Induction of a DNA adduct detectable by 32P-postlabeling in the dorsolateral prostate of NBL/Cr rats treated with estradiol-17{beta} and testosterone

Treatment with estradiol-17ß and testosterone inducesepithelial dysplasia and, subsequently, adenocarcinoma in thedorsolateral prostate of NBL rats. The purpose of this studywas to determine whether this carcinogenic effect is mediatedby genotoxicity. Analogous to adducts produced by estrogensin the male hamster kidney, a target of estrogen carcinogenicity,induction of DNA adducts detectable by ³²P-postlabeling wasinvestigated in the prostate target tissue. NBL rats were treatedwith separate Silastic tubing implants containing testosteroneand estradiol-17ß. Control animals received emptyimplants. Animals were killed at 8, 16 and 24 weeks after initiationof treatment, and accessory sex glands were sampled for adductanalysis. DNA of the dorsolateral and ventral prostate and thecoagulating gland (= anterior prostate) was isolated and analyzedby nuclease Pl-enhancement of the ³²P-postlabeling assay. DNAadducts were quantitated by Cerenkov counting. An adduct occurredselectively in DNA of the dorsolateral prostate of rats treatedwith estradiol plus testosterone for 16 or 24 weeks with relativeadduct level values of     

Genistein suppresses mammary cancer in rats

Female Sprague-Dawley CD rats were injected s.c. with 5 mg genistein,a soy phytoestrogen, or 20 µl of the vehicle, dimethylsulfoxide(DMSO), on days 2, 4 and 6 postpartum. At day 50, they wereexposed to 80 µg dimethyl-benz[a]anthracene (DMBA)/g bodywt. Animals treated neonatally with genistein as compared toDMSO had increased latency and reduced incidence and multiplicityof DMBA-induced mammary adenocarcinomas. Mammary whole mountanalysis showed that 50 day old female rats treated neonatallywith genistein had fewer terminal end buds. Cell proliferationstudies revealed that 50 day old genistein-treated rats hadlower percentages and total numbers of cells in the S-phaseof the cell cycle in terminal end buds, terminal ducts, lobulesI and lobules II. In genistein-treated as compared to vehicle-treatedfemale rats, vaginal openings occurred earlier, the estrus cyclewas disrupted and the uterine-ovarian weights were smaller.In 50 day old genistein-treated females there were atretic antralfollicles, fewer corpora lutea, and lower circulating progesteronebut not estradiol-17ß concentrations. In 21 day oldrats treated neonatally with genistein, mammary glands werelarger and there were more terminal end buds and terminal ducts,and more proliferative activity in all terminal ductal structures.It appears that neonatal genistein-treatment exerted its chemopreventionaction by acting directly to enhance maturation of terminalductal structures and by altering the endocrine system to reducecell proliferation in the mammary gland.     

Effects of 17{beta}-estradiol on radiation transformation in vitro; inhibition of effects by protease inhibitors

We have investigated the effects of 17ß-estradiol,given both alone and with X-irradiation, on the induction ofmalignant transformation in vitro. Treatment with 10^–6M17ß-estradiol for 6 weeks, or 10^–5M 17ß-estradiolfor only 5 days, induced malignant transformation in C3H 10Tcells. Estradiol also acted as a cocarcinogen for X-ray inducedtransformation; the results indicate an additive effect whenthe cells were exposed to both agents together. The proteaseinhibitors antipain and leupeptin suppressed estradiol inducedtransformation as well as the additive effect observed for estradiol- radiation transformation.     

Estradiol-17{beta} as an initiation modifier for radiation-induced mammary tumorigenesis of rats ovariectomized before puberty

This investigation evaluated the roles of estradiol-17ß,progesterone and prolactin in the initiation of mammary tumorigenesisby irradiation. Sixty day old Wistar-MS rats ovariectomizedbilaterally at 23 days of age were injected daily with oliveoil, estradiol-3-benzoate (E₂B), progesterone or haloperidolfor 14 days, and were then irradiated with     

Gene Expression Profiling Identifies Lobe-Specific and Common Disruptions of Multiple Gene Networks in Testosterone-Supported, 17β-Estradiol- or Diethylstilbestrol-Induced Prostate Dysplasia in Noble Rats

The xenoestrogen diethylstilbestrol (DES) is commonly believed to mimic the action of the natural estrogen 17β-estradiol (E₂). To determine if these two estrogens exert similar actions in prostate carcinogenesis, we elevated circulating levels of estrogen in Noble (NBL) rats with E₂/DES-filled implants, while maintaining physiological levels of testosterone (T) in the animals with T-filled implants. The two estrogens induced dysplasia in a lobe-specific manner, with E₂ targeting only the lateral prostate (LP) and DES impacting only the ventral prostate (VP). Gene expression profiling identified distinct and common E₂-disrupted versus DES-disrupted gene networks in each lobe. More importantly, hierarchical clustering analyses revealed that T + E₂ treatment primarily affected the gene expression pattern in the LP, whereas T + DES treatment primarily affected the gene expression profile in the VP. Gene ontology analyses and pathway mapping suggest that the two hormone treatments disrupt unique and/or common cellular processes, including cell development, proliferation, motility, apoptosis, and estrogen signaling, which may be linked to dysplasia development in the rat prostate. These findings suggest that the effects of xenoestrogens and natural estrogens on the rat prostate are more divergent than previously suspected and that these differences may explain the lobe-specific carcinogenic actions of the hormones.     

CANCER BIOLOGY: Effects of dietary {beta}-carotene and selenium on initiation and promotion of pancreatic carcinogenesis in azaserine-treated rats

In the present study the effects of 0.1 or 1.0 g ß-carotene/kg diet (LßC or HßC) and 1.0 mg or 2.5 mgselenium/kg diet (LSel or HSel), as well as combinations ofthe respective low and high concentrations of ß-caroteneand selenium (LMix or HMix) on the initiation/early promotionphase or on the late promotion phase of pancreatic carcinogenesisin azaserine-treated rats, were investigated using cell proliferationand volumetric data of atypical acinar cell foci (AACF) as parameters.The present results indicate chemo-preventive effects of dietaryselenium, dietary ß-carotene and of their combinationon the development of acinar pancreatic lesions induced in ratsby azaserine. The inhibitory effect was most pronounced whenß-carotene and/or selenium were added to the dietsduring the late promotion phase of the carcinogenic process,although inhibition was also observed with these compounds whenthey were added to the diets during the first 5 weeks of thestudy only (initiation/early promotion phase). Neither in theinitiation/ early promotion phase nor in the late promotionphase was a dose-related trend observed. The multiplicitiesof AACF with a diameter over 1.0 mm and of carcinomas in situ(CIS), as well as the incidence of CIS were not significantlydifferent among the groups. However, in the late promotion experimenta dose-related decline in multiplicity could be observed inthe selenium supplemented groups and in the groups receivingcombinations of ß-carotene and selenium. Cell proliferationin azaserine-induced AACF, as estimated by the bromodeoxyuridine(BrdU) labeling index, was significantly higher in HßC,HSel, LMix and HMix groups (initiation/early promotion phase)as well as in HßC, LSel, HSel, LMix and HMix groupsGate promotion phase) than in high fat controls. From the presentresults it can be concluded that: (i) ß-carotene andselenium have inhibitory effects on pancreatic carcinogenesisinduced in rats by azaserine; (ii) the most clear effects wereobserved when selenium was given as such, or in combinationwith ß-carotene during the late promotion phase; and(iii) ß-carotene and selenium stimulate cell proliferationin AACF.     

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.     

MOLECULAR EPIDEMIOLOGY AND CANCER PREVENTION: Prepubertal genistein exposure suppresses mammary cancer and enhances gland differentiation in rats

Genistein, a component of soy, was administered to pre-pubertalfemale Sprague-Dawley CD rats and investigated for chemopreventionagainst mammary cancer. Genistein, at 500 µg/g body wtor an equivalent volume of the vehicle, dimethylsulfoxide (DMSO),was injected (s.c) on days 16, 18 and 20 post-partum. At day50 post-partum all animals were exposed to 80 µg dimethylbenz[a]anthracene(DMBA) per g body wt. Animals treated prepubertally with genisteinas compared to DMSO had reduced incidence and significantlyfewer adenocarcinomas per animal. Mammary whole mount analysisshowed that prepubertal genistein treatment resulted in mammaryglands of 50-day-old rats developing fewer terminal end budsand more lobules II. Cell proliferation studies with bromodeoxyuridine(BrdU) showed that terminal end buds from mammary glands of50-day-old females treated prepubertally with genistein hadsignificantly fewer cells in S-phase of the cell cycle. Serumgenistein concentrations in 21- and 50-day-old females followingprepubertal genistein treatment were 4.2 ± 0.6 µMand 102 ± 30 nM, respectively. Animals treated prepubertallywith genistein as compared to vehicle spent more time in theestras phase of the estrus cycle, although all animals did cycle.In 50-day-old females, circulating estradiol-17ß andprogesterone concentrations were not significantly altered bythe prepubertal genistein treatment Oocyte/follicle counts andnumbers of atretic follicles and corpora lutea were not significantlydifferent between the genistein- and vehicle-treated animals.We conclude that genistein treatment during the prepubertalperiod can suppress the development of chemically-induced mammarycancer without significant toxicity to the endocrine/reproductivesystem.     

Involvement of transforming growth factor {alpha} (TGF{alpha}) and epidermal growth factor receptor (EGFR) in sex hormone-induced prostatic dysplasia and the growth of an androgen-independent transplantable carcinoma of the prostate

We previously reported the induction of dysplasia, a putativeprecursor of carcinoma, in the dorsolateral prostates (DLPs)of Noble rats by the combined administration of testosterone(T) and estradiol-17ß (E₂) for 16 weeks. Additionally,we demonstrated growth of the AIT, a DLP-derived, androgen-independent,transplantable solid tumor, in castrated syngeneic hosts. Inthis investigation, using Northern blot hybridization, radioimmunoassaysand radioligand assays, we showed that transforming growth factor-     

Enhancement of thyroid and hepatocarcinogenesis by 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene in rats at doses that cause maximal induction of CYP2B

To investigate the promoting effects of 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene(TCPOBOP) on liver and thyroid carcinogenesis of rats at dosesthat cause maximal induction of hepatic CYP2B, 5-week-old maleF344 rats were given either a single i.p. dose of 75 mg N-nitrosodiethylamine(NDEA)/kg body wt in saline or saline alone. After 2 weeks therats were fed control diet or a diet containing 330 or 1000p.p.m. TCPOBOP or 500 p.p.m. phenobarbital (PB; a positive controlgroup). A total of four sequential sacrifices (9, 30, 52 and79 weeks of age) was performed. At 30 weeks the mean volume(mm³) of hepatocellular foci in NDEA-initiated rats exposedto either dose of TCPOBOP or to PB was significantly increasedas compared with rats exposed to NDEA followed by control diet(P < 0.05). In addition, the volume percentage of liver occupiedby foci was significantly greater in NDEA-initiated/1000 p.p.m.TCPOBOP-promoted rats as compared with rats exposed to NDEAalone (P < 0.05, n = 6). At 52 weeks of age the incidences(and multiplicities, in units of tumors per tumor-bearing rat)of hepatocellular adenomas were 0, 83 (2.6 ± 1.3), 100(3.4 ± 2.1) or 67% (2.5 ± 1.9) in rats exposedto NEDA alone or NDEA followed by 330 or 1000 p.p.m. TCPOBOPor 500 p.p.m. PB respectively (n = 12). Hepatocellular carcinomaswere found only in rats given 1000 p.p.m. TCPOBOP (17% incidence)or PB (8% incidence) following NDEA initiation. The incidencesof thyroid follicular cell adenomas were 0, 17, 33 or 8% inrats exposed to NDEA alone or NDEA followed by 330 or 1000 p.p.m.TCPOBOP or 500 p.p.m. PB respectively. Between 53 and 79 weeksof age 38% of rats treated with NDEA alone developed multiple(1.5 ± 0.8) hepatocellular adenomas. This incidence wasenhanced to 100% in rats exposed to NDEA followed by either330 or 1000 p.p.m. TCPOBOP. Multiplicities of hepatocellularadenomas were also increased significantly (10.5 ± 3.9,10.4 ± 7.0 and 10.1 ± 6.7 respectively) in ratspromoted with 330 or 1000 p.p.m. TCPOBOP or 500 p.p.m. PB. Noneof the rats exposed to NDEA alone developed hepatocellular carcinomas,while multiple hepatocellular carcinomas occurred in 38% ofthe rats exposed to 330 p.p.m. and 78% of the rats given 1000p.p.m. TCPOBOP following NDEA initiation. Thyroid follicularcell tumors occurred at 79 weeks in more than 40 and 50% incidencesin rats exposed to NDEA followed by 330 or 1000 p.p.m. TCPOBOPrespectively. Also, a significant decrease in serum levels oftriiodothyronine and thyroxine were observed in non-initiated79-week-old rats fed 1000 p.p.m. TCPOBOP, compared with age-matcheduntreated controls (n = 6). Increases in hepatic CYPC2B-mediatedbenzyloxy-resorufin O-dealkylase activity detected in rats exposedto 330 and 1000 p.p.m. TCPOBOP for 2 or 23 weeks were similarin magnitude to those caused by 500 p.p.m. PB. Thus TCPOBOPat maximal CYP2B induction doses exhibits a strong promotingactivity for both liver and thyroid of rats.     

High survial rate of hamsters given intratracheal instillations of benzo[a]pyrene and ferric oxide and kept on a high {beta}-carotene diet

The study described in this paper was primarily conducted toidentify the cell types involved in the formation, progressionand regression of metaplastic changes in the respiratory tractepithelium of hamsters after intratracheal intubations withbenzo[a]pyrene Furthermore, the role of vitamin A and ß;-carotenein these processes was studied. In the course of the study aremarkable effect of dietary ß;-carotene on survivalof hamsters became a subject of investigation. Hamsters werefed diets with various levels of vitamin A or ß-caroteneand were treated intratracheally with a suspension of benzo[a]pyrenewith ferric oxide in saline. The tumour response of the respiratorytract was very low (2.8%) and hyper- and metaplasia of respiratoryepithelium were virtually absent. However, an interesting observationwas an exceptionally low mortality of only 2% after 69 weeksin the group of hamsters fed a high ß-carotene diet(1% w/w), whereas in the other groups mortality after 69 weeksamounted to 25%. Although the exact cause of death of most ofthe hamsters could not be established, a 40% reduction of lipidperoxidation in the livers was found in the high ß-carotenegroup. Moreover, In this group the degree and incidence of nephroslsand of focal mineralization of kidneys and heart were lowerthan in the other groups. These favourable effects of the highß-carotene diet may have contributed to the unusuallyhigh survival rate in hamsters fed this diet. Further studiesare planned to verify and study this observation.     

Stability of drug metabolizing enzymes during the incubation conditions of the liver microsomal assay with non-induced and induced mouse liver S-9 fractions

The purpose of this work was to study the relative activitiesand stabilities of phase-I and phase-II drug metabolizing enzymesin incubation mixtures used in in vitro genotoxicity testingin order to optimize the conditions of the assay, increase sensitivityand eliminate false negative results. Cytochrome P-450, NADPH-cytochromeP-450 (cytochrome c) reductase activity and various phase-Iand phase-II enzyme activities of the drug-metabolizing systemwere determined in incubation mixtures used in liver microsomalassays. The behaviour of aminopyrine N-demethylase and p-nitroanisoleO-demethylase activities as phase-I markers have been reportedpreviously. Other activities measured were glutathione S-transferase,glutathione S-epoxide transferase and epoxide hydrase, and lipidperoxidation (LP) was determined. The experiments were carriedout on liver S9 fractions derived from non-induced mice or miceinduced with sodium phenobarbital (PB), and/or ß-naphthoflavone(ß-NF). The phase-II enzymes were much more stable(70–90% residual activity) than phase-I enzyme activities(35–60%) in all conditions tested. The residual cytochromeP-450 was 70% stable and the remaining activity of NADPH-cytochromec-reductase about 80%, indicating that this latter enzyme doesnot limit the rate of the monooxygenase system in these conditions.Phase-II enzymes were induced to a smaller extent (about 2 times)than in phase-I enzymes (5–6 times) by ß-NF+ PB. NADPH-cytochrome c-reductase behaved as phase-II enzymesin this respect as well as for stability. LP was appreciablyhigher in non-induced than in induced animals. Treatment withthe ß-NF + PB mixture, however, showed that inducedenzymes were more stable than those obtained by simple inductionwith either ß-NF or PB alone. These results lead tothe conclusion that prolonged incubation times in mutagenicityassays are unnecessary when considering the relative stabilitiesof the various phase-I and phase-II enzyme activities in thedrug-metabolizing system.     

Different inhibition of DNA synthesis by transforming growth factor {beta} and phenobarbital on GST-P-positive and GST-P-negative hepatocytes

Hepatocyte resistance against inhibition of DNA synthesis bytransforming growth factor ß₁ (TGF-ß₁) wasstudied in vitro. Hepatocytes were isolated from rats that hadreceived diethylnitrosamine (DEN) for 6 weeks. The effect ofTGF-ß₁ and phenobarbital (PB) on DNA synthesis indifferent cell populations was studied using bromodeoxyuridine(BrdU) incorporation and placental glutathione S-transferase(GST-P) as markers. It was found that GST-P-positive cells wereresistant to the growth inhibitory effect of TGF-ß₁and PB, whereas GST-P-negative cells were inhibited. It is concludedthat resistance to TGF-ß₁-dependent growth controlmay develop early during DEN-induced hepatocarcinogenesis.     

Growth suppression of transformed BALB/c 3T3 cells by transforming growth factor {beta}1 occurs only in the presence of their normal counterparts

Transforming growth factor ß1 (TGF-ß1) enhancesthe yield of transformed foci of BALB/c 3T3 cells, but the continuouspresence of TGF-ß1 after foci formation inhibits thegrowth of transformed foci. The focus-forming ability of Ha-ras-,v-src-and PyMT-transformed cells growing on a monolayer of non-transformedcells was completely suppressed by TGF ß1, whereasgrowth of the transformed cells was little inhibited by TGF-ß1in the absence of their normal counterparts. The inhibitionby TGF-ß1 of focus formation by transformed BALB/c3T3 cells on a normal cell monolayer remained when TGF-ß1was removed from the culture medium after 2 weeks. However,the transformed cells were not killed, since they grew in cultureconditions under which only transformed cells are able to grow(soft agar). These results suggest that TGF-ß1 suppressesgrowth of transformed cells in the presence of normal cells.Furthermore, when non-transformed cells were treated with TGF-ß1before co-culture with Ha-ras-transformed cells, formation oftransformed foci was inhibited. When normal and transformedcells were cultured in the same dish but separated physically,focus formation was still Inhibited. On the other hand, TGF-ß1enhanced the growth and changed the morphology of non-transformedcells only in the presence of transformed counterparts. Thegrowth inhibitory effect of TGF-ß1 on transformedcells and its growth stlmulatory effect on non-transformed cellsin co-culture conditions suggest the induction of reciprocalparacrine growth regulatory factors. As TGF-ß1 inhibitsthe growth of transformed BALB/c 3T3 cells only in the presenceof their normal counterparts, a paracrine negative growth controlmechanism appears to be operating.     

Metabolic oxidation of diethylstilbestrol to diethylstilbestrol-4', 4"-quinone in Syrian hamsters

Diethylstilbestrol-4', 4"-quinone (DES Q) has previously beenpostulated to be a reactive intermediate in diethylstilbestrol(DES) metabolism. DES is oxidized to DES Q in vitro, but theoccurrence of the quinone metabolite in vivo has not yet beendemonstrated due to its instability and chemical reactivity.In this report, the characteristics of in vitro formation ofDES Q and the isolation of ³H-labeled DES Q from tissue extractsof hamsters injected with radiolabeled DES is described. Invitro, the time-dependent formation of DES Q as a function ofmicrosomal protein, cofactor or substrate concentrations wasdemonstrated. The microsome-mediated oxidation of DES to quinonewas inhibited by various compounds that also effectively inhibitthe peroxidatic activity of cytochrome P-450. In vivo, the formationof DES Q occurred in all tissues investigated, livers and kidneysof male and female adult hamsters, neonates and fetuses, andin uterus and placenta. Concentrations of quinone metabolitein liver and kidney of adult hamsters after injection of 75µmol/kg DES were 76 and 20 pmol/g tissue respectively.In neonates and fetus, concentrations of DES Q after the samedose of DES were markedly less than those in adults (0.026 and0.047% of adult levels in neonatal liver and kidney and 0.013and 0.016% of adult levels in fetal liver and kidney respectively).Since DES Q was also formed by fetal liver homogenate in vitro,fetal oxidizing enzymes appear to be the source of the quinonemetabolite in this tissue. DES Q concentrations were also examinedafter injection of DES into hamsters pretreated with vitaminC or -naphthoflavone, substances known to inhibit DES-inducedrenal carcinogenesis. Quinone metabolite levels were cut inhalf in response to vitamin C in correlation with the 50% decreasein DES-induced renal tumors reported previously. -Naphthoflavonepretreatment decreased renal and hepatic DES Q concentrationsby 70 and 17% respectively, also in correlation with the knownprevention of kidney tumors by this flavone. These data supporta role of DES Q in DES-induced carcinogenesis. Since there isno correlation between DES Q concentrations and target sitespecificity of DES induced tumors, the oxidation of DES to DESQ and the genotoxicity of this metabolite may be a necessarybut not sufficient event in tumor development. Hormone-dependentgrowth of initiated cells may also be necessary for the occurrenceof cancers.     

Suppression of testosterone and estradiol-17beta-induced dysplasia in the dorsolateral prostate of Noble rats by bromocriptine

We, and others, have previously described the histological changes that occur in the prostate gland of intact Noble (NBL) rats following prolonged hormonal treatment. Dysplasia, a pre-neoplastic lesion, develops specifically in the dorsolateral prostates (DLPs) of NBL rats treated for 16 weeks with a combined regimen of testosterone (T) and estradiol-17beta (E2) (T + E2-treated rats). Concurrent with DLP dysplasia induction, the dual hormone regimen also elicits hyperprolactinemia, in addition to an elevation of nuclear type II estrogen binding sites (type II EBS), no alteration in estrogen receptors (ER), and marked epithelial cell proliferation in the dysplastic foci. The aim of this study was to investigate whether the dual hormone action is mediated via E2-induced hyperprolactinemia. Bromocriptine (Br), at a dose of 4 mg/kg body wt per day, was used to suppress pituitary prolactin (PRL) release. Serum PRL levels were lowered from values of 341 +/- 50 ng/ml in T + E2-treated rats to 32 +/- 10 ng/ml in Br co-treated animals. The latter values were comparable to those in untreated control rats. In addition, Br co-treatment effectively inhibited the evolution of dysplasia (six out of eight rats) and the often associated inflammation (five out of eight rats) in most animals. In contrast, Br co-treatment did not suppress the T + E2- induced type II EBS elevation nor alter ER levels in the DLPs of these rats, when compared with T + E2-treated rats. These data extend the many previous studies that have detailed marked influences of PRL on rat prostatic functions. However, the current study is the first to implicate PRL in prostatic dysplasia induction in vivo.      

Regulation of the formation of the major diethylstilbestrol--DNA adduct and some evidence of its structure

Diethylstilbestrol (DES) induces kidney tumors in hamsters.In previous studies, DES has been shown by ³²P-postlabelinganalysis to bind covalently to DNA in vivo and in vitro andDES—DNA adduct formation has been suggested to play akey role in DES-induced carcinogenicity. In this study, we haveexamined the influence of the dose of DES, age of animals andorgan specificity on adduct formation in hamsters. In addition,we examined the characteristics of DES-DNA adduct formationin vitro and the structure of the major adduct DES-DNA adductswere detected in liver and kidney of hamsters treated with atleast 20 mg/kg DES. Adduct concentrations were higher at higherdoses or in older compared to younger animals. The covalentbinding of DES to DNA catalyzed by hamster liver microsomesrequired cumene hydroperoxide as cofactor, whereas with NADPH,adducts were barely detectable, presumably because the reactivemetabolic intermediate DES quinone was reduced to DES. The majorDES—DNA adduct formed in vitro was purified by semipreparativeand analytical high pressure liquid chromatography. It is concludedthat DES—DNA adducts are formed from DES quinone at verylow rates in vitro and occur at low levels in vivo, even whenhamsters receive very large doses of DES. The dependence ofDES—DNA adduct concentrations in vitro on organic hydroperoxidecofactors required for cytochrome P450-mediated DES quinoneformation indicates that stilbene-DNA adduction may occur onlyunder conditions of oxidative stress.     

Phenobarbital promotes the development of adenocarcinomas in the accessory sex glands of MNU-inoculated L-W rats

Aged Lobund-Wistar (L-W) rats develop: (i) spontaneous and inducedmetastasizing adenocarcinomas in the prostate and seminal vesicle(P-SV) complex; and (ii) spontaneous hepatomas and hepatocarcinoinas.Within the time-frame of 14 months, similar adenocarcinomaswere induced in the P-SV complex in 70–90% of youngerL-W rats by a single i.v. inoculation of methylmtrosourea (MNU)which was followed by slow release s.c. implants of testosteroneproplonate (TP). Within the same time-frame, neither MNU norTP alone induced signillcant incidences of P-SV tumors; anduntreated control L-W rats were disease-free. Methylnitrosoareaor TP and combinations thereof did not induce liver tumors.However, when MNU-inoculated L-W rats were fed phenobarbital(PB), they developed (i) metastasizing adenocarcinomas in theP-SV complex and (ii) altered cellular foci and nodules in thelivers. Methylnitrosourea induced a high incidence of benignlung adenomas which progressed to lung cancers in numbers whichwere of marginal significance. Thus, dormant MNU-initiated cellsin the P-SV complex were activated by phenobarbital, to produceadenocarcinomas in that complex.     

Loss of p53 enhances the induction of colitis-associated neoplasia by dextran sulfate sodium

Loss of p53 function is an early event in colitis-associatedneoplasia in humans. We assessed the role of p53 in a mousemodel of colitis-associated neoplasia. Colitis was induced inp53^–/–, p53^+/– and p53^+/+ mice using threeor four cycles of dextran sulfate sodium (DSS) followed by 120days of water. Mice were examined for incidence, multiplicityand types of neoplastic lesions. Lesions were examined for mutationsin ß-catenin (exon 3), K-ras (codons 12/13) and p53(exons 5–8) by sequencing and for cellular localizationof ß-catenin by immunohistochemistry. The incidenceof neoplastic lesions was 57, 20 and 20% in p53^–/–,p53^+/– and p53^+/+ mice, respectively (P = 0.013). p53^–/–mice had a greater number of total lesions (P < 0.0001),cancers (P = 0.001) and dysplasias (P = 0.009) per mouse thaneither p53^+/– or p53^+/+ mice. Flat lesions were associatedwith the p53^–/– genotype, whereas polypoid lesionswere associated with the p53^+/– and p53^+/+ genotypes (P< 0.0001). ß-Catenin mutations were present in75% of lesions of p53^+/+ mice and absent in lesions from p53^–/–mice (P = 0.055). Nuclear expression of ß-cateninwas seen only in polypoid lesions (91%). No K-ras or p53 mutationswere detected. These data indicate that loss of p53 enhancesthe induction of colitis-associated neoplasia, particularlyflat lesions, and dysregulation of ß-catenin signalingplays an important role in the formation of polypoid lesionsin this mouse model. As observed in humans, p53 plays a protectiverole in colitis-associated neoplasia in the DSS model. Abbreviations: CRC, colorectal cancer; DSS, dextran sulfate sodium; PCR, polymerase chain reaction; UC, ulcerative colitis; PGE₂, prostaglandin E₂ Received January 24, 2007; revised May 11, 2007; accepted May 29, 2007.