Procarcinogen activation by cytochrome P450 3A4 and 3A5 expressed in Escherichia coli and by human liver microsomes

Recent studies indicate that cytochrome P450 (P450) 3A4 playsimportant roles in the activation of procarcinogens such asaflatoxin B₁ and sterigmatocystin, as well as in the oxidationof a number of structurally diverse chemicals and endogenouscompounds. Since P450 3A5 has been reported to be present atsignificant levels in liver microsomes in     

Metabolic activation of arylhydroxamic acids by N-O-acyltransferase of rat mammary gland.

The lactating mammary glands of rats contain an arylhydroxamic acid N,O-acyltransferase that catalyzes the formation of arylamine-substituted nucleic acid on incubation with N-hydroxy-N-2-acetylaminofluorene or N-hydroxy-N-4-acetylaminobiphenyl and transfer RNA. The acyltransferase activity migrates as a single component with a molecular weight of 28,000 on gel filtration on Sephadex G-100. Acyltransferase activities of the lactating mammary glands of Sprague-Dawley animals are approximately twice those of the less susceptible Fischer strain as determined by assay with either hydroxamic acid. The fluorene substrate was 15 times as efficient as the biphenyl compound in promoting adduct formation. Ribosomal RNA adducts formed in vivo after administration of N-hydroxy-N-2-acetylaminofluorene were consistent with an acyltransferase mechanism of activation in that the adducts did not retain the acetyl group.     

Mutational activation of c-Ha-ras genes in intraductal proliferation induced by N-nitroso-N-methylurea in rat mammary glands

Although administration of a single dose of N-nitroso-N-methylurea (NMU) to young virgin rats induces a high rate of mammary carcinomas, precise histogenesis of the carcinomas has not been well characterized. In this study, we investigated the alterations of H-ras gene in early focal lesions as well as carcinomas in the mammary glands of F344 rats treated with NMU. At 2 weeks after treatment, intraductal proliferation (IDP) was occasionally observed, and mammary carcinomas emerged at 12 and 36 weeks. The individual lesions of IDP and carcinomas were scooped out from the tissue sections under a stereomicroscope, and the DNA-sequence-spanning codon 12 of H-ras gene was amplified from the tissue sections by polymerase chain reaction (PCR). The analysis of amplified DNA by oligonucleotide hybridization revealed that 65% (11/17) of IDP and 89% (16/18) of carcinomas had a point mutation (G-to-A transition) at the 2nd position of H-ras codon 12. However, the DNA amplified from the areas, which appear histologically normal, never showed such mutation. These results indicate that IDP is a very early change for NMU-induced mammary carcinogenesis.     

Interspecies comparison of human and rat mammary epithelial cell-mediated mutagenesis by polycyclic aromatic hydrocarbons

In order to compare the interactions of procarcinogens with mammary cells from humans and rats, a uniform set of mediated mutagenesis assays has been established. In these assays, species-specific mammary epithelial cells activate procarcinogens, and specific locus mutations are quantitated in cocultured V-79 cells. Mutations were induced in the rat mammary cell coculture system exposed to 7,12-dimethylbenz(a)anthracene but not benzo(a)pyrene. In contrast, in the human mammary cell coculture system benzo(a)pyrene was much more effective than 7,12-dimethylbenz(a)anthracene in the induction of mutations. These results suggest caution in extrapolating carcinogenesis data between rodents and humans. They also suggest that the relationship between the ubiquitous environmental xenobiotic benzo(a)pyrene and the etiology of human breast cancer requires further exploration.     

Effect of autologous mammary tumour extracts on human leukocyte migration in vitro

In eight out of 22 patients with mammary carcinoma, extract of tumour tissue induced inhibition of autologous leukocyte migration in vitro, demonstrating a state of cellular (delayed type) hypersensitivity. Non-tumorous tissue did not inhibit migration. The effect of the carcinoma extracts was not due to non-specific toxicity, since the migration of leukocytes from matched controls was not affected by the extracts. In nine cases of benign mammary lesions, extracts of autologous mammary tissue did not induce inhibition of migration. Preliminary results of follow-up studies in some of the carcinoma patients are reported.     

Chemical carcinogen activation in the rat mammary gland: intraorgan cell specificity

Cells from 50–55 day old virgin Sprague-Dawley femalerat mammary gland were divided into parenchymal (epithelial)and stromal enriched populations. The ability of these cellsto activate carcinogens was quantitated employing a mediatedmutagenesis assay. In addition, the populations' ability toproduce water soluble metabolites from these carcinogens wasestimated. We report that the potent mammary carcinogen 7,12-dimethylbenz[a]anthracenewas activated by both mammary parenchymal and stromal cells,while the non-mammary carcinogen aflatoxin B₁ was not activatedby either cell type. However, the weak mammary carcinogen benzo[a]pyrenewas activated by the stromal cells and not by the parenchymalcells from which mammary carcinomas arise. The data suggestthat the intra-organ relationship between cell types that activatea carcinogen, and cell types that undergo neoplastic transformation,may in part help explain the organ specific potency of a carcinogen.     

Chorionic gonadotropin inhibits rat mammary carcinogenesis through activation of programmed cell death

Human chorionic gonadotropin (hCG) inhibits the progression of 7,12- dimethylbenz[a]anthracene (DMBA) induced mammary carcinomas. In order to determine whether this phenomenon was mediated by induction of programmed cell death or apoptosis, 45-day-old virgin Sprague-Dawley rats received 8 mg DMBA/100 g body weight; 20 days later they were injected daily with 100 IU hCG for 40 days (DMBA + hCG group). Age- matched untreated, hCG- and DMBA + saline treated rats were used as controls. Tissues were collected at the time of DMBA administration and at 5, 10, 20 and 40 days of hCG injection. RNA from mammary glands, adenocarcinomas and ovaries was probed for transforming growth factors (TGF) alpha and beta, and the apoptotic genes TRPM2, ICE, bcl2, bcl-XL, bcl-XS, p53 and c-myc. The mammary glands of hCG-treated animals with or without DMBA exhibited elevated expression of TRPM2, ICE, bcl-XS, c- myc and p53; and elevation in the apoptotic index. Mammary adenocarcinomas developed in those animals treated with hCG showed an elevation in the expression of p53, c-myc and ICE genes in comparison with the levels detected in the adenocarcinomas developed by the animals treated with DMBA alone. No significant alterations in the expression of any of the genes tested was observed in ovarian RNAs. These results led us to conclude that hCG induces programmed cell death in the mammary gland initiated in the carcinogenic process, that this process is p53 dependent, and is modulated by c-myc expression. Our data also indicate the possibility that a cell death program dependent on the bcl2 family exists, because of the potential involvement of p53, bcl-XS and Bax in apoptosis. This additional mechanism of tumor inhibition makes hCG treatment a useful approach for the prevention and therapy of breast cancer.      

Methylation silencing of angiopoietin-like 4 in rat and human mammary carcinomas

Aberrant DNA methylation is deeply involved in the development and progression of human breast cancers, but its inducers and molecular mechanisms are still unclear. To reveal such inducers and clarify the molecular mechanisms, animal models are indispensable. Here, to identify genes silenced by promoter DNA methylation in rat mammary carcinomas, we took a combined approach of methylated DNA immunoprecipitation (MeDIP)-CpG island (CGI) microarray analysis and expression microarray analysis after treatment with epigenetic drugs. MeDIP-CGI microarray revealed that among 5031 genes with promoter CGI, 465 were methylated in a carcinoma cell line induced by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), but not in normal mammary epithelial cells. By treatment of the cell line with 5-aza-2'-deoxycytidine and trichostatin A, 29 of the 465 genes were shown to be re-expressed. In primary mammary carcinomas, five (Angptl4, Coro1a, RGD1304982, Tmem37 and Ndn) of the 29 genes were methylated in one or more of 25 samples. Quantitative expression analysis revealed that Angptl4 had high expression in normal mammary glands, but low expression in primary carcinomas. Also in humans, ANGPTL4 was unmethylated and expressed in normal mammary epithelial cells, but was methylated in 11 of 91 (12%) primary breast cancers. This is the first study to identify genes aberrantly methylated in rat mammary carcinomas, and Angptl4 is a novel methylation-silenced gene both in rat and human mammary carcinomas. The combination of the MeDIP-CGI microarray analysis and expression microarray analysis after treatment with epigenetic drugs was effective in reducing the number of methylated genes that are not methylation silenced.     

DNA adduct formation by tamoxifen with rat and human liver microsomal activation systems

Using microsomal preparations from rat and human liver, we investigatedthe activation of the anti-estrogen compound tamoxifen (TMX)to form DNA adducts. Pretreatment of rats with phenobarbitalincreased DNA adduct formation by microsomal activation of TMX3- to 6-fold, depending on the cofactors used. When reducednicotinamide-adenine dinucleotide phosphate (NADPH) was usedas a cofactor in human and rat microsomal activation systems,the relative DNA adduct levels were 2.9 and 5.2 x 10^–8respectively and 1-3 TMX-DNA adducts were detected by ²³P-postlabeling;DNA adduct 1 was the same in both microsomal systems. When cumenehydroperoxide (CuOOH) was used as a cofactor, activation ofTMX produced four major DNA adducts and several minor DNA adductsin both rat and human liver microsomes; the relative adductlevels were 11.1 and 23.1 xlO^–8 respectively. TMX-DNAadducts 1, 4, 5 and 6 were similar in both human and rat microsomalsystems with CuOOH as the cofactor. The TMX-DNA adducts formedwith NADPH as the cofactor were clearly different from thoseformed with CuOOH as the cofactor, which implies that the metabolitesleading to the individual DNA adducts were different. Additionof a P450 inhibitor, either n-octylamine or     

Modulation of rat mammary carcinogenesis by indomethacin

Indomethacin, a nonsteroidal antiinflammatory agent which inhibits prostaglandin biosynthesis, has significant activity in inhibiting the growth and/or inducing the regression of transplantable tumors. The present study was designed to determine if, in addition to its chemotherapeutic effects, indomethacin also acts as a cancer chemopreventive agent. Fifty-day-old virgin female Sprague-Dawley rats were given a single intragastric dose of either 8 or 16 mg of 7,12-dimethylbenz(a)anthracene (time 0). Basal diet was supplemented with 25 or 50 mg of indomethacin per kg of diet by the following protocol: (a) -2 to +1 week; (b) +1 week to end; or (c) none. Administration of indomethacin by both protocols resulted in an inhibition of mammary tumorigenesis; however, the effect of -2 to +1 week indomethacin exposure was primarily on the induction of benign mammary tumors, while +1 week to end indomethacin administration inhibited the induction of both benign mammary tumors and mammary cancers. These data indicate that indomethacin has significant protective activity when administered either during the "early" stage (comprising the carcinogen-target cell interaction) or the "late" stage (postcarcinogen tumor development) of mammary carcinogenesis in rats. Possible mechanisms of indomethacin action include both local and systemic effects.     

Inhibition of rat mammary carcinogenesis by monoterpenoids

We have previously demonstrated the cancer chemopreventive activities of the monocyclic unsaturated monoterpene d-limonene. In the present work we report data evaluating the chemopreventive effects of limonene and five other monoterpenes with various chemical structures using a 7,12-dimethylbenz[a]anthracene (DMBA)-induced rat mammary carcinogenesis model. The terpenes tested include: oxygenated [(-)-menthol] and non-oxygenated (d-limonene) monocyclic forms, oxygenated (1,8-cineole) and non-oxygenated [(+/-)-alpha-pinene] bicyclic forms and oxygenated [(+/-)-linalool] and non-oxygenated (beta-myrcene) acyclic forms. Dietary additions of each of the monocyclic terpenes, d-limonene or (-)-menthol resulted in a significant inhibition of mammary carcinogenesis. Furthermore, menthol was found to be a more potent chemopreventive agent than limonene during the DMBA initiation of rat mammary tumors. The acyclic and bicyclic terpenes had no significant chemopreventive activities at the dose levels used in these studies.     

Mutagenicity and DNA-excision repair induced by isoniazid after metabolic activation by isolated human and rat hepatocytes

The mutagenic potency of isoniazid (INH) (a widely used antitubercular drug) towards Salmonella typhimurium strain hisG46 was studied in the Salmonella/hepatocyte suspension-assay, comprising isolated human or rat hepatocytes as metabolic system. The potency of INH to induce DNA-excision repair in these hepatocytes was also measured. With rat hepatocytes, INH appeared to be only weakly mutagenic and did not induce significant increases in hepatocellular DNA-excision repair. With isolated hepatocytes of two human subjects, INH appeared also only weakly mutagenic. However, with hepatocytes of two other human subjects, INH was found to be highly mutagenic. Comparable results were obtained for the induction of hepatocellular DNA-excision repair.     

Relation between hormone dependency and the estrogen receptor of mammary carcinoma--comparative study of rat and human mammary tumors

A comparative study of rat and human mammary tumors was undertaken to clarify the mechanism of hormone dependency by the PAP method. In normal mammary glands, histological estrogen receptor ER (+) cells were observed in the ductal epithelium and acinal cells, while only ER (-) cells were noted in HAN. After overiectomy, 55.6% of the ER (+) cells changes to ER (-). Furthermore, histological changes were glandular atrophy, anaplastic changes, (with) the remaining ER (+) cells (being in the) insular group. Those ER (+) cancer cells seem to be different in hormone sensitivity. As for human mammary glands, proliferation of ER (+) cells was observed in papillary lesions as duct papillomatosis. In the cancer tissues as a whole, ER (+) and ER (-) cancer cells were mixed, showing a so-called mosaic pattern.     

Aberrant activation of benzo(a)pyrene in cultured rat mammary cells in vitro and following direct application to rat mammary glands in vivo

Primary cultures of epithelial cell aggregates and fibroblasts derived from mammary tissue from female Wistar rats were treated with benzo(a)pyrene (BP), and their DNA was isolated and analyzed. At least seven BP-DNA adducts were detected in DNA hydrolysates by high-performance liquid chromatography, none of which had the chromatographic properties characteristic of adducts formed by the putative ultimate carcinogen r-7, t-8-dihydroxy-t-9, 10-oxy-7, 8,9,10-tetrahydrobenzo(a)pyrene (anti-BP-7, 8-diol-9, 10-oxide) or other known electrophilic metabolites of BP. Similar profiles of adducts were obtained from mammary DNA of rats that had been treated with BP by injection into their mammary fat pads. Chromatography on boronate columns indicated that five of the seven adducts contained cis-hydroxyl groups. In contrast, when BP was administered by i.p. injection to female Wistar rats, anti-BP-7,8-diol-9,10-oxide-DNA adducts were detected in each of seven tissues, including mammary gland, that were examined.     

DNA damage in breast epithelial cells: detection by the single-cell gel (comet) assay and induction by human mammary lipid extracts

The presence of DNA damage in primary cultures of human mammary epithelial cells (HMECs), and the ability of extracts of human mammary lipid to cause such damage, has been investigated. Lipid extracts, prepared by a solid-phase procedure, and HMECs were obtained from breast tissue removed from healthy women (ages 18-50 years) who were resident in the UK and undergoing elective reduction mammoplasties. DNA single strand breaks (SSBs) were detected using the single-cell gel assay (comet assay) with alkaline electrophoresis (pH 12.3) and quantified by measuring comet tail length (CTL) (microm). Untreated HMECs and HMECs incubated (30 min, 37 degrees C) with a mammary lipid extract, with or without DNA-repair inhibitors hydroxyurea (HU) and cytosine arabinoside (ara-C), were examined. Ionizing radiation was used as a positive control. An active lipid extract gave a linear dose- response over the range 2.0-12.2 g equivalents. When MCL-5 cells, a line of metabolically-competent human lymphoblastoid cells, were used to compare the DNA-damaging properties of lipid extracts from six different donors, significant interindividual variations (median CTLs were 15.0, 53.5, 32.5, <4.0, <4.0 and 77.5 microm respectively) were observed. In eight subjects, the donors' HMECs were examined both before and after treatment with extracts of that donor's own lipid. Pre- existing DNA damage was detected in untreated HMECs from some donors (median CTLs 22.0-37.5 microm) that was not present in others (median CTLs 4.0-11.5 microm), and increases in CTL could be induced by incubation with the matching lipid extract (8 g equivalent) in more than half (five out of eight) the subjects examined (median CTL up to 111.0 microm). There was a tendency for the most active lipid extracts to be those obtained from donors whose HMECs also contained the most pre-existing DNA SSBs. The results of this pilot study may prove to be significant in relation to the initiation of breast cancer.