Use of short-term assays to evaluate the potential toxicity of two new biosoluble glasswool fibers.

Two new glasswools were developed for optimal biosolubility in the lung: JM 902, for insulation and filtration; and JM 901F, for standard thermal and acoustical insulation. Both were tested for lung biopersistence and their potential to induce persistent pulmonary inflammation in rats. Their dissolution rate constants (k(dis)) were estimated in vitro. Results for 902 were: in vitro k(dis) (pH 7.4) = 150 ng/cm2/h; after 5 days of fiber inhalation (IH), lung clearance of fibers > 20 microm length (F > 20 microm) indicated a weighted half-time (WT(1/2)) of 6.8 days and 90% clearance time (T90) of 33 days; following intratracheal instillation (IT), lung clearance half-time (T(1/2)) for F > 5 microm was 20 days. Results for 901F were: k(dis) (pH 7.4) = 500-560; after 5 days of fiber inhalation exposure, WT(1/2) (F > 20 microm) = 8.1 days and T90 = 38 days. After 5 days of fiber inhalation, both fibers induced initial pulmonary inflammation followed by return to normal within 3 wk postexposure. Lung clearance half-times for 902 and 901F passed the European Union (EU) criteria for noncarcinogenic fibers (IH WT(1/2) F > 20 microm was < 10 days); 902 passed the noncarcinogenic criterion of the German government (IT T(1/2) F > 5 microm was < 45 days). Thus, carcinogenicity labeling is not required for either fiber in the EU. Short-term test results for 902 and 901F were similar to results for synthetic vitreous fibers (SVFs) that were innocuous in rodent chronic inhalation studies, but short-term test results for 902 and 901F differed sharply from results for other SVFs that were pathogenic in chronic studies. Thus, these short-term tests indicate that 902 and 901F are biosoluble fibers and would be nonpathogenic in the rat exposed by inhalation.

Nephrotoxicity and hepatotoxicity induced by inhaled bromodichloromethane in wild-type and p53-heterozygous mice.

Bromodichloromethane (BDCM) is a common municipal drinking water disinfection by-product, resulting in widespread trace human exposure via ingestion and inhalation. The present studies were designed to define organ-specific, BDCM-induced toxicity in wild type (p53(+/+)) and heterozygous (p53(+/-)) mice on both the FVB/N and C57BL/6 genetic backgrounds. Mice were exposed to BDCM vapor daily for 6 h/day and 7 days/week at concentrations of 0, 1, 10, 30, 100, or 150 ppm for 1 week and at 0, 0.3, 1, 3, 10, or 30 ppm for 3 weeks. In the 1-week exposure study, dose-dependent mortality and morbidity were observed at concentrations of 30 ppm and above and were as high as 100% at 150 ppm. In the 3-week exposure study, mortality and morbidity were found only in the 30-ppm exposure groups and were 0, 17, 67, and 33% for the wild-type C57BL/6, p53(+/-) C57BL/6, wild-type FVB/N, and p53(+/-) FVB/N mice, respectively. BDCM was a particularly potent kidney cytotoxicant. Dose-dependent tubular degeneration, necrosis, and associated regenerative cell proliferation greater than 10-fold over controls were seen at concentrations as low as 10 ppm in the kidneys of all strains at 1 week. Similar dose-dependent increases in hepatic necrosis, degeneration, and regenerative cell proliferation were observed but were induced only at concentrations of 30 ppm and higher. Pathological changes were more severe in the FVB/N compared to the C57BL/6 mice and were more severe in the heterozygotes compared to the wild-type mice. However, recovery and return of the percentage of kidney cells in S-phase to control levels was seen at 3 weeks. The estimated maximum tolerated dose for longer-term exposures was 15 ppm, based on mortality, induced kidney pathology, and regenerative cell proliferation. A one-year cancer bioassay was initiated with doses of 0, 0.5, 3, 10, and 15 ppm, based on this information. No pathological changes in the livers were found at the 13-week time point of that study. At 13 weeks, the kidney lesions and regenerative cell proliferation seen at the 1-week time point at doses of 10 ppm and above had resolved, and the cell proliferation rates had returned to baseline. Differences in toxicity indicate that caution be used in substituting wild-type mice for transgenic mice for range-finding studies to select doses for p53(+/-) cancer studies. Resolution of the kidney lesions indicates that periods of very high regenerative cell proliferation, potentially important in the carcinogenic process, may not be observed if measurements are taken only at 3 weeks of exposure or later.

alpha 2u-Globulin nephropathy, renal cell proliferation, and dosimetry of inhaled tert-butyl alcohol in male and female F-344 rats.

tert-Butyl alcohol (TBA) has been shown to cause kidney tumors in male rats following chronic administration in drinking water. The objective of the present study was to determine whether TBA induces alpha 2u-globulin (alpha 2u) nephropathy (alpha 2u-N) and enhanced renal cell proliferation in male, but not female, F-344 rats, and whether the dosimetry of TBA to the kidney is gender specific. Male and female F-344 rats were exposed to 0, 250, 450, or 1750 ppm TBA vapors 6 h/day for 10 consecutive days to assess alpha 2u-nephropathy and renal cell proliferation and for 1 and 8 days to evaluate the dosimetry of TBA following a single and repeated exposure scenario. Protein droplet accumulation was observed in kidneys of male rats exposed to 1750 ppm TBA, with alpha 2u-globulin immunoreactivity present in these protein droplets. A statistically significant increase in alpha 2u concentration in the kidney, as measured by an enzyme-linked immunosorbent assay, was observed in male rats exposed to 1750 ppm TBA with a exposure-related increase in renal cell proliferation. Renal alpha 2u concentration was positively correlated with cell proliferation in male rat kidney. No histological lesions or increased renal cell proliferation was observed in female rats exposed to TBA compared to controls. The TBA kidney:blood ratio was higher at all concentrations and time points in male rats compared with female rats, which suggests that TBA is retained longer in male rat kidney compared with female rat kidney. Together these data suggest that TBA causes alpha 2u-N in male rats, which is responsible for the male rat-specific increase in renal cell proliferation.

Carcinogenicity of a nephrotoxic metabolite of the "nongenotoxic" carcinogen hydroquinone.

Hydroquinone (HQ) is a potential human carcinogen to which many people are exposed. HQ generally tests negative in standard mutagenicity assays, making it a "nongenotoxic" carcinogen whose mechanism of action remains unknown. HQ is metabolized to 2,3,5-tris(glutathion-S-yl)HQ (TGHQ), a potent toxic and redox active compound. To determine if TGHQ is a carcinogen in the kidney, TGHQ was administered to Eker rats (2 months of age) for 4 or 10 months. Eker rats carry a germline mutation in the tuberous sclerosis 2 (Tsc-2) tumor suppressor gene, which makes them highly susceptible to the development of renal tumors. As early as 4 months after the initiation of treatment (2.5 micromol/kg, i.p.), TGHQ-treated rats developed numerous toxic tubular dysplasias of a form rarely present in vehicle-treated rats. These preneoplastic lesions are believed to represent early transformation within tubules undergoing regeneration after injury by TGHQ, and adenomas subsequently arose within these lesions. After treatment for 10 months (2.5 micromol/kg for 4 months followed by 3.5 micromol/kg for 6 months), there were 6-, 7-, and 10-fold more basophilic dysplasias, adenomas, and renal cell carcinomas, respectively, in TGHQ-treated animals than in controls. Most of these lesions were in the region of TGHQ-induced acute renal injury, the outer stripe of the outer medulla. Loss of heterozygosity (LOH) at the Tsc-2 locus was demonstrated in both the toxic tubular dysplasias and tumors from rats treated with TGHQ for 10 months, consistent with TGHQ-induced loss of tumor suppressor function of the Tsc-2 gene. Thus, although HQ is generally considered a nongenotoxic carcinogen, our data suggest that HQ nephrocarcinogenesis is probably mediated by the formation of the quantitatively minor yet potent nephrotoxic metabolite TGHQ, which induces sustained regenerative hyperplasia, loss of tumor suppressor gene function, and the subsequent formation of renal adenomas and carcinomas. In addition, our data demonstrate that assumptions regarding mechanisms of action of nongenotoxic carcinogens should be considered carefully in the absence of data on the profiles of metabolites generated by these compounds in specific target organs for tumor induction.

Advances in uterine leiomyoma research: conference overview, summary, and future research recommendations.

Uterine leiomyomas (fibroids, myomas) are the most common tumors occurring in the genital tract of women over 30 years of age. These benign uterine smooth-muscle tumors are estimated to be clinically significant in at least 25% of the American female population during their reproductive years. Furthermore, when thorough pathologic examination of hysterectomy specimens has been performed in patients with or without clinical history of myomatous uteri, the incidence of fibroids is 77%, suggesting that these tumors are far more prevalent than estimated by clinical cases. In spite of their high prevalence, little is known concerning the etiology or the molecular basis of their development and growth. It is well known that leiomyoma growth is regulated by ovarian steroid hormones, yet the exact molecular pathway(s) involved in tumor growth and the role of genetic susceptibility/predisposition and the environment are unclear. This article is an overview of some of the topics addressed at the conference on Women's Health and the Environment: The Next Century--Advances in Uterine Leiomyoma Research. A summary of research needs and recommendations for future research directions based on conference discussions are also presented.

Preclinical evidence for therapeutic efficacy of selective estrogen receptor modulators for uterine leiomyoma.

OBJECTIVE: Uterine leiomyoma are the most common gynecologic neoplasm and a primary cause of hysterectomy in premenopausal women. Preclinical studies were conducted in the Eker rat model to investigate the potential efficacy of selective estrogen receptor modulators (SERMs) as therapeutic agents for this tumor. METHODS: Twelve-month-old Eker rats were randomized into five treatment arms including tamoxifen, placebo, LY 326315, vehicle, and no treatment. Additional animals received ovariectomy or sham surgery at 4 months of age to determine the effect of ovarian ablation on tumor development. The study was terminated after 2 to 4 months of treatment, and tumor incidence, size, proliferative and apoptotic indices were determined. Size and incidence data were subjected to chi-square analysis. One-way analysis of variance and Fisher's least significant difference tests were used to compare proliferative and apoptotic indices. RESULTS: Ovariectomy virtually ablated leiomyoma development, indicating that these tumors were dependent on ovarian hormones for growth and development. Treatment with tamoxifen or raloxifene analog LY 326315 reduced leiomyoma incidence by 40-60% and reduced the size of remaining tumors. The effect of SERMs on leiomyomas was mediated by a decrease in cell proliferation without a decrease in apoptotic index. CONCLUSION: SERMs have been shown to be therapeutically efficacious against breast cancer and to reduce tumor incidence in women at increased risk for this disease. The present data indicate that therapeutic efficacy may also be extended to uterine leiomyoma and demonstrate the utility of this animal model for preclinical studies to identify new therapeutic modalities.

Promotion by sodium barbital induces early development but does not increase the multiplicity of hereditary renal tumors in Eker rats.

Induced cell proliferation is important in the mode of action of many non-genotoxic renal carcinogens. Since Tsc2 mutant (Eker) rats are genetically predisposed to the development of renal cell tumors, they provide a useful animal model in which to study the action of renal carcinogens. Sodium barbital was used as a model non-genotoxic renal carcinogen to test whether a concentration that increased renal tubular proliferation without severe nephrotoxicity would enhance tumor induction in a hereditary tumor model. First, a subchronic concentration-response study was conducted in wild-type male Long-Evans rats to determine increased cell proliferation without severe nephrotoxicity. Rats were dosed with sodium barbital in the feed at 0, 50, 250, 500, 1000, 2000 or 4000 p.p.m. for 3 or 8 weeks. Cell proliferation within the cortex and nephrotoxicity were quantitated. Enhanced proliferation with minimal nephrotoxicity occurred at 500 p.p.m. A second study was conducted in male Tsc2 mutant rats given sodium barbital in the feed at 0, 100 or 500 p.p.m. from 9 weeks of age to either 6 or 12 months of age. An additional group of rats was treated with sodium barbital for 6 months and then provided control feed until 12 months of age. Rats necropsied at 6 months of age had a concentration-dependent increase in preneoplastic and total renal lesions. Sodium barbital-treated rats necropsied at 12 months of age had numbers of lesions that were not different from controls. Total combined preneoplastic and neoplastic lesions in the 6 month, high dose group was the same as the 12 month control group. These data show that sodium barbital caused progression to the stage of spontaneous renal lesions in Tsc2 mutant rats but did not increase their overall number. These data suggest that enhanced cell proliferation without significant cytotoxicity exerted a promotional influence in this hereditary model.

Metabolism of chloroform by cytochrome P450 2E1 is required for induction of toxicity in the liver, kidney, and nose of male mice.

Chloroform is a nongenotoxic-cytotoxic liver and kidney carcinogen and nasal toxicant in some strains and sexes of rodents. Substantial evidence indicates that tumor induction is secondary to events associated with cytolethality and regenerative cell proliferation. Therefore, pathways leading to toxicity, such as metabolic activation, become critical information in mechanism-based risk assessments. The purpose of this study was to determine the degree to which chloroform-induced cytotoxicity is dependent on the cytochromes P450 in general and P450 2E1 in particular. Male B6C3F(1), Sv/129 wild-type (Cyp2e1+/+), and Sv/129 CYP2E1 knockout (Cyp2e1-/- or Cyp2e1-null) mice were exposed 6 h/day for 4 consecutive days to 90 ppm chloroform by inhalation. Parallel control and treated groups, excluding Cyp2e1-null mice, also received an i.p. injection (150 mg/kg) of the irreversible cytochrome P450 inhibitor 1-aminobenzotriazole (ABT) twice on the day before exposures began and 1 h before every exposure. Cells in S-phase were labeled by infusion of BrdU via an implanted osmotic pump for 3.5 days prior to necropsy, and the labeling index was quantified immunohistochemically. B6C3F(1) and Sv/129 wild-type mice exposed to chloroform alone had extensive hepatic and renal necrosis with significant regenerative cell proliferation. These animals had minimal toxicity in the nasal turbinates with focal periosteal cell proliferation. Administration of ABT completely protected against the hepatic, renal, and nasal toxic effects of chloroform. Induced pathological changes and regenerative cell proliferation were absent in these target sites in Cyp2e1-/- mice exposed to 90 ppm chloroform. These findings indicate that metabolism is obligatory for the development of chloroform-induced hepatic, renal, and nasal toxicity and that cytochrome P450 2E1 appears to be the only enzyme responsible for this cytotoxic-related metabolic conversion under these exposure conditions.

Comparison of pulmonary and pleural responses of rats and hamsters to inhaled refractory ceramic fibers.

The present study was designed to determine whether pleural fiber burdens or subchronic pleural fibroproliferative and inflammatory changes can help explain the marked interspecies differences in pleural fibrosis and mesothelioma that are observed following long-term inhalation of RCF-1 ceramic fibers by rats and hamsters. Fischer 344 rats and Syrian golden hamsters were exposed to RCF-1 for 4 h per day, 5 days per week, for 12 consecutive weeks. Lung and pleural fiber burdens were characterized during and after exposure. For all time points, approximately 67% of fibers associated with lung tissues from both rats and hamsters were longer than 5 microns in length. In comparison, fibers longer than 5 microns recovered from the pleural compartment, following a 12-week exposure and 12 weeks of recovery, accounted for 13% (hamsters) and 4% (rats) of the distribution. In the 12 weeks after the cessation of exposure, the number of fibers longer than 5 microns in length remained constant in the hamster at approximately 150 fibers per cm2 pleura. This was 2 to 3 times the corresponding fiber surface density in the rat. Significant pulmonary and pleural inflammation was detected at all time points and for both species. DNA synthesis by pleural mesothelial cells was quantified by bromodeoxyuridine uptake following 3 days of labeling. Labeling indices were higher in hamsters than in rats, both for RCF-1-exposed and filtered air-control animals and was highest for the parietal surface of the pleura. Significantly greater collagen deposition was measured in the visceral pleura of hamsters 12 weeks post-exposure but was not significantly elevated in rats. These findings demonstrate that subchronic inhalation exposure to RCF-1 induces pleural inflammation, mesothelial-cell turnover, pleural fibrosis, and an accumulation of fibers with a length greater than 5 microns in the hamster. The accumulation of long fibers in the pleural space may contribute to the pathology observed in the hamster following chronic inhalation of RCF-1, whereas the presence of short, thin fibers may play a role in the acute-phase biological response seen in both species.

Estrogen treatment enhances hereditary renal tumor development in Eker rats.

Hormonal influences are known to affect the development of renal cell carcinoma in man and laboratory animal models. We tested the hypothesis that estrogen treatment or ovariectomy of rats modulates renal tumor development using tuberous sclerosis 2 (Tsc2) heterozygous mutant (Eker) rats in which a germline mutation predisposes the animals to renal cell tumor development. Two-month-old female wild-type and Eker rats were ovariectomized or sham-operated and treated with placebo or 5 mg 17beta-estradiol in s.c. pellets for 6 or 10 months. Rats were examined at 8 or 12 months of age, at which time the numbers of renal tumors and preneoplastic foci were quantitated and the severity of nephropathy was assessed. In contrast to what may have been expected, prolonged estrogen treatment enhanced the development of hereditary renal cell tumors, with a 2-fold greater number of preneoplastic and neoplastic renal lesions compared with untreated Eker rats. Ovariectomized Eker rats had 33% fewer renal lesions than the unmanipulated control group. No tumors or preneoplastic lesions were present in wild-type rats at either time point. Estrogen treatment increased the severity of nephropathy in both wild-type and Eker rats, whereas ovariectomy was protective against nephropathic changes. Although estrogen is not a rat renal carcinogen, it enhanced the development of hereditary renal cell tumors when administered to Eker rats. Eker rats heterozygous for a mutation in the Tsc2 locus provide a good model in which to study how genetic and hormonal factors contribute to the development of renal cell tumors and to understand the influence genetic susceptibility has on the development of renal cell carcinoma.

Long-term mutagenicity studies with chloroform and dimethylnitrosamine in female lacI transgenic B6C3F1 mice.

The weight of evidence indicates that chloroform induces cancer in the female B6C3F1 mouse liver via a nongenotoxic-cytotoxic mode of action. However, it is probable that DNA damage occurs secondary to events associated with cytolethality and regenerative cell proliferation. The purpose of the present study was to evaluate the potential mutagenic activity of chloroform in the B6C3F1 lacI transgenic mouse liver mutagenesis assay including mutagenic events that might occur secondary to cytolethality. The positive control, dimethylnitrosamine (DMN) is a DNA-reactive mutagen and carcinogen. DMN-induced mutations were anticipated to require only a brief exposure and without further treatment were predicted to remain unchanged over time at those frequencies. Chloroform-induced mutations secondary to toxicity were anticipated to require longer exposure periods and to occur only under conditions that produced sustained cytolethality and regenerative cell proliferation. Female B6C3F1 lacI transgenic mice were treated with daily doses of 2, 4, or 8 mg/kg of DMN by gavage for 4 days and then held until analysis 10, 30, 90, and 180 days postexposure. Livers from DMN-treated mice exhibited a dose-related 2- to 5-fold increase over control mutant frequencies and remained at those levels for 10 through 180 days postexposure. Thus, following the initial induction by DMN no selective mutation amplification or loss was seen for this extended period of time. Female B6C3F1 lacI mice were exposed daily for 6 hr/day 7 days/week to 0, 10, 30, or 90 ppm chloroform by inhalation, representing nonhepatotoxic, borderline, or overtly hepatotoxic chloroform exposures. Timepoints for determination of lacI mutant frequency were 10, 30, 90, and 180 days of exposure. No increase in lacI mutant frequency in the liver was observed at any dose or timepoint with chloroform, indicating a lack of DNA reactivity. DNA alterations secondary to toxicity either did not occur or were of a type not detectable by lacI mutant frequency analysis, such as large deletions.

Altered expression of transforming growth factor-alpha: an early event in renal cell carcinoma development.

Transforming growth factor-alpha (TGF-alpha) is a multifunctional cell regulatory protein with a wide range of effects on cell growth and differentiation and has been implicated in the neoplastic transformation of a variety of cell types. Altered expression of TGF-alpha and its cognate receptor (epidermal growth factor receptor) is enhanced in human and rat renal cell carcinomas. The objective of the study reported here was to determine whether altered TGF-alpha expression is an early or late event in renal tubular oncogenesis. The immunohistochemical expression of TGF-alpha was studied in preneoplastic renal tubular lesions in a rat model of hereditary renal cell carcinoma. Strong TGF-alpha immunoreactivity was present at all stages of renal cell tumor development, including the earliest detectable dysplasias. In contrast, the non-neoplastic regenerating tubular epithelium of rat degenerative nephropathy did not stain for TGF-alpha, although this tissue exhibited a proliferative capacity similar to that observed in the dysplastic and neoplastic lesions. This study indicated that altered TGF-alpha expression was detectable early in the development of renal cell tumors and may be an important feature of the transformed phenotype.

Chrysotile asbestos and H2O2 increase permeability of alveolar epithelium.

The alveolar epithelium contains tight junctions and provides a barrier to passage of potentially injurious substances into the pulmonary interstitium. Alveolar epithelial injury is hypothesized to be an important early event in the pathogenesis of asbestosis. Mechanisms that may contribute to alveolar epithelial cell injury following asbestos exposure include the physicochemical interactions between asbestos fibers and cells, and the generation of reactive oxygen species such as hydrogen peroxide (H2O2). The present study examined changes in transepithelial resistance (Rt) (a measure of barrier function) and permeability of alveolar epithelium after chrysotile asbestos and H2O2 exposure. Alveolar epithelial cell monolayers, obtained from isolation of rat alveolar type II cells and grown on porous supports, were exposed to chrysotile asbestos or polystyrene beads (control) at concentrations of 5, 10, and 25 micrograms/cm2 for 24 h. In separate experiments, monolayers were exposed to H2O2 at concentrations of 50, 75, and 100 microM for 1 h Rt was measured using a voltohmmeter. Prior to treatment, monolayers had a high Rt (> 2000 ohms.cm2). Permeability was assessed by measuring flux of [3H]sucrose from apical to basolateral compartments. Cytotoxicity was evaluated by lactate dehydrogenase (LDH) and preincorporated [14C]adenine release. The morphological integrity of the monolayers was evaluated by scanning electron microscopy. Chrysotile asbestos and H2O2 exposure resulted in dose-dependent decrease in alveolar epithelial Rt and increases in permeability under conditions that did not result in over cytotoxicity. These results demonstrate that both chrysotile asbestos and H2O2 have effects on alveolar epithelial Rt and permeability and suggest a potential role for the alveolar epithelium in mediation of asbestos-induced pulmonary interstitial disease.

Pulmonary and pleural responses in Fischer 344 rats following short-term inhalation of a synthetic vitreous fiber. I. Quantitation of lung and pleural fiber burdens.

The pleura is an important target tissue of fiber-induced disease, although it is not known whether fibers must be in direct contact with pleural cells to exert pathologic effects. In the present study, we determined the kinetics of fiber movement into pleural tissues of rats following inhalation of RCF-1, a ceramic fiber previously shown to induce neoplasms in the lung and pleura of rats. Male Fischer 344 rats were exposed by nose-only inhalation to RCF-1 at 89 mg/m3 (2645 WHO fibers/cc), 6 hr/day for 5 consecutive days. On Days 5 and 32, thoracic tissues were analyzed to determine pulmonary and pleural fiber burdens. Mean fiber counts were 22 x 10(6)/lung (25 x 10(3)/pleura) at Day 5 and 18 x 10(6)/lung (16 x 10(3)/pleura) at Day 32. Similar geometric mean lengths (GML) and diameters (GMD) of pulmonary fiber burdens were observed at both time points. Values were 5 microns for GML (geometric standard deviation GSD approximately 2.3) and 0.3 micron for GMD (GSD approximately 1.9), with correlations between length and diameter (tau) of 0.2-0.3. Size distributions of pleural fiber burdens at both time points were approximately 1.5 microns GML (GSD approximately 2.0) and 0.09 micron GMD (GSD approximately 1.5; tau approximately 0.2-0.5). Few fibers longer than 5 microns were observed at either time point. These findings demonstrate that fibers can rapidly translocate to pleural tissues. However, only short, thin (< 5 microns in length) fibers could be detected over the 32-day time course of the experiment.

Hereditary renal cell carcinoma in the Eker rat: a unique animal model for the study of cancer susceptibility.

A class of genes, the so-called tumor suppressor genes or anti-oncogenes, was originally identified as being responsible for germ-line transmission of cancer susceptibility in humans. Tumor suppressor genes are recessive at the cellular level with respect to oncogenesis but often manifest as dominantly inherited familial cancer syndromes. This type of cancer syndrome arises in the Eker rat due to a genetic defect in the tuberous sclerosis 2 (Tsc2) gene. The Eker rat familial cancer syndrome serves as a unique animal model in which to study the molecular pathways of renal tubular epithelial carcinogenesis as well as a valuable tool for studies that examine how chemical carcinogens interact with cancer susceptibility genes.

Estrogen stimulation and tamoxifen inhibition of leiomyoma cell growth in vitro and in vivo.

Uterine leiomyomas (fibroids) are the most common gynecological neoplasms and may be associated with significant morbidity. Recently, we described a rat model (Eker rat) of fibroid development in which reproductive tract leiomyomas develop spontaneously with high frequency. The present studies describe the estrogen and antiestrogen responsiveness of an Eker rat leiomyoma-derived cell line in vitro and a nude mouse xenograft system in vivo. In this cell line, estradiol stimulated growth in estrogen-depleted medium, whereas the nonsteroidal antiestrogen tamoxifen maximally inhibited cell proliferation in medium containing 10% charcoal-stripped serum. Proliferation was also decreased by the biologically active tamoxifen metabolite 4-hydroxytamoxifen; the metabolite was more effective than the parent compound in exerting this growth inhibition. Compared to placebo-treated controls, estradiol increased the size of tumors that developed in a nude mouse xenograft system, whereas tamoxifen increased tumor latency and decreased tumor size. This study of leiomyoma cells in a well defined system suggests that antiestrogens may prove efficacious in the treatment of this clinically important neoplasm.

Allelic loss at the tuberous sclerosis 2 locus in spontaneous tumors in the Eker rat.

Somatic events leading to the inactivation of tumor suppressor genes often involve chromosomal alterations that can be detected as loss of heterozygosity (LOH). In the Eker rat, spontaneous tumors of the kidney, uterus, and spleen develop as a result of a germline mutation of the tuberous sclerosis 2 (Tsc2) gene. We examined the pattern and frequency of LOH at the predisposing locus in 77 primary tumors and cell lines to gain an understanding of the role of Tsc2 allelic loss in the pathogenesis of Eker-derived tumors. Although most renal and uterine tumors (primary and cell lines) displayed LOH, splenic hemangiosarcomas did not. Although the presence of normal tissue may account for some of this difference, the possibility exists that an alternative mechanism, such as subtle mutation or gene dosage effects, may be involved during splenic tumorigenesis. Northern analysis confirmed that LOH resulted in loss of the wild-type transcripts for the Tsc2 gene. Thus, the inactivation of both alleles plays an important role in renal and uterine tumor development, in keeping with Knudson's two-hit hypothesis. In addition, renal tumors that retained the wild-type allele also did not express the normal transcript, suggesting that the remaining Tsc2 alleles had acquired subtle mutations resulting in loss of gene function.

Expression of insulin-like growth factor II in spontaneously immortalized rat mesothelial and spontaneous mesothelioma cells: a potential autocrine role of insulin-like growth factor II.

Insulin-like growth factors (IGFs) are polypeptides that play an important role in cellular proliferation and differentiation. The present study examines the role of IGFs in the growth of mesothelial cells. Cell lines derived from normal rat mesothelium as well as lines derived from spontaneous rat mesotheliomas were found to express RNA transcripts for IGF-II. In contrast, cell lines derived from asbestos-induced rat mesotheliomas did not express this growth factor. All cell lines expressed receptors for IGF-I and IGF-II, as well as insulin receptors. Coexpression of IGF-II and its cognate receptor suggested that IGF-II was acting as an autocrine growth factor in the spontaneously immortalized cells and the cells derived from the spontaneous tumors. The biological activity of IGF-II secreted by the cell lines into conditioned medium could be neutralized using an IGF-II-specific antibody. Growth was inhibited in a dose-dependent manner; at the highest antibody concentration used (100 micrograms anti-IGF-II/ml), cell growth was decreased to 47% of control values. This inhibition was partially reversible by treatment of the cultures with IGF-II (91% of the control). These data suggest that IGF-II expression may be involved in the spontaneous alteration of rat mesothelial cells and may function as an autocrine or paracrine growth factor to modulate the growth of these cells in vitro and in vivo. Ubiquitous expression of IGF-II by cells that have not been exposed to asbestos and the lack of IGF-II expression by asbestos-transformed cells suggest that the mechanisms of changes in growth factor expression differ in mesothelial cells transformed by different pathways.

Preneoplastic and neoplastic lesions of rat hereditary renal cell tumors express markers of proximal and distal nephron.

Long-Evans (Eker) rats carry a mutation that predisposes them to develop spontaneous renal cell tumors of two morphologic patterns: solid chromophilic masses or cystic lesions lined by eosinophilic cells. Previous studies have suggested that these tumors arise from the proximal tubules. In the present study, lectin-binding characteristics and cytokeratin expression of various stages of hereditary rat renal epithelial neoplasia were examined to localize the portion of the nephron from which tumors arise. Lectin-binding histochemistry has been used as a marker of cell surface glycoprotein expression, thought to be important in the differentiation of benign from malignant epithelial lesions and in the determination of their cell of origin. The presence or absence of keratin intermediate filaments in the rat nephron has been used to identify nephron segments. The polyclonal antibody to high- and low-molecular-weight cytokeratin stained the cells of the collecting ducts but not the proximal or distal tubules. Binding to the proximal tubules by the lectins Conavalia ensiformis (Con A), Dolichas biflorus, Ricinus communis (RCA-1), and Triticum vulgare and to the distal tubules by Con A, RCA-1, Arachis hypogaea (PNA) with and without neuraminidase, and the antibody for cytokeratins was demonstrated. The lectin binding and cytokeratin staining patterns of rat hereditary renal cell carcinoma, adenoma and the preneoplastic lesions of atypical tubules and hyperplasias suggest that cystic adenomas arise from the distal nephron, principally the collecting duct, whereas the solid atypical tubules, hyperplasias, and adenomas arise from the proximal nephron, principally the proximal tubule.