Comparative action of 1,4-phenylenebis(methylene)selenocyanate and its metabolites against 7,12-dimethylbenz[a]anthracene-DNA adduct formation in the rat and cell proliferation in rat mammary tumor cells

1,4-phenylenebis(methylene)selenocyanate (p-XSC) inhibits 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary carcinogenesis and DMBA-DNA binding in the rat mammary gland. Tetraselenocyclophane (TSC) was identified in rat feces as a metabolite of p-XSC. This led us to postulate the metabolic pathway: p-XSC-->glutathione conjugate (p-XSeSG)-->aromatic selenol (p-XSeH)-->TSC. Whether p-XSC or one of its metabolites is responsible for cancer prevention is the focus of this study. We utilized the DMBA-DNA binding assay with p-XSC as a positive control to evaluate the chemopreventive potential of p-XSC metabolites at dietary selenium levels of 10 ppm. Rats were fed AIN-76A diet supplemented with various selenium compounds for 1 week prior to the oral administration of a single dose of [3H]DMBA (5 mg per rat, specific activity 51.3 mCi/mmol). The rats were sacrificed 24 h later and DNA was isolated from the mammary fat pads. Relative levels of total binding were: [pmol/mg DNA, mean +/- S.D., n=6]; DMBA [7.2 +/- 1.6]; DMBA+p-XSC [3.5 +/- 2.7]; DMBA+p-XSeSG [2.2 +/- 1.1]; DMBA+TSC [5.6 +/- 2.9]. All selenium compounds, except TSC, significantly inhibited DMBA-DNA adduct formation; however, the difference between p-XSC and p-XSeSG was not statistically significant. The inhibition of total binding was attributed to a reduction in the formation of the three major adducts derived from bay-region diol epoxides of DMBA. On the basis of their chromatographic characteristics, these were identified as anti-diol-epoxide:deoxyguanosine, syn-diol-epoxide:deoxyadenosine, and anti-diol-epoxide:deoxyadenosine. Our results suggest that p-XSeSG, but not TSC, is the likely inhibitor of mammary cancer. Selenium levels measured by atomic absorption spectroscopy in the target organ (mammary fat pads) and in plasma following the dietary administration of selenium compounds were in the order of p-XSeSG congruent with p-XSC>TSC. These results appear to be consistent with their order of inhibitory effects on total DMBA-DNA binding. Further in vitro studies of the effect of selenium compounds on cell proliferation suggest that, depending on the dose and time point selected, p-XSC is comparable to or better than p-XSeSG; but both are more effective than TSC. Collectively, our in vivo and in vitro results indicate that p-XSC and its conjugate are better candidates than TSC for future studies on mammary cancer chemoprevention.     

Influence of age and parity on the susceptibility of rat mammary gland epithelial cells in primary cultures to 7,12-dimethylbenz(a) anthracene

Summary Mammary gland epithelial cells from rats of different ages or with different reproductive histories vary in their proliferative properties and susceptibility to dimethylbenz(a)anthracene (DMBA) carcinogenesis in vivo. The present study was carried out to determine whether these differences are maintained under in vitro conditions. Primary cultures of mammary gland epithelial cells of young virgin, old virgin, and parous rats were treated with various doses of DMBA. Growth rates, DNA synthesis, and dose-response curves were determined; the toxicity of DMBA was measured by its effect on cell growth. Cell morphology was studied by transmission and scanning electron microscopy. Epithelial cells from the mammary gland of young virgin rats adapted rapidly to the culture conditions, behaving as if the cells were in the logarithmic phase of growth prior to plating. Mammary gland epithelial cells from old virgin and parous rats required a lag period prior to cell growth during which the proliferating cells adapted to the culture conditions. Cells from each group had comparable doubling times, and DNA synthesis peaked approximately 1 d after initiation of growth in culture. The numbers of proliferating cells decreased with increasing age and parity of the donor. Mammary gland epithelial cells of young virgin rats were more susceptible to both low and high doses of DMBA than those of old virgin and parous rats when the carcinogen was added either 24 h after plating or at the peak of DNA synthesis. These results indicate that age and parity influence the proliferative status of the cells and their susceptibility to DMBA in vitro, simulating in that way the in vivo situation. Supported by Public Health Service Grants CA-23539 and CA-27026 from the National Cancer Institute and by an Institutional grant from the United Foundation of Greater Detroit.     

Formation of 7,12-dimethylbenz[alpha] anthracene-DNA adducts in 7,8-benzoflavone-treated hamster embryo cells.

Pretreatment of secondary cultures of Syrian hamster embryo cells with 7,8-benzoflavone (7,8-BF) inhibited both the metabolism of 7,12-dimethylbenz[alpha] anthracene (DMBA) and the formation of DMBA-DNA adducts. The DMBA-deoxyribonucleoside adducts from 7,8-BF-treated cultures had the same elution profiles on Sephadex LH-20 columns as those from cultures exposed to DMBA alone, but 7,8-BF-treated cultures contained smaller amounts of DMBA-DNA adducts per mg DNA. As the concentration of 7,8-BF was increased, the decrease in the amount of DMBA-DNA adducts per mg DNA was logarithmic with respect to the decrease in the amount of DMBA metabolized. The results suggest that more than one metabolic step is required for the binding of DMBA to DNA in hamster embryo cells.     

7,12-Dimethylbenz[a]anthracene-DNA adduct formation in Wistar rat and Syrian hamster embryo cell cultures

The binding of 7,12-dimethylbenz[a]anthracene (DMBA) to DNA was examined in Syrian hamster and Wistar rat embryo cell cultures exposed to DMBA for 5, 24, 48 and 72 h. The level of binding of DMBA to DNA was about twice as great in the hamster embryo cells as in the rat embryo cells at all times. Analysis of the DMBA-deoxyribonucleoside adducts by immobilized boronate chromatography demonstrated that the ratio of adducts with no cis vicinal hydroxyl groups to those containing cis vicinal hydroxyl groups was much greater in the rat embryo cells (from 2.2:1 to 2.9:1) than in the hamster embryo cells (from 1.3:1 to 1.6:1). The hamster embryo cells contained three major DMBADE-DNA adducts: based upon their chromatographic behavior and comparison with the three major DMBA-DNA adducts described by Dipple et al. in mouse embryo cell cultures (Biochemistry, 24 (1985) 2291), two were tentatively identified as resulting from the reaction of anti-DMBADE (the isomer of 1,2-epoxy-3,4-dihydroxy-1,2,3,4-tetrahydro-DMBA with the epoxide and benzylic hydroxyl on the opposite faces of the molecule) with deoxyguanosine and deoxyadenosine and one adduct resulted from reaction of syn-DMBADE (epoxide and benzylic hydroxyl on the same face of the molecule) with deoxyadenosine. The anti-DMBADE-deoxyguanosine, syn-DMBADE-deoxyadenosine, and anti-DMBADE-deoxyadenosine adducts were present in hamster embryo cell DNA in a ratio of 1.2:2:1. The Wistar rat embryo cell DNA contained a much larger proportion of the syn-DMBADE-deoxyadenosine adduct. The relative proportions of the three major DMBA-DNA adducts in Syrian hamster embryo cells were similar at all times, but the proportion of syn-DMBADE-deoxyadenosine adduct decreased slightly with time in the rat embryo cells. These results indicate that there are species specific differences in the stereospecificity of activation of DMBA to DNA-binding diol epoxides which parallel those observed for benzo[a]pyrene (BaP). The high proportion of deoxyadenosine adducts suggests that they may have an important role in the induction of biological effects by DMBA.     

The binding of 7,12-dimethylbenz[a]anthracene to mammary gland macromolecules in the hamster:effects of Enovid feeding or transplacental exposure to diethylstilboestrol

The covalent binding of 7,12-[3H]dimethylbenz[a]anthracene ([3H--DMBA) to mammary gland macromolecules was studied in hamsters fed a contraceptive mixture, Enovid, those exposed transplacentally to diethylstilboestrol (DES), and controls. Compared with rats, hamsters are relatively resistant to DMBA mammary carcinogenesis, but susceptibility is increased by either of the above treatments with Enovid or DES. The amount of DMBA bound to DNA and protein ws 4-5 times greater than to RNA, but only DNA binding was persistent. Fifty-three percent of the DNA-bound DMBA was still present after 8 days. The amount of DMBA bound to hamster mammary DNA and its persistence was similar to that found in rats. Neither Enovid nor DES treatment altered the levels of binding to mammary macromolecules, nor their persistence. These results indicate that the species differences in the susceptibility to DMBA-induced mammary carcinogenesis in hamsters and rats, and modification of the former by hormones, is not due to differences in the activation of carcinogens. The role of hormones such as prolactin in the promotion phase of mammary gland carcinogenesis may explain these differences.     

Inhibition of mammary tumorigenesis in virgin rats by adoptive transfer of splenocytes from parous donors

Summary Splenocytes from parous rats have been previously found to have cytotoxic activity against mammary tumor cells in vitro. Experiments were carried out to determine if this pregnancy-induced cytotoxic nature of the splenocytes is inherent and transferable. Splenocytes from parous rats were adoptively transferred to a group of virgin rats. Another group of age-matched, virgin rats received splenocytes from virgin donors in a similar way. After a period of rest, at the age of 55 days, the rats belonging to both of the groups, received 7,12-dimethylbenz(a)anthracene (DMBA) intragastrically. A third group of untreated virgin rats were also given the chemical carcinogen the same way as above and were considered as intact controls. The rats were monitored for development and growth of mammary tumor from 60 days of DMBA administration. After 4 months of DMBA administration the rats were sacrificed and mammary glands were examined for tumors. Mammary glands with no visible tumors were taken for whole mount preparation, to be examined for microscopic lesions. The results showed that 33 of 41 intact control rats, developed tumor and 27 of the 34 rats that received spleen cells from virgin rats developed tumors. Of the rats that received spleen cells from parous rats, only 18 out of 37 rats developed tumors, indicating an inhibition of tumor induction in these rats. Growth rate of the tumors in this group was also slower than in the control groups.This research was supported by USPHS grant CA 3613906 awarded by the National Cancer Institute     

Binding of polycyclic aromatic hydrocarbons to DNA of cells in culture: a rapid method for its analysis using hydroxylapatite column chromatography.

A rapid procedure to study the interaction of carcinogens with DNA in cultured cells has been developed. The cells, which are labeled with 7,12-[3H]dimethylbenz[a] anthracene ([3H]DMBA), are lysed with 0.24 M phosphate buffer (pH 6.8), 1% sodium dodecyl sulfate (SDS), 8 M urea and 0.01 M ethylenediamine-tetraacetate (EDTA) and sonicated. The cell lysates are fractionated on columns of hydroxylapatite. Proteins and RNA are removed with 8 M urea in 0.24 M phosphate buffer (pH 6.8). DMBA-bound DNA is eluted with 0.4 M phosphate buffer (pH 6.8). DMBA-DNA isolated by this procedure is virtually free from proteins and RNA. Thermal stability, ultraviolet spectra and the density of DNA is not altered by DMBA binding. The uptake of DMBA by mouse epidermal cells is rapid and the binding of DMBA to DNA is linear for the first 8 h of exposure. DMBA binds to DNA in all phases of the cell cycle. However, the highest binding occurs immediately following maximum DNA synthesis.     

Charles River Sprague Dawley rats lack early age-dependent susceptibility to DMBA-induced mammary carcinogenesis

Developmental stages of mammary glands influence their susceptibility to initiating events related to carcinogenesis. The "window of susceptibility" to mammary carcinogenesis is classically defined as the time in early puberty when the mammary gland morphology is most sensitive to initiation events. Administration of the polyaromatic hydrocarbon, 7,12-dimethylbenz(a)anthracene (DMBA), in a single oral dose yields maximal mammary tumor formation when administered in this "window". We examined the DMBA treated mammary glands, precursor lesions, and morphology of the uninvolved mammary epithelium for the first 100 days of life for Charles River Sprague Dawley CD(R) IGS. Our goal was to determine the DMBA dose at which 50% of the rats (IC50) developed carcinoma in situ (CIS) within three months of dosing. Here we demonstrate, rather than the classical U-shaped dose curve in which there is maximum sensitivity for DMBA at 50 days, there is an increasing degree of sensitivity with age in the CD(R) IGS rat. Additionally, we report that vehicle-treated animals developed mammary CIS without any known initiator, and 100 day virgin animals demonstrated lactational changes, independent of DMBA exposure or dose. Lastly, we demonstrate this strain of virgin female rats has elevated pituitary prolactin immunoreactivity independent of the level of mammary differentiation. We conclude this strain of Charles River Sprague Dawley rats has prolactin-induced pituitary stimulation, and therefore, the window of susceptibility for mammary tumorigenesis is absent.     

Eugenol inhibit 7,12-dimethylbenz[a]anthracene-induced genotoxicity in MCF-7 cells: Bifunctional effects on CYP1 and NAD(P)H:quinone oxidoreductase

Typically, chemopreventive agents either inhibit the cytochrome P450s (CYPs) that are essential for the metabolism of carcinogens or induce phase II detoxifying enzymes. This study examined the chemopreventive effect of eugenol on 7,12-dimethylbenz[a]anthracene (DMBA)-induced DNA damage in MCF-7 cells. Eugenol inhibited the formation of the DMBA-DNA adduct in a dose dependent manner. CYP1A1 and CYP1B1 activity, which catalyze the biotransformation of DMBA, were strongly inhibited by eugenol. Eugenol also suppressed the CYP1A induction by DMBA through decreased aryl hydrocarbon receptor activation and subsequent DNA binding. Furthermore, eugenol increased the expression and activity of NAD(P)H:quinone oxidoreductase (QR), a major detoxifying enzyme for DMBA, through NF-E2 related factor2 binding to antioxidant response element in QR gene. Therefore, eugenol has a potent protective effect against DMBA-induced genotoxicity, presumably through the suppression of the DMBA activation and the induction of its detoxification. These results suggest that eugenol has potential as a chemopreventive.     

Inhibition of DMBA Induced Rat Mammary Duct Damage by Novel Synthetic Organoselenium Compounds.

The balance between prooxidants and antioxidants is crucial to the survival and functioning of aerobic organisms. Partially reduced derivatives of oxygen, which are produced in aerobic organisms as part of normal physiological and metabolic processes, are toxic species, oxidizing numerous biomolecules, which initiate tissue injury and cell death. DMBA (7,12-dimethylbenz[a]anthracene) is a polycyclic aromatic hydrocarbon (PAH) known to cause tumors in rats. DMBA is known to generate DNA-reactive species, which may enhance oxidative stress in cells, during its metabolism. Besides the formation of DNA adducts, oxidative products derived from mutagen metabolism, such as DMBA, might impair vital cellular functions by damaging proteins and lipid membranes. Synthetic organoselenium compounds inhibit the initiation phase of carcinogenesis by inhibiting DMBA-DNA adduct formation in the target organ in vivo. Because of the health problems induced by many environmental pollutants, many efforts have been undertaken to evaluate the relative antioxidant potential of selenium and synthetic organoselenium compounds. We undertook the present study to evaluate the chemopreventive potential of the novel synthetic organoselenium compounds (1-isopropyl-3-methylbenzimidazole-2-selenone (SeI) and 1,3-di-p-methoxybenzylpyrimidine-2-selenone (SeII)) in the well-established DMBA-treated rat model by monitoring the extent of lipid peroxidation and mammary duct damage. In this study, adult female Wistar rats were treated with DMBA and the novel organoselenium compounds (SeI and SeII) in determined doses. In DMBA-treated rats, the effects of the organoselenium compounds on malondialdehyde (MDA) levels and histological changes in the rat mammary lactiferous duct were studied. The ability of the organoselenium compounds to prevent oxidative damage induced by DMBA in rat mammary ducts was demonstrated. Protection against lipid peroxidation measured as MDA in the SeI and SeII treated groups was provided by the novel synthesized organoselenium compounds. SeI and SeII both provided chemoprevention against DMBA-induced oxidative stress in the rat mammary duct.     

Mechanistic studies on the inhibitory action of dietary dibenzoylmethane, a beta-diketone analogue of curcumin, on 7,12-dimethylbenz[a]anthracene-induced mammary tumorigenesis

Dietary factors play important roles in the carcinogenic process. The results of epidemiological data and some laboratory animal studies indicate that certain naturally occurring and synthetic components are able to block the carcinogenic process and inhibit the development of certain cancers. Dibenzoylmethane (DBM), a curcumin-related beta-diketone analogue has been reported to exhibit a remarkable inhibitory effect on 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary tumorigenesis in Sencar mice. The present study investigated the possible mechanisms of inhibitory action of DBM on DMBA-induced mammary tumorigenesis in mice. The summarized results indicate that: (1) in in-vitro studies. DBM inhibited DMBA metabolism and the formation of DMBA-DNA adducts in a dose-dependent manner; (2) in the assay of competitive binding to estrogen receptors with [3H]-estradiol in vitro, DBM showed weak binding affinity; (3) in vivo, feeding of 1% DBM in the diet of immature Sencar mice for 4 -5 weeks decreased the uterine and parametrial fat pad weights, and lowered the serum estrogen and triglyceride levels. This study provides insight into the mechanisms involved in the inhibitory action of DBM in mouse mammary tumorigenesis.     

Characteristics of rat normal mammary epithelial cells and dimethylbenzanthracene-induced mammary adenocarcinoma cells grown in monolayer culture

Summary The characteristics of normal mammary epithelial and 7,12-dimethylbenz[a]anthracene (DMBA)-induced adenocarcinoma cells derived from rats and grown in monolayer culture were compared. Normal mammary epithelial cells exhibited different morphology and agglutinability by plant lectins, slower growth rate, and lower saturation density and cloning efficiency. In addition, the normal cells were sensitive to the toxic effect of DMBA, and were unable to grow in soft agar or to form tumors, when inoculated into newborn Sparague-Dawley rats. The converse was true in each case for the adenocarcinoma cells. Supported by Public Health Service Research Grant CA 01237603 from the National Cancer Institute Portions of this paper were presented at the 65th Annual Meeting of the American Association for Cancer Research at Houston, Texas, 1974.     

Mammary gland growth in the hypophysectomized pregnant rat (38522).

Sprague-Dawley-Rolfsmeyer rats were hypophysectomized on days 11, 12 or 15 of pregnancy and sacrificed on day 20 to determine the extent of mammary development, as assessed by determination of nucleic acid content. The DNA of six abdominal-inguinal glands in the hypophysectomized groups was not significantly different from that in the sham-operated pregnant or intact pregnant control groups. All groups maintaining pregnancy had significantly higher DNA contents in mammary glands than virgin control or hypophysectomized aborted groups. In order to determine the minimal numbers of placental-fetal units required to maintain pregnancy and mammary gland growth, fetuses and placentas were removed on day 12 of pregnancy in addition to the pituitary so that only one fetus and one placenta remained in the uterus of a group of 6 rats with other groups having 2, 3, 4, 5 remaining. Pregnancy was maintained with only one placental-fetal unit, but mammary gland proliferation was significantly lower than the control group on day 20 of pregnancy. Three to five conceptuses supported mammary proliferation during the latter half of pregnancy at a level not significantly different from intact or sham-operated control groups. Removal of placental units on day 12 in rats having pituitaries intact resulted in no mammary DNA change when 1-5 units remained. Removal of pitutaries on day 12 and placental-fetal units on day 14 also resulted in no change in mammary DNA with as little as two placentas (minus all fetuses),while only one placenta remaining resulted in a significantly lower mammary DNA than in groups wtih 2 or more placentas.     

Expression, activity, and localization of hormone-sensitive lipase in rat mammary gland during pregnancy and lactation

We examined the presence of hormone-sensitive lipase (HSL) in mammary glands of virgin, pregnant (12, 20, and 21 days), and lactating (1 and 4 days postpartum) rats. Immunohistochemistry with antibody against rat HSL revealed positive HSL in the cytoplasm of both alveolar epithelial cells and adipocytes. In virgin rats, immunoreactive HSL was observed in mammary adipocytes, whereas diffuse staining was found in the epithelial cells. Positive staining for HSL was seen in the two types of cells in pregnant and lactating rats. However, as pregnancy advanced, the staining intensity of immunoreactive HSL increased in the epithelial cells parallel to their proliferation, attaining the maximum during lactation. An immunoreactive protein of 84 kDa and a HSL mRNA of 3.3. kb were found in the rat mammary gland as in white adipose tissue. Both HSL protein and activity were lower in mammary glands from 20 and 21 day pregnant rats than from those of virgin rats, although they returned to virgin values on days 1 and 4 of lactation. Mammary gland HSL activity correlated negatively to plasma insulin levels. Immunoreactive HSL and HSL activity were found in lactating rats' milk. The observed changes indicate an active role of HSL in mammary gland lipid metabolism.     

The use of thioesterase II as a rat mammary epithelial cell-specific marker

The avidin-biotin-peroxidase complex immunoperoxidase technique was employed to determine the intercellular distribution of thioesterase II in rat mammary glands. This enzyme is responsible for shifting the product specificity of the fatty-acid synthetase enzyme complex from long to medium chain fatty acids. Thioesterase II was found exclusively in the cells lining the lumen of the ductal and alveolar structures in glands from mature virgin (150 days old) and pregnant rats. The ductal cell staining intensity was considerably less than that of the alveolar cells in the mature virgin rat glands. No immunoreactive thioesterase II was found in the stromal, adipose, vascular, or myoepithelial components of the gland in the developmental stages examined. In the glands from immature virgin rats (40-45 days old) thioesterase II was again found only in the epithelial cells lining the lumen of the ductal and end-bud structures although this layer was usually more than one cell thick. Quantitative determination of thioesterase II activity in cytosol preparations revealed similar levels in mammary fragments from enzymatically-dissociated glands obtained from mature virgins and in end buds derived from immature virgins, but somewhat higher levels in mammary structures derived from late-pregnant animals. These immunohistological and biochemical results demonstrate thioesterase II's usefulness as a mammary epithelial cell-specific marker.     

Immunohistochemical localization of ornithine decarboxylase in the rat ovary

Summary The present report describes the immunocytochemical localization of ornithine decarboxylase in the prepubertal rat ovary after administration of human chorionic gonadotropin (HCG). Numerous ornithine decarboxylase immunoreactive cells appeared in the thecal layer as well as in the interstitial gland tissue after treatment with HCG. The granulosa cells, the ovum and the ovarian stroma were devoid of immunoreactive ornithine decarboxylase. In contrast to the ovary of HCG-treated rats, the ovary of prepubertal rats given the vehicle alone contained only a few weakly immunoreactive cells.     

Evidence for the involvement of a diol-epoxide in the binding of 7,12-dimethylbenz(a)anthracene to DNA in cells in culture.

1,1,1-Trichloropropene 2,3-oxide (TCPO), a known inhibitor of the enzyme epoxide hydrase, inhibits binding of the carcinogen, 7,12-dimethylbenz(a)anthracene (DMBA), to the DNA of secondary mouse embryo cell cultures under conditions which do not appreciably decrease the overall metabolism of this carcinogen. This suggests that the formation of a transdihydrodiol is a necessary step in the metabolic pathway leading to DNA binding and that binding probably occurs through the generation of a reactive diol-epoxide. In concert with this, the major DMBA-DNA product isolated by chromatography on Sephadex LH-20 eluted with a methanol-water gradient is resolved into two separate components in a methanol-sodium borate solution gradient suggesting that, as is known for benzo(a)pyrene, two stereoisomeric diol-epoxides are involved in the binding of DMBA to DNA.     

Quantification of epithelial cell differentiation in mammary glands and carcinomas from DMBA- and MNU-exposed rats

Rat mammary carcinogenesis models have been used extensively to study breast cancer initiation, progression, prevention, and intervention. Nevertheless, quantitative molecular data on epithelial cell differentiation in mammary glands of untreated and carcinogen-exposed rats is limited. Here, we describe the characterization of rat mammary epithelial cells (RMECs) by multicolor flow cytometry using antibodies against cell surface proteins CD24, CD29, CD31, CD45, CD49f, CD61, Peanut Lectin, and Thy-1, intracellular proteins CK14, CK19, and FAK, along with phalloidin and Hoechst staining. We identified the luminal and basal/myoepithelial populations and actively dividing RMECs. In inbred rats susceptible to mammary carcinoma development, we quantified the changes in differentiation of the RMEC populations at 1, 2, and 4 weeks after exposure to mammary carcinogens DMBA and MNU. DMBA exposure did not alter the percentage of basal or luminal cells, but upregulated CD49f (Integrin α6) expression and increased cell cycle activity. MNU exposure resulted in a temporary disruption of the luminal/basal ratio and no CD49f upregulation. When comparing DMBA- or MNU-induced mammary carcinomas, the RMEC differentiation profiles are indistinguishable. The carcinomas compared with mammary glands from untreated rats, showed upregulation of CD29 (Integrin β1) and CD49f expression, increased FAK (focal adhesion kinase) activation especially in the CD29hi population, and decreased CD61 (Integrin β3) expression. This study provides quantitative insight into the protein expression phenotypes underlying RMEC differentiation. The results highlight distinct RMEC differentiation etiologies of DMBA and MNU exposure, while the resulting carcinomas have similar RMEC differentiation profiles. The methodology and data will enhance rat mammary carcinogenesis models in the study of the role of epithelial cell differentiation in breast cancer.     

Electron microscopy of isolated rat testicular cells grown in vitro

Summary Suspensions of viable testicular cells obtained from two groups of rats (one group treated for ten days with 50 I. U. of serum gonadotropins (HCG) daily; one group not previously treated) were cultured for ten days. Numerous cells adhered to the glass to form a confluent monolayer and remained in good condition after ten days. This monolayer contained two cell types identified by electron microscopy: fibroblastic cells and Leydig cells. The relative proportion by which these two cells contributed to the monolayer was related to the condition of the donor when the culture was initiated. Fibroblastic cells were more abundant when the animal was not previously treated with HCG. However Leydig cells almost exclusively formed the monolayer when cultures were begun with testicular cells of HCG-treated rats. Gonadotropins on the other hand did not seem capable of acting in vitro.     

Analysis of mammary tumors for cytochemical evidence of endogenous mammary peroxidase.

The purpose of this study was to determine whether or not endogenous mammary peroxidase can serve as a cytochemical marker to distinguish ovarian hormone-dependent from ovarian hormone independent mammary tumors. Spontaneous mammary tumors arising in virgin C3H and GR mice (hormone independent tumors) and hormone-dependent mammary tumors arising during pregnancy in GR mice were examined. None of these tumors contained mammary peroxidase. Mammary tumors induced in Sprague-Dawley rats with methylnitrousourea (MNU) and dimethylbenzanthracene (DMBA) were also examined. These tumors included hormone-dependent and hormone independent ones. Several of the DMBA-induced hormone-dependent tumors contained a few peroxidase-positive cells, but the hormone independent tumors were negative. All of the MNU-induced tumors examined were negative for mammary peroxidase. Twenty human breast tumors (malignant and non-malignant) removed from women at surgery, were also negative for mammary peroxidase. Our results indicate that endogenous mammary peroxidase cannot be used to distinguish hormone-dependent from hormone independent mammary tumors.