Oxidative stress specifically downregulates survivin to promote breast tumour formation.

BACKGROUND: Breast cancer, a heterogeneous disease has been broadly classified into oestrogen receptor positive (ER+) or oestrogen receptor negative (ER-) tumour types. Each of these tumours is dependent on specific signalling pathways for their progression. While high levels of survivin, an anti-apoptotic protein, increases aggressive behaviour in ER- breast tumours, oxidative stress (OS) promotes the progression of ER+ breast tumours. Mechanisms and molecular targets by which OS promotes tumourigenesis remain poorly understood. RESULTS: DETA-NONOate, a nitric oxide (NO)-donor induces OS in breast cancer cell lines by early re-localisation and downregulation of cellular survivin. Using in vivo models of HMLE(HRAS) xenografts and E2-induced breast tumours in ACI rats, we demonstrate that high OS downregulates survivin during initiation of tumourigenesis. Overexpression of survivin in HMLE(HRAS) cells led to a significant delay in tumour initiation and tumour volume in nude mice. This inverse relationship between survivin and OS was also observed in ER+ human breast tumours. We also demonstrate an upregulation of NADPH oxidase-1 (NOX1) and its activating protein p67, which are novel markers of OS in E2-induced tumours in ACI rats and as well as in ER+ human breast tumours. CONCLUSION: Our data, therefore, suggest that downregulation of survivin could be an important early event by which OS initiates breast tumour formation.

High incidence of hepatocellular carcinomas after synthetic estrogen administration in Syrian golden hamsters fed alpha-naphthoflavone: a new tumor model.

P8 80-100% incidence of multinodular hepatocellular carcinomas was observed in castrated male hamsters following synthetic estrogen treatment in the presence of 0.2-0.4% alpha-naphthoflavone (ANF) in the diet after 8.5-10 months. Induction of these liver tumors was detected as early as 3.5-4.0 months in low frequency. Of the synthetic estrogens studied, ethynylestradiol (CAS: 57-63-6) was a more potent inducer of these hepatic carcinomas than either diethylstilbestrol (CAS: 56-53-1) or hexestrol (CAS: 84-16-2). ANF, considered an inhibitor of P450-dependent multisubstrate monooxygenases, did not produce any liver tumors when administered alone for up to 12 months. Neither concomitant androgen nor progesterone (CAS: 57-83-0) treatment resulted in any hepatic carcinomas in animals maintained on ANF. Moreover, beta-naphthoflavone (CAS: 6051-87-2) treatment alone or in combination with these synthetic estrogens also resulted in no hepatic tumors. This new estrogen-induced liver tumor model could be useful to elucidate the casual relationship that exists between estrogenic hormones and hepatic tumors in humans.

Steroid hormone receptors in the rat mammary adenocarcinoma induced by N-hydroxy-N-2-fluorenylacetamide.

The steroid hormone receptors in N-2-fluorenylacetamide (2-FAA)- and N-hydroxy-2-FAA-induced mammary adenocarcinomas in outbred Sprague-Dawley rats were analyzed. Both 8S and 4S estrogen-binding components have been detected in cytosols of these tumors following sucrose gradient sedimentation in low salt. Competitive binding analyses of this binder indicated a specificity profile expected of an estrogen receptor. Both androgen and progesterone receptors were also present in the cytosols of these mammary tumors. While the androgen receptor sedimented in the 8S region of the gradient, the progestin binder appeared only as a 4S moiety under similar conditions. The relative concentrations of these receptors (expressed in fmol/mg protein +/- SE) were: 17 beta-estradiol (28.6 +/- 4.1) greater than 5 alpha-dihydrotestosterone (8.5 +/- 2.2) greater than progesterone (5.0 +/- 1.3). The progesterone receptor was increased at least eightfold in the mammary adenocarcinomas from ovariectomized rats that were treated with diethylstilbestrol for 6 days. Binding equilibrium data indicated Ka = 1.2-1.8 X 10(9) M-1 for the above cytoplasmic hormone receptor complexes (Ka, association constant). Although cytosols prepared from lactating mammary gland contained appreciable quantities of glucocorticoid receptor, only trace amounts were found in the mammary adenocarcinoma.

Estrogen 2- and 4-hydroxylase activity, catechol estrogen formation, and implications for estrogen carcinogenesis in the hamster kidney.

Estrogen 2- and 4-hydroxylase (ESH), a microsomal enzyme which mediates the formation of catechol estrogens, has been studied in the kidneys of castrated male Syrian hamsters, a species uniquely susceptible to induction of renal carcinomas by both steroidal and stilbene estrogens. The apparent Km for estrone was 17.0 microM, and Vmax was 0.5 pmol per mg protein per min for ESH in renal microsomes derived from castrated hamsters. Different steroidal estrogen substrates exhibited decreasing catechol formation with hamster kidney microsomal preparations in the following order: estrone greater than d-equilenin greater than 17 beta-estradiol greater than equilin greater than ethynyl estradiol greater than estriol. Except for beta-dienestrol, the stilbene estrogens revealed levels of catechol formation that were similar to 17 beta-estradiol. These findings provide a rationale for the weak carcinogenic activity of ethynyl estradiol, estriol, and beta-dienestrol, since they were poor substrates for hamster renal ESH and for the relatively potent carcinogenic activity of the distal metabolite of diethylstilbestrol, indenestrol B/A, which exhibited substantial levels of o-hydroxylation when used as a substrate. Interestingly, ESH activity was significantly greater in the hamster kidney compared to corresponding rat tissue, and catechol estrogen formation was found to be 2.5- to 19-fold higher in the hamster kidney compared to the rat, using various steroidal and stilbene estrogen substrates. Moreover, the finding that a 3.5- to nearly 6-fold decrease, compared to untreated levels, in catechol formation in kidneys but not in livers of alpha-naphthoflavone-exposed hamsters, depending on the steroidal or stilbene estrogen substrate used, is consistent with the belief that the catechol estrogen pathway is pertinent to events leading to estrogen-induced renal tumorigenesis in the hamster.

Nuclear retention of all steroid hormone receptor classes in the hamster renal carcinoma.

The estrogen-induced hamster renal carcinoma contains appreciable amounts of all cytosolic steroid receptor classes sedimenting as 7 to 8S moieties following sucrose gradient centrifugation. Their relative concentrations, expressed in fmol/mg protein +/- S.E. are progesterone (1496 +/- 23) greater than estradiol (218 +/- 3) greater than 5 alpha-dihydrotestosterone (154 +/- 7) greater than dexamethasone (138 +/- 3) greater than aldosterone (40 +/- 2). No cross-competition is apparent for either estrogen or progesterone receptors, but progesterone competes for both androgen and adrenocorticoid binding. These hormone-receptor complexes undergo nuclear translocation using purified tumor nuclei and in tissue minces at elevated temperatures. Salt-extractable nuclear receptors for estrogen (5S), androgen (3.2S), progesterone (2.7S), and corticosteroid (3.5S) have been identified. The existence of five specific steroid receptors within a single tissue is unique and provides a novel model for studying the interrelated actions of all steroid hormones and their therapeutic responses in a hormone-dependent neoplasm.

Receptor characteristics of specific estrogen binding in the renal adenocarcinoma of the golden hamster.

Linear sucrose gradient analyses reveal that all estrogen-induced and -dependent primary renal tumor cytosols examined contain an 8 S and variable amounts of 4 S receptor in low ionic buffer concentrations. Similar results were obtained with extracts of primary metastases of these tumors. Sucrose gradients containing high salt (0.4 M KCl) convert the 8 S receptor in both the hamster renal tumor and uterus to a 4 to 5 S complex. Scatchard plot analysis reveals that the renal tumor cytosol estradiol-receptor complex has a Ka of 1.7 X 10(9) M-1 and 9.2 X 10(-10) M binding sites. Competition for the tritiated 17beta-estradiol binding sites in the renal tumor was similar to that in the uterus with respect to estrogenic compounds. Nonestrogenic steroids exhibited minimal competition at the same concentrations or higher. Substitution in the ring structure, particularly in position 3 of the phenolic A-ring, resulted in a considerable loss in the ability of such compounds to compete for these receptors. Aniestrogens were effective competitors for these estrogen receptors only at higher concentrations relative to the tritiated estradiol.

Carcinogenic activities of various steroidal and nonsteroidal estrogens in the hamster kidney: relation to hormonal activity and cell proliferation.

The therapeutic use of estrogens has been associated with an increased risk of some of the most predominant, as well as less prevalent, cancers in women. The estrogen-induced renal tumor is one of the primary animal models to evaluate the carcinogenic properties of estrogens. Correlations were made with various estrogens by using parameters of estrogenicity end points such as competitive binding, progesterone receptor induction, and alterations in prolactin levels; in vitro renal proximal cell proliferation; and in vivo estrogen-induced carcinogenicity. The most potent estrogens were Moxestrol (MOX), diethylstilbestrol (DES), and 17 beta-estradiol, followed by indenestrol B, 16 alpha-hydroxyestrone, and 11 beta-methoxyestradiol with moderate estrogenic activities, whereas 11 beta-methylestradiol, 17 alpha-estradiol, indanestrol, and deoxoestrone were all relatively weaker. As expected, hydrolyzed Premarin (unconjugated estrogens) was strongly estrogenic. Of the estrogens tested, MOX was the most potent carcinogenic estrogen in the hamster kidney. Both 16 alpha-hydroxyestrone and 11 beta-methoxyestradiol induced intermediate tumor incidences with distinctly lower frequencies of renal tumor foci compared to the most potent carcinogenic estrogens. However, hamsters treated for 9.0 months with 11 beta-methylestradiol, 17 alpha-estradiol, deoxoestrone, and indanestrol exhibited no tumors. In contrast, treatment with estrone, equilin plus d-equilenin, and hydrolyzed Premarin for the same time period resulted in 100% renal tumor incidences and numerous tumor foci. Cell proliferation studies of cultured hamster kidney proximal tubule cells were carried out at varying estrogen concentrations (0.01-100 nM). Exposure to MOX resulted in consistently high renal cell proliferative response over a concentration range of 0.1-10 nM. Strongly carcinogenic estrogens such as estrone had a maximal renal cell proliferation response (2.4-fold above untreated control levels) between 0.1 and 10 nM, DES and 17 beta-estradiol responded at 1.0 nM, and 4-hydroxyestradiol responded at 10 nM. Interestingly, exposure to ethinylestradiol, a potent estrogen, at similar or higher doses as those used for DES and 17 beta-estradiol, yielded only a 10% renal tumor incidence and induced only a 1.7-fold increase in proximal tubule cell proliferation. In contrast, 17 alpha-estradiol, deoxoestrone, indanestrol, and 11 beta-methylestradiol, all weakly estrogenic and noncarcinogenic agents, had relatively little effect on tubule cell proliferation. The hydrolyzed Premarin exhibited a maximal 2.0-fold cell proliferative response at 10 nM. The present results provide clear evidence that, in the hamster kidney, the degree of carcinogenicity of a given estrogen correlates with its ability to induce proximal tubule cell proliferation in vitro.(ABSTRACT TRUNCATED AT 400 WORDS)

Estrogen-induced proto-oncogene and suppressor gene expression in the hamster kidney: significance for estrogen carcinogenesis.

Chronic administration of estrogen to male Syrian hamsters for 7.0 to 9.0 months induces a high frequency of estrogen-dependent renal cancers. We have proposed a sequential multistage scheme involving tubular cell damage, regenerative cell proliferation, aneuploidy, chromosomal imbalance, genetic instability, gene alteration, and amplification as essential steps for estrogen carcinogenesis in this model. A systematic study was undertaken to assess the expression of nuclear proto-oncogenes, c-myc, c-fos, and c-jun, and suppressor genes, p53 and WT-1, by Northern blot analysis to further support this scheme. Hamster kidney RNA, taken at monthly intervals (1.0 to 6.0 months) from diethylstilbestrol (DES)-treated castrated male hamsters and corresponding age-matched untreated controls was used in these studies, as well as primary estrogen-induced renal tumor RNA, for reference. Although no significant changes in the expression of these proto-oncogenes were detected in the first 4 months of estrogen treatment relative to age-matched controls, 2.1-kb c-myc expression was elevated 2.8- and 4.1-fold at 5.0 and 6.0 months, respectively. Moreover, the expression of 2.2-kb c-fos transcript rose 4.6- and 4.8-fold; and 3.2- and 2.7-kb c-jun expression increased 2.8- and 5.1-fold at these same respective estrogen treatment time intervals. Tumor suppressor gene expression, p53 and WT-1, was also evaluated in similar estrogen-exposed hamsters. Although no significant changes were found in hamster kidney p53 expression in the first 5.0 months of DES treatment, it rose 1.8-fold at 6.0 months of estrogen treatment and more than 2.0-fold in the primary renal tumor. In contrast, no detectable changes in WT-1 expression were found during the first 6.0 months of DES treatment. However, a dramatic 7.0-fold increase in WT-1 expression was observed in the primary renal tumor. It is evident that two WT-1 transcripts reside in the hamster kidney; a lower molecular weight transcript was found in the normal adult kidney, and a higher molecular weight 3.2-kb transcript was observed in the renal tumor, similar to that seen in the newborn mouse kidney. In summary, the estrogen-induced inappropriate gene expression, including p53, reported herein, is consistent with the view that the elevations seen in gene expression contribute to proliferative advantages of certain proximal tubular interstitial cells necessary for estrogen-driven tumor formation in the hamster.

Relative carcinogenic activity of various synthetic and natural estrogens in the Syrian hamster kidney.

Both synthetic and natural estrogens have been studied for their ability to induce renal carcinomas in castrated male hamsters after 9.0 months of treatment. Tumor foci were detected in frozen serial sections stained histochemically for estrase activity. Both diethylstilbestrol (DES) and 17 beta-estradiol had equal ability (100%) to induce renal tumors [approximately 20.5 +/- 3 (S.E.) tumor foci] in these animals. Hexestrol induced the same incidence and number of renal carcinoma foci as DES or 17 beta-estradiol. However, alpha -dienestrol and DES 3,4-oxide showed an 86 to 88% incidence of renal tumors in hamsters (approximately 10.8 +/- 3). When equilin and d-equilenin, components of therapeutic conjugated estrogens, were tested, only equilin had a 76% incidence of renal tumor foci (5.5 +/- 0.9). The ability of these stilbene and steroidal estrogens to compete for renal tumor estrogen receptor generally correlated well with their ability to cause renal tumorigenesis in the hamster with one notable exception. Although ethinyl estradiol competed as well as did DES or 17 beta-estradiol for estrogen receptor, had similar ability to induce renal progesterone receptor, and led to similar high serum prolactin levels as either DES or 17 beta-estradiol, it had only weak carcinogenic activity (21%) in the hamster kidney (0.6 +/- 0.5 foci). These data represent the first detailed analysis of the relative carcinogenic activity of different estrogens within a given tumor-inducing system, and based on the carcinogenicity data of hexestrol and alpha-dienestrol presented herein, they suggest that epoxidation of the olefinic double bond and the p-quinone metabolite of DES probably are not involved significantly in its carcinogenic activity. Moreover, the poor carcinogenic activity of ethinyl estradiol in this system, despite strong estrogenicity, suggests that estronic activity alone may not be sufficient to effect renal tumorigenesis in the hamster.

Overexpression and amplification of c-myc in the Syrian hamster kidney during estrogen carcinogenesis: a probable critical role in neoplastic transformation.

An estrogen receptor-driven, multistep process for estrogen carcinogenesis in the Syrian hamster kidney is proposed. Because in this species the reproductive and urogenital tracts arise from the same embryonic germinal ridge, it is evident that the kidney has carried over genes that are responsive to estrogens. Using in situ hybridization, overexpression of early estrogen-response genes, i.e., c-myc and c-fos, has been shown to be localized preferentially in early renal tumor foci after 3.5-4.0 months of estrogen treatment. This event coincides with an increased number of S-phase proliferating cell nuclear antigen-labeled cells in these tumor foci, along with a rapid rise in aneuploid frequency in the kidney. Western blot analyses of c-MYC and c-FOS protein products support the overexpression of these genes. Amplification of c-myc, 2.4-3.6-fold, but not of c-fos, was detected in 67% of the primary renal tumors examined, by Southern blot analyses. Consistent chromosomal gains, common to both diethylstilbestrol- and estradiol-induced renal neoplasms, were observed in chromosomes 1, 2, 3, (6), 11, (13), 16, 20, and 21 (chromosome number alterations are indicated in parentheses). Using fluorescence in situ hybridization, the c-myc gene was localized to hamster chromosome 6qb. Chromosome 6 exhibited a high frequency of trisomies and tetrasomies in the kidney after 5.0 months of estrogen treatment and in primary renal tumors. The data presented indicate that estrogen-induced genomic instability may be a key element in carcinogenic processes induced by estrogens.

Health care and social service professionals' perceptions of a home-visit program for young, first-time mothers.

INTRODUCTION: Little is known about health care and social service professionals' perspective on the acceptability of long-term home-visit programs serving low-income, first-time mothers. This study describes the experiences and perspectives of these community care providers involved with program referrals or service delivery to mothers who participated in the Nurse-Family Partnership (NFP), a targeted nurse home-visit program. METHODS: The study included two phases. Phase I was a secondary qualitative data analysis used to analyze a purposeful sample of 24 individual interviews of community care providers. This was part of a larger case study examining adaptations required to increase acceptability of the NFP in Hamilton, Ontario, Canada. In Phase II (n = 4), themes identified from Phase I were further explored through individual, semi-structured interviews with community health care and social service providers, giving qualitative description. RESULTS: Overall, the NFP was viewed as addressing an important service gap for first-time mothers. Providers suggested that frequent communication between the NFP and community agencies serving these mothers could help improve the referral process, avoid service duplication, and streamline the flow of service access. The findings can help determine key components required to enhance the success of integrating a home-visit program into an existing network of community services. CONCLUSION: The function of home-visit programs should not be viewed in isolation. Rather, their potential can be maximized when they collaborate and share information with other agencies to provide better services for first-time mothers.

TITRE: Points de vue de professionnels de la santé et des services sociaux sur un programme de visites à domicile destiné aux jeunes mères d'un premier enfant. INTRODUCTION: On sait peu de choses sur ce que pensent les professionnels de la santé et des services sociaux des programmes à long terme de visites à domicile pour les nouvelles mères à faible revenu. Cette étude fait état des expériences et des points de vue de fournisseurs de services communautaires qui orientent les mères participant au Nurse-Family Partnership (NFP) ­ un programme de visites à domicile par une infirmière auprès de cette population cible ­ vers le programme lui-même ou qui interviennent directement dans le cadre de celui-ci. MÉTHODOLOGIE: L'étude s'est déroulée en deux phases. La phase I a consisté en une analyse secondaire de données qualitatives issues d'un échantillon de 24 entrevues individuelles dirigées avec des fournisseurs de soins communautaires, cette démarche s'inscrivant dans le cadre d'une étude de cas plus vaste réalisée à Hamilton (Ontario, Canada) et destinée à examiner les moyens à mettre en oeuvre pour accroître la recevabilité du NFP. La phase II, à laquelle ont pris part 4 participants, a consisté en une description qualitative des 3 thèmes relevés lors de la phase I, thèmes qui ont été approfondis au moyen d'entrevues individuelles semi-structurées menées auprès de certains fournisseurs de soins de santé et de services sociaux communautaires. RÉSULTATS: Le NFP est perçu dans l'ensemble comme un programme comblant une lacune importante dans les services dispensés aux nouvelles mères. Des échanges fréquents entre le NFP et les organismes communautaires offrant déjà des services à ces mères pourraient contribuer à en améliorer le processus d'orientation, à éviter leur chevauchement et à en faciliter l'accès. Ces résultats contribuent à mieux définir les composantes nécessaires au succès de l'intégration d'un programme de visites à domicile au réseau déjà en place de services communautaires. CONCLUSION: Le rôle des programmes de visites à domicile ne doit pas être envisagé isolément. Au contraire, le potentiel de ceux-ci est optimisé par la collaboration et l'échange d'information avec d'autres organismes afin d'offrir de meilleurs services aux mères d'un premier enfant.

Effect of androgen and estrogen treatment on hamster liver and kidney estrogen 2-/4-hydroxylase activity.

The activity of microsomal estrogen 2-/4-hydroxylase enzyme (ESH), which mediates the formation of catechol estrogens, was determined in the hamster kidney and liver under different endocrine states and after treatment with diethylstilbestrol (DES) and 5 alpha-dihydrotestosterone alone or in combination. Our results indicate that at least 64% of the renal ESH activity is localized in the kidney cortex. Employing either estrone or 17 beta-estradiol as substrate, a significant decline in renal ESH activity was observed after castration, with estrone remaining the more active substrate. In contrast, hepatic ESH activity, which is about 2.0- to 2.5-fold higher than the kidney enzyme, was not altered after gonadectomy using either estrogen substrate. A further reduction in renal ESH activity was found in DES-treated castrated hamsters when estrone was used. Androgen treatment resulted in a nearly 2-fold increase in kidney ESH activity using either estrogen substrate. Animals treated concomitantly with DES and 5 alpha-dihydrotestosterone exhibited catechol estrogen formation similar to untreated castrate hamster kidney microsomes. In contrast, hamster liver ESH activity was unaffected by androgen treatment. HPLC profiles of the catechol estrogen monomethyl ethers confirm these changes. Hamster kidney ESH activity in females was only 5-7% of that in intact males. Ovariectomy resulted in a 3-fold increase in the activity of this microsomal enzyme with either estrogen substrate. ESH activity was substantially increased in uteri of intact animals after androgen treatment. These data clearly demonstrate that ESH activity is under androgen control, particularly in the hamster kidney of both sexes, and may be pertinent in understanding the antagonism of this hormone in estrogen-induced renal tumorigenesis.

Characterization of specific glucocorticoid receptor in the Syrian hamster testis.

Cytosols of whole testicular homogenates from the Syrian golden hamster contained specific binding sites for [3H]triamcinolone acetonide that exhibited limited capacity and high affinity binding characteristic of glucocorticoid receptors in other target tissues. The receptor complex sedimented as an 8.6S binder in low salt 5-20% linear sucrose gradients and as 6.2S and 4.0S moieties in 0.15M and 0.4 M KCl, respectively. The Ka at equilibrium was 3.1-3.3 X 10(9) M-1 at 4 C in intact and adrenalectomized males. The testicular glucocorticoid binder was vulnerable to proteolytic degradation while being completely resistant to the action of RNase and DNase. In addition the binding protein exhibited the usual steroid specificities for type I glucocorticoid receptor: triamcinolone acetonide greater than dexamethasone greater than cortisol greater than corticosterone greater than progesterone greater than aldosterone greater than prednisone greater than 5 alpha-dihydrotestosterone greater than diethylstilbestrol. Unexpectedly, 17 beta-estradiol competed for receptor binding to the same extent as prednisone. A 3.2 S nuclear receptor was extracted from purified testicular nuclei after incubation of whole suspensions in culture media containing 5 nm radiolabeled triamcinolone acetonide at 32 C. Although the glucocorticoid receptor concentrations in prepubertal, adrenalectomized, and hypophysectomized animals were markedly higher in the testis compared to the concentration in the normal adult hamster (52 +/- 4 fmol/mg cytosol protein), the greatest total amount of receptor per testis was found in the mature intact animal. Moreover, under the conditions studied, the concentration of glucocorticoid receptor substantially exceeded the levels of either androgen or estrogen receptor when determined simultaneously. In contrast, no measurable cytoplasmic [3H]triamcinolone acetonide binding was detected in adjacent urogenital organs such as the epididymis and seminal vesicle. It is therefore unlikely that the testicular glucocorticoid receptor is associated with the spermatid or present as a secretory product in the seminiferous tubule lumen.

Biochemical comparison of estrogen receptors of the hamster hypothalamus and uterus.

Comparison of the soluble estradiol receptor proteins in the hypothalamus and uterus of the golden Syrian hamster revealed that both have sedimentation coefficients of 8S in a low ionic strength buffer and migrate similarly on analytical disc-gel electrophoresis in both 5% and 7% acrylamide gels. The competition of [3H]-17beta-estradiol binding by unlabeled estrogenic as well as nonestrogenic compounds was similar with the receptors present in the two tissues. The affinity constants for the hypothalamic and uterine receptors were 4.3 X 10-9M-1 and 1.6 X 10-10M-1, respectively; the number of binding sites when expressed on an equivalent protein basis (i.e. 1 mg/ml) was 0.18 X 10-10M and 3.1 X 10-10M, respectively. Identical amounts of soluble receptors with similar sedimentation characteristics were found in hypothalami from male and female animals gonadectomized 64 h prior to use. A titration of binding sites in the uteri of intact and ovariectomized animals revealed decreased binding in the supernatant preparation from intact animals, probably due to the presence of bound endogenous unlabeled hormone.

ER and PR in renomedullary interstitial cells during Syrian hamster estrogen-induced tumorigenesis: evidence for receptor-mediated oncogenesis.

The estrogen-induced and -dependent Syrian hamster renal tumor is the most intensively studied model in estrogen carcinogenesis. Yet, it remains confounding that the kidney of this species behaves as an estrogen target tissue. As both reproductive and urinary systems arise from the same germinal ridge, we propose that some of the germinal cells, normally destined for the uterus, migrate and establish themselves in the renal corticomedullary region in this hamster strain. These ectopically located germinal cells remain dormant unless exposed to estrogen. Supporting this contention, a subset of renal interstitial cells, primarily located in the corticomedullary region, express PR after only 2 wk and ER alpha after 1.5--3.0 months of estrogen treatment. As treatment continues, groups of cells of the renal interstitium and small and large renal tumors show ER alpha(+) and PR(+) staining. Although ER alpha and PR isoform profiles in estrogen-treated hamster kidneys are distinctly different from corresponding uterine patterns, both receptor isoform profiles in primary renal tumors closely resemble those seen in hamster uteri. Renal ER alpha protein and mRNA expression increased after 2.0 and 4.0 months of estrogen treatment and in all renal tumors examined. Using nuclear image cytometry, both early small and large renal tumors were highly aneuploid, indicating that genomic instability is probably a critical early event in estrogen carcinogenesis.

Prevention of solely estrogen-induced mammary tumors in female aci rats by tamoxifen: evidence for estrogen receptor mediation.

There is increasing evidence that both endogenous and exogenously ingested estrogens play a primary role in sporadic breast cancer causation. To establish further that solely estrogen-induced mammary oncogenesis in female ACI rats is an estrogen receptor (ERalpha)-driven process, we show for the first time that concomitant treatment with the antiestrogen, tamoxifen citrate (TAMc), completely prevents the induction of 17beta-estradiol (E(2))-induced mammary gland tumors (MGTs). This finding is also supported by the reduced mammary gland (MG) lobulo-alveolar development and proliferative activity observed in TAMc+E(2)-treated animals compared with MGs from animals treated with E(2) alone. These data also correlated with a marked decrease in the number of MG cells expressing ERalpha and progesterone receptor (PR) in immunostained MG tissue sections from TAMc+E(2)-treated animals. Additionally, a marked decline in the level of expression of ERalpha 47, 56 and 66 kDa forms, and PR-A and PR-B was seen in TAMc+E(2)-treated MGs, compared with MGs treated solely with E(2). Thus, both ERalpha and PR MG profiles in TAMc+E(2)-treated rats essentially revert to their respective receptor profiles seen in untreated control and TAMc-alone-treated rats. The presence of 56 and 54 kDa isoforms in chronically E(2)-treated MGs and in MGTs respectively may contribute to fostering the enhanced E(2)-dependent growth response of both precursor and frank MGT epithelial cells. These findings are consistent with an ERalpha/PR-mediated mg cell proliferation, a prerequisite for generating the high frequency of chromosomal instability seen in E(2)-induced ductal carcinomas in situ and primary MGTs in female ACI rats reported by us previously.

The effects of steroidal estrogens in ACI rat mammary carcinogenesis: 17beta-estradiol, 2-hydroxyestradiol, 4-hydroxyestradiol, 16alpha-hydroxyestradiol, and 4-hydroxyestrone.

Several investigators have suggested that certain hydroxylated metabolites of 17beta-estradiol (E2) are the proximate carcinogens that induce mammary carcinomas in estrogen-sensitive rodent models. The studies reported here were designed to examine the carcinogenic potential of different levels of E2 and the effects of genotoxic metabolites of E2 in an in vivo model sensitive to E2-induced mammary cancer. The potential induction of mammary tumors was determined in female ACI rats subcutaneously implanted with cholesterol pellets containing E2 (1, 2, or 3 mg), or 2-hydroxyestradiol (2-OH E2), 4-hydroxyestradiol (4-OH E2), 16alpha-hydroxyestradiol (16alpha-OH E2), or 4-hydoxyestrone (4-OH E1) (equimolar to 2 mg E2). Treatment with 1, 2, or 3 mg E2 resulted in the first appearance of a mammary tumor between 12 and 17 weeks, and a 50% incidence of mammary tumors was observed at 36, 19, and 18 weeks respectively. The final cumulative mammary tumor incidence in rats treated with 1, 2, or 3 mg E2 for 36 weeks was 50%, 73%, and 100% respectively. Treatment of rats with pellets containing 2-OH E2, 4-OH E2, 16alpha-OH E2, or 4-OH E1 did not induce any detectable mammary tumors. The serum levels of E2 in rats treated with a 1 or 3 mg E2 pellet for 12 weeks was increased 2- to 6-fold above control values (approximately 30 pg/ml). Treatment of rats with E2 enhanced the hepatic microsomal metabolism of E2 to E1, but did not influence the 2- or 4-hydroxylation of E2). In summary, we observed a dose-dependent induction of mammary tumors in female ACI rats treated continuously with E2; however, under these conditions 2-OH E2, 4-OH E2, 16alpha-OH E2, and 4-OH E1 were inactive in inducing mammary tumors.

Influence of estrogens on peroxidase activity in the Syrian hamster liver, kidney, and renal adenocarcinoma.

Only very low levels of peroxidase activity were detected in castrated male hamster kidneys [1.0 +/- 0.8(S.E.) units/g protein], and chronic estrogen administration, either diethylstilbestrol (DES) or 17 beta-estradiol, for 1-5 months did not result in any appreciable increase in this activity. In contrast, hamster liver peroxidase activity was initially 10- to 20-fold higher than kidney levels, and chronic estrogen treatment for similar periods resulted in up to a 9-fold elevation in the activity of this enzyme. Moreover, the level of liver peroxidase activity in both intact and in 5 alpha-dihydrotestosterone-treated castrate hamsters was 2-fold higher than castrate-untreated values. Pure renal carcinoma induced after 9 months of estrogen treatment exhibited peroxidase values similar to those found in hamster livers [124 +/- 27 (S.E.) units/g protein] following estrogen treatment. When administered concomitantly with DES, tamoxifen significantly reduced the elevated levels of liver peroxidase activity observed after 2 months of DES treatment alone. A high affinity (KA = 0.10 X 10(9)M-1) estrogen receptor was found in liver cytosols of DES-treated hamsters which had increased slightly from untreated castrate levels.

Effect of steroid hormone treatment on aryl hydrocarbon hydroxylase activity in the Syrian hamster kidney.

Aryl hydrocarbon hydroxylase (AHH) activity was determined in castrate and intact male Syrian hamster kidney and liver microsomes following in vivo treatment with either diethylstilbestrol (DES) or 17 beta-estradiol as well as other steroid hormones. After 1 month of estrogen treatment, there was a 5-fold decline in AHH activity in castrated hamster kidneys compared with untreated castrate levels. The amount of AHH activity in the kidney was depressed more than 75% of untreated castrate levels even after the estrogen had been withdrawn for 6 days. Consistent with a nearly 2.5-fold higher renal AHH activity observed in intact male hamsters compared to castrates was the finding of a 1.7-fold elevation in the activity of this enzyme after treatment of castrated animals with androgen[5 alpha-dihydrotestosterone (5 alpha-DHT)] for 1 month. Moreover, following withdrawal of estrogen from intact hamsters, the increase in AHH activity in the kidney essentially paralleled the rise in serum testosterone levels. In castrated animals, the depression of AHH activity by estrogen was partially reversed by concomitant 5 alpha-DHT treatment. However, no appreciable changes were seen in liver AHH activity with androgen treatment in the presence or absence of estrogen. Similarly, the level of AHH activity, which was nearly 7- and 14-fold higher than intact and castrate kidney levels, respectively, was not altered by estrogen treatment. Neither progesterone nor cortisone had any effect on the levels of AHH activity in either the kidney or liver. Therefore, AHH activity in the male hamster kidney, but not the liver, is responsive to both estrogens and androgenic hormone.

Estrogen carcinogenesis in the hamster kidney: role of cytotoxicity and cell proliferation.

Both natural and synthetic estrogens are capable of inducing renal neoplasms in Syrian hamsters with an incidence approaching 100%. Neither the sequence of events nor the mechanisms involved in estrogen carcinogenesis in this model have been established. Results presented here indicate that estrogen induces renal tubular damage in the hamster kidney that is progressive and cumulative. Tubular injury was evident both as abnormal or lost microvilli, accumulation of cytoplasmic lipid droplets, vacuolization, and increases in secondary and tertiary lysosomes after 1.5 months of diethylstilbestrol (DES) treatment. Increasing tubular damage was evidence by the detachment of tubular cells, cell debris, and occluded renal tubular lumens. In an effort to repair proximal tubular damage in the hamster kidney elicited by estrogens, a 4.0-fold increase in proximal tubule BrdU labeling was evident at 4 months of DES or 17 beta-estradiol (E2) treatment and in earlier estrogen treatment periods (1-3 months). During this period, there was a significant increase in aneuploid cells in the hamster kidney, the near diploid frequency increased more than 6.0-fold, and the near tetraploid frequency increased at least 3.0-fold between 1.5 and 3.5 months of estrogen treatment. Based on these data, the early sequence of events leading to estrogen-induced renal neoplastic transformation in the hamster is presented.