Post-initiation treatment of rats with indole-3-carbinol or β-naphthoflavone does not suppress 7,12-dimethylbenz[a]anthracene-induced mammary gland carcinogenesis

Indole-3-carbinol (I3C) and β-naphthoflavone (β-NF), blocking agents of 7,12-dimethylbenz[a]anthracene (DMBA)-initiated mammary gland carcinogenesis, were examined as potential post-initiation suppressing agents. Treatment of female Sprague–Dawley rats with I3C (250 mg/kg body weight (b.w.)), β-NF (20 mg/kg b.w.) or the vehicle ethanol:corn oil (2:3) (2.5 ml/kg b.w.), three times weekly by gavage, started 3 weeks after the initiation with one oral dose of DMBA (20 mg/rat at 7 weeks of age) and continued for up to 12 weeks. I3C- or β-NF- or vehicle-treated groups did not differ significantly in the overall outcome of mammary tumorigenesis including cumulative mammary tumor incidences and multiplicities, latent periods and number and weight of mammary tumors per tumor-bearing rat for malignant, benign and/or malignant + benign tumors. A tendency of the I3C-treated rats to develop fewer mammary adenocarcinomas with a greater average weight per tumor per rat (2.32±1.50 g) than in the β-NF- (1.52±1.58 g) or vehicle- (1.55±1.53 g) treated groups suggests an effect, yet to be confirmed, of I3C on tumor development and growth. A 12-week treatment with I3C or β-NF significantly increased the P450-dependent activities of ethoxy-, methoxy-, benzyloxy- and pentoxy-(with I3C only) resorufin O-dealkylase in hepatic microsomes indicating induction of several P450s. The alterations in the P450 complement may affect endogenous estrogen metabolism and mammary gland and tumor characteristics at the molecular level, e.g. estrogen receptor status and/or proliferative activity, which require further studies.     

Suppression of mammary gland carcinogenesis by post-initiation treatment of rats with tamoxifen or indole-3-carbinol or their combination.

This study examined whether suppression of mammary gland carcinogenesis elicited by low doses of tamoxifen (TAM) can be enhanced by concomitant treatment of rats with indole-3-carbinol (I3C), a component of cruciferous vegetables and a dietary supplement used for its putative antiestrogenicity. Two weeks after one oral dose of 7,12-dimethylbenz[a]anthracene (DMBA) at 65 mg/kg body weight, female Sprague-Dawley rats started treatment with TAM (10 microg/rat) by subcutaneous injection, I3C (250 mg/kg body weight) by oral gavage, TAM+I3C or their respective vehicles three times per week, for up to 20 weeks. Significant increases in the median latency of malignant mammary tumors and decreases in the mean tumor mass per rat were due to TAM. Significant decreases in the mean tumor number per rat in TAM, I3C and TAM+I3C-treated rats indicated a cooperative effect of the two compounds. In both DMBA-initiated and uninitiated rats, significant increases in the ratios of liver to body weight in I3C and TAM+I3C-treated groups coincided with I3C-dependent increases of hepatic cytochrome P450 levels and activities (1A1, 1A2 and 2B1/2). The ratios of uterus to body weight decreased with the number of treatments and the decreases effected by TAM were greater than those by I3C. The levels of circulating estrone were increased in response to I3C treatment and were greater in DMBA-initiated rats than in uninitiated rats, which may contribute to the preventive effect of I3C. Chemoprevention may be accomplished through up-regulation of apoptotic enzyme (caspase) activities in the mammary gland or mammary tumors. Treatment with TAM, I3C or TAM+I3C had no effect on caspase-3&7, caspase-6, caspase-8 and caspase-9 activities in the mammary tumors or mammary gland of tumor-bearing rats or that of uninitiated rats. In the mammary gland of DMBA-initiated tumor-free rats, however, I3C treatment increased the levels of caspase-3&7 and caspase-9 activities, suggesting an I3C-mediated protective effect. Even though I3C alone is a much less effective suppressing agent of mammary carcinogenesis than TAM, I3C in combination with TAM does not weaken but may foster the benefits of chemoprevention with TAM.     

Role of β-carotene on the changes in activity patterns and levels of biotransforming enzymes in transplantable murine lymphoma

The differential levels of induction of hepatic microsomal cytochrome P-450 (cyt. P-450), UDP-glucuronyl transferase (UDPGT) and cytosolic glutathione-S-transferase (GST) activities were evaluated over various periods of time, following tumor transplantation in male Swiss albino mice in the presence and absence of β-carotene supplementation in their basal diet (100 mg/kg). An increase in the total hepatic microsomal cytochrome P-450 and UDP-glucuronyl transferase and cytosolic GSH-transferase activities (1.5 to 2 fold) occurred during the later stage of tumor progression (22 ± 2 days onwards). However, β-carotene supplementation throughout the study increased or decreased the random activity trends of the above markers significantly (P < 0.05−< 0.01). Finally, β-carotene supplementation could enhance the survival of the host bearing lymphoma by almost 2-fold (50–60 days) over and above the lymphoma controls (30–35 days).     

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.     

Metabolism of nitrofluoranthenes by rat lung subcellular fractions

The nitrofluoranthene (NF) family of compounds includes thepotent pulmonary carcinogen 3, 9-dinitrofluoranthene (3, 9-DNF)and the weak carcinogen 3-nitrofluoranthene (3-NF). Althoughthe specific molecular mechanisms involved in this differencein sensitivity for the induction of lung tumors in rats by 3,9-DNF and 3-NF have not been defined, these compounds most likelyinduce carcinogenesis by metabolic activation to electrophilicmetabolites that bind DNA. The purpose of these investigationswas to determine the activation pathways in the rat lung forthe metabolism of the di-(3, 9-DNF) and mono-nitroisomers (3-NF,8-NF, 2-NF) of NFs. The metabolic rates of NFs were comparedfor lung subcellular fractions of pristine rats as well as ratspreviously treated with 3-methylcholanthrene (3-MC) or phenobarbitalat levels that would induce cytochrome P450 enzymes. One majormetabolite, the amino derivative, was detected by high pressureliquid chromatography following anaerobic incubation of ratlung cytosol with 3-NF, 8-NF, 2-NF or 3, 9-DNF. 3, 9-DNF wasmetabolized to its amino derivative, aminonitrofluoranthene,at a higher rate than 3-NF, 8-NF or 2-NF. Pretreatment of therats with 3-MC or phenobarbital did not affect the metabolicrates of cytosolic reduction. Both 3-NF and 3, 9-DNF were metabolizedanaerobically to their amino derivatives by microsomal reductase(s).3, 9-DNF was metabolized twice as fast as 3-NF. The formationof the aminonitrofluoranthene metabolite was increased 2 timeswith microsomes from 3-MC-induced rats, but was unaffected bymicrosomes from phenobarbital-treated rats. This suggests thatthe cytochrome P450 isozymes and reductase, which are inducedby 3-MC, may be involved in the metabolism of 3-NF and 3, 9-DNF.The metabolic products of 3-NF, formed aerobically, consistedof one major and three minor compounds. The major metabolite,tentatively identified as 3-NF-8-ol, was increased 6 times usingmicrosomes from 3-MC-induced rats. In contrast, 3, 9-DNF metabolismwas not detected aerobically with lung microsomes. Thus, ringhydroxylation was inhibited in the metabolism of 3, 9-DNF, andthe major pathway was nitroreduction. This higher rate of anaerobicmetabolism of 3, 9-DNF over 3-NF and the expected high reactivityof the putative N-acetoxy derivative formed from 3, 9-DNF maybe responsible for the differential potency for lung cancerinduction by these two carcinogens.     

Microsomal metabolism of the carcinogen, N-2-fluorenylacetamide, by the mammary gland and liver of female rats. I. Ring- and N-hydroxylations of N-2-fluorenylacetamide

We determined ring- and N-hydroxybtions of a systemic mammarygland cardnogen, N-2-fluorenylaeetamide (2-FAA), by microsomalfractions of liver and mammary gland of female rats and theeffects of in vivo and/or in vitro modifiers of these oxidations.Pretreatment of lactating rats with 3-methylcholanthrene (3-MC)or ß-naphthoflavone (ß-NF) and non-lactating(50-day old virgin) rats with ß-NF showed similareffects in that the formation of 3-, 5-, 7-, 9- and N-hydroxy-2-FAAby hepatic microsomes was increased manyfold and the formationof 1-hydroxy-2-FAA was induced. In mammary gland microsomes,the formation of 3-, 5- and 7-hydroxy-2-FAA was likewise increased,but of 9-hydroxy-2-FAA was unaffected. Only mammary microsomesof lactating rats had capacity for N-hydroxylation which wasincreased {small tilde}3 times by pretreatment of rats with3-MC or ß-NF. All of the induced increases of metabolitesof 2-FAA in hepatic and mammary microsomes were inhibited by0.1 mM -naphthoflavone (-NF) in vitro. Pretreatment of non-lactatingrats with phenobarbital increased only the formation of 7-hydroxy-2-FAAin hepatic microsomes which was further stimulated by -NF invitro. The latter also stimulated the formation of 7- and 9-hydroxy-2-FAA by hepatic microsomes of the uninduced rats, buthad no effects in mammary microsomes, in which 9-hydroxy-2-FAAwas a major metabolite. Hence, the data showed qualitative andquantitative differences between lactating and non-lactatingrats in metabolism of 2-FAA by mammary microsomes which mayresult from differences in the levels (e.g., of cytochrome P-450)and activities of microsomal enzymes determined herein. In hepaticmicrosomes of these rats, differences in quantities of metabolitesof 2-FAA (3-, 7-, 9- and N-hydroxy-2-FAA) were found in cornoil-treated rats only. The solvent (methanol or acetone) usedfor addition of 2-FAA to the incubation mixtures altered quantitativelythe metabolite profiles in hepatic and mammary microsomes of3-MC or ß-NF treated rats. The formations of 1- and3- or 5- and 7-hydroxy-2-FAA were greater in the presence ofacetone or methanol, respectively. The results of this studysuggest that the formation of phenolic and N-hydroxy metabolitesof 2-FAA in both hepatic and mammary microsomes of lactatingrats is catalyzed by similar form(s) of cytochrome P-450 inducedby pretreatment with 3-MC or ß-NF. Lack of inductionN-hydroxylation of 2-FAA in mammary microsomes of non-lactatingrats supports our earlier conclusion that the formation of aproximate metabolite in mammary tumorigenesis by 2-FAA is accomplishedin the liver.     

Failure of ascorbic acid to inhibit growth of transplantable and dimethylbenzanthracene induced rat mammary tumors

The effect of ascorbic acid on the growth of 7,12-dimethylbenzanthracene (DMBA)-induced mammary tumor and on growth of R3230AC and MT/W9-B transplantable rat mammary tumors was investigated. High doses of ascorbic acid averaging about 540 mg/day/rat administered orally in drinking water had no effect on the growth of both R3230A and MT/W9a-B transplantable mammary tumors. Furthermore, vitamin C was unable to postpone tumor induction, reduce tumor incidence or prolong survival time of rats treated with DMBA.     

Use of monoclonal antibodies to characterize the induction response of the cytochrome P-450-dependent mixed function oxidase system to nitrofluoranthenes

In prior studies with neonatal rats we have suggested that nitratedpolycyclic aromatic hydrocarbons (NPAH) are 3-methylcholanthrene(3-MC) type of inducers of cytochrome P-450. These observationshave been extended by studying the effect of fluoranthene (FL)and its nitrated derivative, 3-nitrofluoranthene (3-NF) anda mixture of nitrated fluor-anthenes (NTs) on the inductionof hepatic and pulmonary monooxygenase activities in adult rats.We have characterized the effect of these compounds on hepaticcytochrome P-450 isozyme(s) using immunoblot analysis. The administrationof 3-NF and NFs to rats resulted in highly significant induction(1.9- to 5.8-fold) of hepatic and pulmonary aryl hydrocarbonhydroxy-tose (AHH) and 7-ethoxvresoruffiri-O-deethylase (ERD)activities. FL was either ineffective or much less effectivein inducing these enzyme activities. The enzyme induction responseto these compounds occurred in the following order: NFs >3-NF > FL. SDS-PAGE of hepatic mkrosomes prepared from FL-,3-NF-and NFs-treated animals revealed a higher content of proteinmigrating in the P-450 region. Characterization of isozymesof P-450 was carried out by Western blot analysis with highlyspecific monoclonal antibodies (MAb) raised against 3-MC-spedficP-450 (MAb 1-7-1) and phenobarbital-spedfic P-450 (MAb-2-66-3)isozymes. Hepatic mkrosomes prepared from 3-NF-and NFs-treatedrats showed two distinct immunopredpitin bands with MAb 1-7-1whereas mkrosomes prepared from FL-treated animals showed asharp band with MAb 2-66-3. MAb 1-7-1 significantly inhibited({small tilde}80%) AHH activity induced by 3-NF and NFs. Onthe other hand FL-induced AHH activity was only moderately ({smalltilde}30%) inhibited by MAb 1-7-1 whereas higher inhibition({small tilde}60%) was observed with MAb 2-66-3. Analysis ofBP metabolites by h.p.l.c. revealed enhanced production of metabolitesby liver microsomes from 3-NF- and NFs-treated animals. Theformation of BP 7,8-diol was 1.8-to 2.4-fold increased followingtreatment of animals with 3-NF and NFs respectively. Additionof MAb 1-7-1 to a microsomal mixture from 3-NF- and NFs-treatedrats inhibited the formation of BP phenols (60—75%) andBP 7,8 diol (52—60%). These inhibitory effects were notobserved with mkrosomes prepared from FL-treated rats. Thesestudies suggest that NFs induce specific monooxygenases in liverand that they are inducers of P-450 isozymes c and d.     

Experimental down-regulation of intermediate biomarkers of carcinogenesis in mouse mammary epithelial cells

Summary The polycyclic aromatic hydrocarbon 7,12-dimethylbenz(a)anthracene (DMBA) is a metabolism-dependent procarcinogen whose tumorigenicity is modified by dietary and endocrine manipulationsin vivo. DMBA initiates molecular and cellular alterations in the mammary tissue, while dietary components and estrogens affect the post-initiational phase of tumorigenic transformation. The mechanism(s) responsible for modulation of tumorigenic transformation remain unclear. This study examines the effects of selected tumor suppressing agents and estradiol (E₂) metabolites onin vitro DMBA carcinogenesis utilizing a newly established mouse mammary epithelial cell line C57/MG. Alteration in DNA repair synthesis, metabolism of E₂ via the C2- and C16-hydroxylation pathways, and acquisition of anchorage-independent growth were utilized as molecular, endocrine, and cellular biomarkers to quantitate the cellular transformation by DMBA and its modulation by tumor suppressing agents and E₂ metabolites. A single 24 hr exposure of 0.78 µM DMBA to C57/MG cells resulted in a 193.9% increase in DNA repair synthesis and a 73.1% decrease in C2/C16 hydroxylation of E₂. The DMBA treated C57/MG cells also exhibited increased anchorage-independencein vitro prior to tumorigenesisin vivo. A simultaneous treatment of cells with DMBA and with the highest non-cytotoxic doses of the tumor suppressing agents 5 µM N-(4-hydroxyphenyl) retinamide (HPR), 50 µM indole-3-carbinol (I3C), or 1 µM tamoxifen (TAM) resulted in a 35.6% to 63.9% decrease in DNA repair synthesis, a 23.8% to 1347.6% increase in C2/C16 hydroxylation of E₂, and a 53.8% to 72.4% decrease in anchorage-independent growth. The E₂ metabolites at the highest non-cytotoxic doses of 0.76 µM estrone (E₁), 0.69 µM 2-hydroxyestrone (2-OHE₁), and 0.66 µM 2-methoxyestrone (2-MeOHE₁) suppressed DMBA-induced DNA repair synthesis by 56.0% to 68.8%. These tumor suppressing agents and E₂ metabolites also effectively suppressed post-initiational, anchorage-independent growth by 24.9% to 72.4%. These results indicate that DMBA induces cellular transformation in part by causing DNA damage, altering C2/C16 hydroxylation in favor of C16-hydroxylation, and inducing anchorage-independent growth prior to tumor development. Effective downregulation of these genotoxic, endocrine and proliferative end points by prototypic tumor suppressing agents and by E₂ metabolites generated via the C2-hydroxylation pathway suggest that these agents may influence mammary tumorigenesis by inhibiting early occurring initiational and/or post initiational events.Abbreviations DMBA 7,12-dimethylbenz(a)anthracene - HPR N-(4-hydroxyphenyl) retinamide - I3C indole-3-carbinol - TAM tamoxifen - E₂ 17-estradiol - E₁ estrone - 2-OHE₁ 2-hydroxyestrone - 2-MeOHE₁ 2-methoxyestrone - 16-OHE₁ 16-hydroxyestrone - E₃ estriol - DME/F12 Dulbecco's modified Eagle's medium - F12 Ham's medium - HU hydroxyurea - PBS phosphate buffered saline - NaOH sodium hydroxide - SDS sodium dodecyl sulfate - TCA trichloroacetic acid - [C2-³H] E₂ estradiol labeled at C2 position - [C16-³H] E₂ estradiol labeled at C16 position - ANOVA analysis of variance     

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     

Effects of 5,6-benzoflavone, indole-3-carbinol (I3C) and diindolylmethane (DIM) on chemically-induced mammary carcinogenesis: is DIM a substitute for I3C?

The abilities of 5,6-benzoflavone (5,6-BF, a synthetic flavonoid), indole-3-carbinol (I3C, a plant derived product) or diindolylmethane (DIM, a condensation product of I3C) to alter the induction of mammary cancers induced by the carcinogens 7,12-dimethylbenzanthracene (DMBA) or N-methyl-N-nitrosourea (MNU) were evaluated. Interestingly, the first two agents act as aryl hydrocarbon receptor (AhR) agonists, while DIM does not. The agents were initially examined for their ability to inhibit DMBA-induced mammary carcinogenesis. Agents were administered for 14 days starting 7 days prior to a single dose of the carcinogen. Evaluated over an extensive range of doses (165, 550 and 1650 ppm in the diet), 5,6-BF caused a dose-dependent decrease of mammary cancers. In addition, 5,6-BF at doses of 1650 and 165 ppm in the diet blocked the induction of DMBA-induced DNA adducts in the mammary gland by approximately 85% and 45%, respectively. In contrast, DIM (180 or 20 mg/kg BW/day) failed to block induction of DMBA tumors. The effect of these agents on the promotion/progression phase of carcinogenesis using the MNU mammary cancer model was also determined. 5,6-BF (1650 or 165 ppm in the diet), I3C (180 or 60 mg/kg BW/day administered by gavage), or DIM (180 or 60 mg/kg BW/day by gavage) were initiated 5 days after the administration of MNU, and continually thereafter. 5,6-BF decreased MNU- induced mammary tumor multiplicity by 40-60%. I3C reduced tumor multiplicity at the high dose, while DIM at either dose had minimal effects on tumor multiplicity. Thus, 5,6-BF and I3C were highly effective against initiation of DMBA-induced mammary carcinogenesis, and were also effective against MNU-induced tumors during the promotion/progression phase of carcinogenesis. In contrast, DIM had minimal effects in either model; arguing that administration of DIM is not analogous to administration of I3C.     

Inhibition of 7,12-dimethylbenz(a)anthracene-induced mammary tumors and DNA adducts by dietary selenite

The present studies were designed to examine the influence of dietary selenite supplementation on the initiation phase of 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary carcinogenesis and to correlate selenite-induced changes in the binding of DMBA metabolites to rat mammary cell DNA with the ultimate tumor incidence. Diets formulated to contain selenium, as sodium selenite at 0.1, 0.5, 1, 2, or 4 micrograms/g were fed for 2 weeks prior to and 2 weeks following treatment with DMBA (5 mg/kg body weight). Food intake and weight gain did not differ among treatments. Tumor incidence correlated inversely to the quantity of selenium consumed (r = -0.99). Final tumor incidences were 52, 32, 24, 14, and 10% for rats fed 0.1, 0.5, 1, and 4 micrograms selenium/g, respectively. In a separate group of rats fed a diet containing 4 micrograms selenium/g during both the initiation and promotion stages the final tumor incidence was 4.8%. Selenite supplementation for 2 weeks markedly depressed the occurrence of individual and total DMBA-DNA adducts. The final mammary tumor incidence correlated positively with total DMBA-DNA adducts (r = 0.99). These studies clearly demonstrate that selenite can inhibit the initiation stage of mammary carcinogenesis. This reduction in tumor incidence is likely due to a reduction in carcinogen metabolism and ultimately adduct formation.     

Induction of mammary cytochromes P-450: an essential first step in the metabolism of 7,12-dimethylbenz[a]anthracene by rat mammary epithelial cells

Rat mammary epithelial cells (RMEC) in culture have been shownto activate polycydlic aromatic hydrocarbon (PAH) carcinogens.This study investigates the role of mammary cyto chrome P-450monooxygenases in these metabolic processes. Monooxygenatlonof 7,12-dimethythenz[a]anthracene (DMBA) by RMEC in cultureexhibited a 6-h lag period before reaching a constant rate.The mechanism for this time-dependent expression of DMBA monooxygenaseactivity was investigated in lysed cells, where both conjugationand in situ induction of P-450 are prevented. Although metabolismof DMBA by untreated RMEC lysates was undetectable (<1 pmol/mgcell protein/h), prior exposure of cultured cells to benz[a]anthracene(BA) induced DMBA metabolism, ({small tilde}100 pmol/mg cellprotein/h). BA pretreatment also eliminated the lag period formetabolism of DMBA by cultured RMEC but did not prevent additionalinduction of DMBA monoxygenase activity by the substrate. Thedistribution of monooxygenated DMBA metabolites formed by BA-inducedcell lysates was clearly different from that obtained with purifiedP-450c, the predominant PAH-inducible isozyme in rat liver.For example, the carcinogen precursor DMBA 3,4-dihydrodiol,which is not formed by P-450c, was a dearly detectable productin RMEC. The low epoxide hydratase activity of BA-induced lysate({small tilde}400-fold lower compared to that in the liver)limited formation of all DMBA dihydrodlols. The formation ofDMBA 3,4-dihydrodiol increased by 5-fold following additionof exogenous purified epoxide hydratase. The DMBA monooxygenaseactivity of BA-induced RMEC lysates was completely inhibitedby -naphthoflavone but was only partially inhibited (50%) bya polyclonal antibody raised against cytochrome P-450c. AntiP-450c completely inhibited formation of some of the metabolites,partially inhibited formation of others and notably stimulatedformation of DMBA 3,4-dihydrodlol by 60%. A polyclonal antibodythat recognized both rat hepatic P-450a and a group of P-450isozymes related to P-450h, and which totally inhibited DMBA3,4-dihydrodiol formation by rat liver microsomes, did not inhibitformation of any DMBA metabolite in RMEC, including DMBA 3,4-dihydrodiol.Western blot analyses of RMEC homogenates demonstrated thatBA pretreatment induces P-450c, but not P-450a or any of theP-450h-related isozymes. We conclude that metabolism of DMBAby RMEC depends on induction of P-450c and at least one additionalform of cytochrome P-450 which is immunochemically distinctfrom rat hepatic P-450a and P-450h related isozymes, but issensitive to a-naphthoflavone.     

Reduction by pituitary isograft of inhibitory effect of large dose of estrogen on incidence of mammary tumors induced by carcinogen in ovariectomized rats

Interaction of prolactin and estrogen on the incidence of mammary tumors induced by 7,12-dimethylbenz (a) anthracene (DMBA) in female Sprague-Dawley rats was studied. Rats were bilaterally ovariectomized at 45 days of age and then treated subcutaneously with 20μg of estradiol benzoate (EB) every 2 days beginning the next morning. They received single intravenous injections of 5mg DMBA at 52 days of age. Two months later they were divided into four groups: Group I received no pituitary grafting: group II was grafted with three isologous pituitaries under the right kidney capsule: groups III and IV received six pituitaries each. EB injections were continued throughout the experiment to every group except group IV. Serum prolactin levels were determined by radioimmunoassay just before pituitary grafting, 20 days, 2 and 3 months after grafting. At the end of 5 months after DMBA injection, the mammary tumor incidence and the number of palpable tumors per tumor-bearing rat apparently increased, and the percentage of completely regressed tumors decreased in groups II and III as compared to group I. Serum prolactin level was significantly higher in groups II and III than in group I at 3 months after pituitary grafting. Only one rat had palpable mammary tumors in group IV and serum prolactin levels were always significantly lower in this group than in the others. These results indicate that prolactin secreted by the grafted pituitaries overcomes the inhibitory action of a large dose of estrogen on the growth of DMBA-induced mammary tumors of the rat and results in an increased incidence of the tumors.     

Evidence of separate pathways for viral and chemical carcinogenesis in c3h/stwi mouse mammary glands

Mammary tumors in mice may arise as the result of exogenous infection with the mouse mammary tumor virus [MMTV(S)], usually via the milk; by the action of endogenous MMTV genes which are transmitted genetically and are sometimes expressed as infectious virus [MMTV(L)]; or by the action of chemical carcinogens. We have examined the etiological relationship between chemical and virus in the induction of mammary cancer in C3H/StWi mice. Mammary tumors were induced with 7.12-dimethylbenz(a)anthracene (DMBA) in virgin C3H/StWi mice infected with exogenous mouse mammary tumor virus (C3H/StMTV) and in uninfected C3H/StWi females. The percent tumor risk in females whose glands were infected with exogenous mouse mammary tumor virus [MMTV(S)] was not different from that of MMTV(S)-negative, C3H/StWi mice following treatment with DMBA. This result suggested that there was no synergistic effect between the two carcinogens, exogenous MMTV and Dmba. All the tumors arising in DMBA-treated C3H/StMTV virgins were positive for MMTV env gene product, gp52 and MMTV gag gene product, p²⁷ by radioimmune competition assay. MMTV(S)-induced hyperplastic alveolar nodules (HAN) were observed in 62 % of the untreated C3H/StMTV glands at 8 months of age. Therefore, MMTV(S) was present and active in the mammary gland during the experimental period but had no enhancing influence on the sensitivity of the gland to carcinogenesis by DMBA. Tumors appearing in DMBA-treated C3H/StWi virgin females were also tested for MMTV gp⁵² and p²⁷ antigens to determine the presence of endogenous MMTV gene activity. Only occasional C3H/StWi tumors were positive and in most of these, p²⁷, but not gp⁵² was detected, suggesting non-coordinate expression of these endogenous MMTV gene products. Both alveolar (HAN) and ductal hyperplasia (DH) were found in DMBA-treated C3H/StMTV glands, whereas only DH were found in DMBA-treated C3H/StWi mice. Nevertheless, the DMBA-induced C3H/StMTV tumor histopathology was remarkably indistinguishable from that in tumors produced by DMBA in C3H/StWi mice, implying that in both groups, tumors arose primarily from the chemically-induced mammary dysplasias. These data taken together appear to support the conclusion that DMBA and Mmtv follow separate pathways to the induction of cancer in the mouse mammary gland.     

Overexpression of 5‐lipoxygenase and its relation with cell proliferation and angiogenesis in 7,12‐dimethylbenz(α)anthracene‐induced rat mammary carcinogenesis

The present study was performed to investigate the critical role of 5‐lipoxygenase (5‐LOX) in 7,12‐dimethylbenz(α)anthracene (DMBA)‐induced rat mammary inflammation associated carcinogenesis. Female Sprague–Dawley rats at 50 days of age were treated with 7,12‐dimethylbenz(α)anthracene (DMBA; 0.5 mg/100 g body weight) by a single tail vein injection, followed by administration of zileuton (2000 mg/kg diet) from week 7 until the termination of the study at 31 wk. 5‐LOX protein expression, 5‐hydroxyeicosatetraenoic acid (5‐HETE), and leukotriene B₄ (LTB₄) production in rat mammary tissue were analyzed at 6, 12, and 24 wk post‐DMBA injection. Rate of cell proliferation was analyzed by bromodioxyuridine labeling index (BrdU‐LI). Microvessel density, level of VEGF, and MMP‐2 were also measured. DMBA induces inflammation in rat mammary gland as early as 6 wk. 5‐LOX is upregulated in DMBA treated rats right from 6 wk when compared with their normal counterparts. An overexpression of 5‐LOX is accompanied with increase in 5‐HETE, LTB₄ production and high BrdU‐LI with an increase of two key angiogenic factors for tumorigenesis; MMP‐2 and VEGF. It was found that 5‐LOX specific inhibitor brought about substantial protection against DMBA‐induced mammary carcinogenesis. Histological findings showed substantial repair of hyperplastic lesions. There was a significant reduction in the rate of cell proliferation and expression of angiogenic factors, MMP‐2 and VEGF. 5‐LOX plays an important role in DMBA‐induced inflammation associated carcinogenesis via activation of MMP‐2 and VEGF. 5‐LOX expression can be considered as a critical event in controlling the process of mammary tumor development. © 2011 Wiley Periodicals, Inc.     

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.

     

Black tea and mammary gland carcinogenesis by 7,12- dimethylbenz[a]anthracene in rats fed control or high fat diets

Epidemiological studies suggest that tea may reduce cancer risk, and in laboratory rodents, chemopreventive effects of tea or purified extracts of tea have been demonstrated in lung, gastrointestinal tract and skin. There is some evidence of chemoprevention by tea in the mammary gland, but the data are not conclusive. In order to evaluate more fully the possible influence of black tea on 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary gland tumors in the female S-D (Sprague-Dawley) rat, three large studies were performed: experiment 1, tumorigenesis in rats fed AIN-76A diet and given 25 mg/kg DMBA and 1.25 or 2.5% whole tea extract or water to drink; experiment 2, tumorigenesis in rats given 15 mg/kg DMBA and the same diet and fluids as in experiment 1; experiment 3, tumorigenesis in rats fed control or HF (high fat, corn oil) diet and given 15 mg/kg DMBA and 2% tea or water to drink. Tea was given throughout the experiment; DMBA was given by gastric gavage at 8 weeks of age. There was no consistent effect of tea on tumorigenesis in rats fed AIN-76A diet; there was, however, evidence in experiment 3 of a reduction of tumorigenesis by tea in rats fed the HF diet. In experiment 3, rats fed the HF diet and given water showed the expected increase in tumor burden (number and weight) compared with rats fed control diet. However, rats fed the HF diet and given 2% tea showed no increase in tumor burden; their tumor burden was significantly lower than in rats fed the HF diet and given water (P < 0.01) and was not different from rats fed control diet and given water or tea. In addition, in experiment 3, the number of malignant tumors per tumor- bearing rat was increased by the HF diet in water-drinking rats (P < 0.01) but not in tea-drinking rats. Therefore, it appears that tea partially blocked the promotion of DMBA-induced mammary tumorigenesis by the HF diet.