradecanoyl phorbol-13-acetate (TPA)-induced tumour promotion response and oxidative stress. Single topical application of DMBA followed by twice weekly applications of croton oil after one week for 20 weeks resulted in 100% incidence of tumours in animals in 15 weeks. However, application of H. rosa sinensis extract 30 minutes prior to the application of croton oil twice weekly for 20 weeks caused significant reduction in the number of tumours per mouse and the percentage of tumour-bearing mice. Also, the latency period for the appearance of the first tumour was delayed on H. rosa sinensis pretreatment. A single topical application of TPA caused significant depletion in reduced glutathione (GSH) content, activities of its metabolizing and antioxidant enzymes, while malondialdehyde (MDA) formation, H2O2 content, ornithine decarboxylase (ODC) activity and DNA synthesis were significantly increased. Interestingly, pretreatment of H. rosa sinensis extract (3.5 mg and 7 mg/kg body weight) and gentisic acid (2.0 microg and 4.0 microg/0.2 ml acetone per animal) restored the levels of GSH, and its metabolizing and antioxidant enzymes (P<0.05). There was also a statistically significant reduction in MDA formation and H2O2 content (P<0.05) at both doses. Although inhibition of ODC activity by gentisic acid was not dose-dependent, thymidine incorporation in DNA (P<0.05) was dose-dependently recovered by the plant extract and its chemical constituent. We therefore propose that gentisic acid has a role in the modulatory activity of H. rosa sinensis extract.     

Two-stage skin carcinogenesis in sensitive and resistant mouse strains

Strains of laboratory mice vary markedly in their susceptibilityto two-stage skin carcinogenesis using 7,12-dimethyl-benz[a]anthracene(DMBA) as the initiating carcinogen and croton oil as promoter.This study has been undertaken in order to clarify the basisof the strain differences. LACA mice were used as the susceptiblestrain and BALB/c mice as the resistant strain. DMBA was a moreeffective complete carcinogen in LACA mice than in BALB/c mice.However, dose-response studies with respect to DMBA in the twostrains in two-stage carcinogenesis suggested that metabolicactivation of DMBA to the active carcinogen was not limitingin the resistant strain. The observed strain differences inresponse to DMBA in one and two stage carcinogenesis may reflectthe ability of DMBA to also act as a promoter in the two strains.The possibility that the strains vary in their ability to repairdamaged DNA has, however, not been eliminated. Unlike polycyclicaromatic hydrocarbon carcinogens such as DMBA, phorbol esterpromoters (the active components in croton oil) do not appearto require metabolic activation. However, they are degradedand inactivated by epidermal and other cells. Experiments inwhich the dose and frequency of application of croton oil wereincreased, and the unrelated promoter anthralin was substitutedfor croton oil, failed to produce any evidence that differencesin promoter degradation contributed to the differences in susceptibilitybetween strains.     

Chemopreventive Action of Syzygium cumini on DMBA-induced Skin Papillomagenesis in Mice

Syzygium cumini L. is widely used for the treatment of diabetes in various parts of India. The protectiveefficacy of S. cumini seed extract (SCE) against peroxidative damage contributing to skin carcinogenesis inSwiss albino mice was tested in the present study. A single topical application of 7,12-dimethyl benz(a)anthracene(100μg/100μl acetone), followed 2 weeks later by repeated application of croton oil (1% in acetone three times aweek) and continued till the end of the experiment (i.e., 16 weeks) caused a 100% tumor incidence. In contrast,mice treated with the SCE (125 mg/ kg/ b.wt./ animal / day)in either the peri (i.e. 7 days before & 7 days after theapplication of DMBA) or post-initiational (i.e. from the day of start of croton oil treatment & continued till theend of the experiment) phases demonstrated significant reduction in cumulative numbers of papillomas andtumor incidence (75%). The average latency period in the SCE treated group was also significantly increased(Pre Group – 11.1 weeks; Post Group – 10.9 weeks) as compared with the carcinogen control group (7.9). Resultsfrom the present study indicate that the anticarcinogenic activity of SCE during DMBA-induced skinpapillomagenesis is mediated through alteration of antioxidant status. Thus, SCE can be considered as a readilyaccessible, promising novel cancer chemopreventive agent.     

Anticlastogenic and Anticarcinogenic Potential of Thai Bitter Gourd Fruits

Thai bitter gourd fruits (Momordica charantia Linn., TBG) has been previously demonstrated to possessphase II detoxificating enzymes inducing properties, as well as the ability to reduce phase I carcinogen activatingenzyme activity in rat liver. In addition, it was partially inhibited 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary gland carcinogenesis in female Sprague-Dawley rats. In this study, we therefore examined theanticlastogenic and anticarcinogenic effect of TBG against clastogens, cyclophosphamide (CYP) and DMBA, inmice using the in vivo erythrocyte micronucleus assay and azoxymethane (AOM)-induced colon carcinogenesisin rats, respectively. For anticlastogenicity test, male mice were fed with modified AIN-76 diets containing6.25% and 12.5% of ground freeze-dried TBG for 2 weeks prior to administration of clastogens till the end ofexperiment. Blood samples were collected and counted for reticulocytes by using the fluorescent microscope. Foranticarcinogeicity test, male Wistar rats were fed with modified AIN-76 diets containing 5% and 10% groundfreeze-dried TBG for 2 weeks prior to, during and 1 week after the completion of AOM administration (15 mg/kgonce a week for 2 weeks). It was found that TBG at 6.25% resulted in a significant reduction in micronucleatedperipheral reticulocytes (MNRETs) induced by only CYP. Study on anticarcinogenic potential demonstratedthat rats fed with TBG diets at the concentration tested developed significantly higher incidence as well as themultiplicities of colon tumors than the control group. These results demonstrated that Thai bitter gourd fruitspossesses anticlastogenic potential against clastogen in the mouse. Interestingly, it had no preventive potentialagainst AOM-induced colon carcinogenesis in rat, rather increasing the incidence of colonic neoplasm whengiving during the initiation stage.     

Antimutagenic activity of anthocyanins isolated from Aronia melanocarpa fruits

Anthocyanins belong to the flavonoid family and are ubiquitous in plants, especially in flower petals and fruit peels. We established that anthocyanins isolated from fruits of Aronia melanocarpa markedly inhibited the mutagenic activity of benzo(a)pyrene and 2-amino fluorene in the Ames test. In the Sister Chromatid Exchanges (SCEs) test with human blood-derived lymphocytes cultured in vitro, a significant decrease of SCEs frequency induced by benzo(a)pyrene was observed in the presence of anthocyanins. In the case of mitomycin C the effect of anthocyanins on SCEs frequency was smaller but still noticeable. Anthocyanins markedly inhibited the generation and release of superoxide radicals by human granulocytes. The results suggest that the antimutagenic influence of anthocyanins is exerted mainly by their free-radicals scavenging action as well as by the inhibition of enzymes activating promutagens and converting mutagens to the DNA-reacting derivatives. These preliminary data seem to be important in the aspect of a possible antimutagenic and anticarcinogenic potency of anthocyanins commonly present in fruits and vegetables.     

Specificity of cDNA-expressed human and rodent cytochrome P450s in the oxidative metabolism of the potent carcinogen 7,12-dimethylbenz[a]anthracene

7,12-Dimethylbenz[a]anthracene (DMBA), a potent carcinogen, requires metabolic activation by cytochrome P450s (P450s) to electrophilic metabolites that result in DNA modification, mutagenicity, and carcinogenicity. In this study, we used eight human forms, four rodent forms, and one rabbit form of P450 expressed from recombinant vaccinia or baculovirus vectors to define their specificity for metabolizing DMBA. Of the eight human P450s, 1A1 was the most active (specific activity = 14.7 nmol/min/nmol of P450) in total metabolism of DMBA and showed approximately 6- to 33-fold more activity than other P450s. 2B6, 2C9, and 1A2 were also capable of metabolizing DMBA (2.0–2.5 nmol/min/nmol of P450), whereas 2C8, 2E1, 3A4, and 3A5 exhibited relatively low activities. Among animal P450s, mouse 1A1 exhibited activity similar to that of human 1A1 and had 5.0- to 37-fold more activity than other rodent and rabbit P450s. In regard to enzyme regioselectivity, most human and rodent P450s predominantly formed the 8,9-diol, but human 2B6 and rat 281 preferentially formed the 5,6-diol. In the production of monohydroxymethyl metabolites, all the enzymes yielded more 7-hydroxymethyl-12-methylbenz[a]anthracene (7HOM12MBA) than 12-hydroxymethyl-7-methylbenz[a]anthracene (7M12HOMBA), except for human 1A1, which presented the reverse selectivity. Human liver microsomes from 10 organ donors were shown to metabolize DMBA and in most circumstances generated the metabolic profile DMBA trans-8,9-dihydrodiol > 7HOM12MBA ≥ DMBA trans-5,6-dihydrodiol ≥ 7,12-dihydroxymethylbenz[a]anthracene > 7M12HOMBA > DMBA trans-3,4-dihydrodiol. Thus, the combined activity of hepatic microsomal 2C9, 1A2, and 2B6 may contribute to the metabolic activation and the metabolism of DMBA in normal human liver. © 1996 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Chemopreventive potential of Annona muricata L leaves on chemically-induced skin papillomagenesis in mice

Annona muricata L (Annonaceae), commonly known as soursop has a long, rich history in herbal medicine with a lengthy recorded indigenous use. It had also been found to be a promising new anti-tumor agent in numerous in vitro studies. The present investigation concerns chemopreventive effects in a two-stage model of skin papillomagenesis. Chemopreventive effects of an ethanolic extract of A. muricata leaves (AMLE) was evaluated in 6-7 week old ICR mice given a single topical application of 7,12-dimethylbenza(α)anthracene (DMBA 100 μg/100 μl acetone) and promotion by repeated application of croton oil (1% in acetone/ twice a week) for 10 weeks. Morphological tumor incidence, burden and volume were measured, with histological evaluation of skin tissue. Topical application of AMLE at 30, 100 and 300 mg/kg significantly reduced DMBA/croton oil induced mice skin papillomagenesis in (i) peri-initiation protocol (AMLE from 7 days prior to 7 days after DMBA), (ii) promotion protocol (AMLE 30 minutes after croton oil), or (iii) both peri-initiation and promotion protocol (AMLE 7 days prior to 7 day after DMBA and AMLE 30 minutes after croton oil throughout the experimental period), in a dose dependent manner (p<0.05) as compared to carcinogen-treated control. Furthermore, the average latent period was significantly increased in the AMLE-treated group. Interestingly, At 100 and 300 mg/ kg, AMLE completely inhibited the tumor development in all stages. Histopathological study revealed that tumor growth from the AMLE-treated groups showed only slight hyperplasia and absence of keratin pearls and rete ridges. The results, thus suggest that the A.muricata leaves extract was able to suppress tumor initiation as well as tumor promotion even at lower dosage.     

High promutagen activating capacity of yeast microsomes containing human cytochrome P-450 1A and human NADPH-cytochrome P-450 reductase

Yeast Saccharomyces cerevisiae strains have been constructedthat co-express cDNAs coding for the human cytochrome P-450enzymes CYP1A1 or CYP1A2 in combination with human NADPH-cytochromeP-450 reductase (oxidoreductase). Microsomal fractions preparedfrom the strains were able to efficiently activate various drugsto Salmonella mutagens. These experiments demonstrated thata functional interaction occurred between the respective humanenzymes in the yeast microsomes. For every drug tested, themicrosomes containing CYP enzymes and oxidoreductase were 2-to 4-fold better in activation than the corresponding microsomesthat contained CYP alone. Interestingly, co-expression of CYP1A2with oxidoreductase resulted in a decrease of 7-ethoxyresorufin-O-deethylaseactivity, a problem which is related to this specific substrate.Using the microsomes, it was demonstrated that aflatoxin B₁,was activated to a mutagen not only by CYP1A2 but also by CYP1A1.In contrast, benzo[a]pyrene was exclusively activated by CYP1A1whereas CYP1A2 was inactive. The drug 3-amino-1-methyl-5H-pyrido[4,3-b]indole(Trp-P-2) was activated by CYP1A2 and to a lesser extent byCYP1A1. A strong substrate specificity was observed with thetwo structurally related heterocyclic arylamines 2-amino-3,4-dimethylimidazo[4,5-f]quinoline(MeIQ) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQ_x).MeIQ_x was activated efficiently by both CYP enzymes, whereasMeIQ was only activated by CYP1A2 and not by CYP1A1. The factthat microsomes from vector transformed control strains wereunable to activate any of the drugs studied underlines the suitabilityof these microsomes for metabolic studies. Moreover, the presenceof suitable marker genes in the yeast strains will enable usto study mitotic recombination and gene conversion events inducedby drugs that require metabolic activation.     

Evaluation of Anticarcinogenic and Antimutagenic Potential of Bauhinia variegata Extract in Swiss Albino Mice

Background: Infusions from the bark of Bauhinia is used to treat various diseases in the traditional medicalsystem of India and decoction of the roots is used in dyspepsia and act as an antidote to snake poison. Itschemopreventive potential for cancer was the subject of the present study. Materials and methods: To evaluatethe anticarcinogenicity and antimutagenicity of Kachanar extract a skin carcinogenesis and melanoma tumourmodel was used, along with micronucleus and chromosomal aberration tests, in Swiss albino mice. Results: Inthe skin papilloma model, significant prevention, with delayed appearance and reduction in the cumulative no.of papillomas was observed in the DMBA + Kachanar + croton oil treated group as compared to the DMBA +Croton Oil group. C57 Bl mice which received a 50 % methanolic extract of Kachanar extract at the doses of500 and 1000 mg/ kg body weight for 30 days showed increase in life span and tumour size was significantlyreduced as compared to controls. In antimutagenicity studies,a single application of Kachanar extract at dosesof 300, 600 and 900 mg/kg dry weight, 24 hours prior the i.p. administration of cyclophosphamide (at the 50 mg/kg) significantly prevented micronucleus formation and chromosomal aberrations in bone marrow cells ofmice, in a dose dependent manner. Conclusions: Our results suggest that Kachanar extract exerts anticarcinogenicand antimutagenic activity.