The Role of the IACUC in the Design and Conduct of Animal Experiments that Contribute to Translational Success.

Institutional Animal Care and Use Committees (IACUCs) have a mandated role under the Animal Welfare Act and under Public Health Service Policy to assure the ethical and humane use of research animals in experiments conducted in the United States. The IACUC by virtue of its mandated functions is well positioned to help nurture an institutional culture of optimized animal use since this Committee is often responsible in large part for the culture of animal use that evolves within an institution. In addition to fostering a culture of humane care for research animals and a culture of working with the concepts of the 3Rs (refinement, reduction, replacement), the IACUC can help foster a culture of optimized animal use that encourages high quality reproducible studies that contribute to translational success. In part this is achieved when the IACUC is successful in encouraging interdisciplinary collaboration early and often within the animal use community it serves. Unfortunately in some instances the institutional research community may envisage the IACUC as a bureaucratic burden, regulatory necessity, and compliance tool more than a group that enhances the methodology and quality of animal experiments. A well-functioning IACUC should strive to nurture an institutional culture that places value in enhancing the scientific quality of research to help assure the reproducibility of animal studies and translational success of animal models. This is integral to both high quality science as well as excellence in the supporting animal care and use.

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.

Acute pulmonary toxicity of particulate matter filter extracts in rats: coherence with epidemiologic studies in Utah Valley residents.

Epidemiologic reports by C.A. Pope III et. al. demonstrated that in the Utah Valley, closure of an open-hearth steel mill over the winter of 1987 was associated with reductions in respiratory disease and related hospital admissions in valley residents. To better examine the relationship between plant-associated changes in ambient particulate matter (PM) and respiratory health effects, we obtained total suspended particulate filters originally collected near the steel mill during the winter of 1986 (before closure), 1987 (during closure), and 1988 (after plant reopening). PM subcomponents were water-extracted from these filters and Sprague-Dawley rats were intratracheally instilled with equivalent masses of extract. Data indicated that 24 hr later, rats exposed to 1986 or 1988 extracts developed significant pulmonary injury and neutrophilic inflammation. Additionally, 50% of rats exposed to 1986 or 1988 extracts had increased airway responsiveness to acetylcholine, compared to 17 and 25% of rats exposed to saline or the 1987 extract, respectively. By 96 hr, these effects were largely resolved except for increases in lung lavage fluid neutrophils and lymphocytes in 1986 extract-exposed rats. Analogous effects were observed with lung histologic assessment. Extract analysis using inductively coupled plasma-mass spectroscopy demonstrated in all three extracts nearly 70% of the mass appeared to be sodium-based salts derived from the glass filter matrix. Interestingly, relative to the 1987 extract, the 1986/1988 extracts contained more sulfate, cationic salts (i.e., calcium, potassium, magnesium), and certain metals (i.e., copper, zinc, iron, lead, strontium, arsenic, manganese, nickel). Although total metal content was (3/4) 1% of the extracts by mass, the greater quantity detected in the 1986 and 1988 extracts suggests metals may be important determinants of the pulmonary toxicity observed. In conclusion, the pulmonary effects induced by exposure of rats to water-based extracts of local ambient PM filters were in good accord with the cross-sectional epidemiologic reports of adverse respiratory health effects in Utah Valley residents.

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.

Protein Carbonyls in Bronchoalveolar Lavage Fluid in Mice, Rats and Hamsters Following Inhalation of Pigmentary Titanium Dioxide Particles.

Elevation of protein carbonyls has been implicated in the clinical setting as a result of oxidant damage associated with a number of disease states in both humans and laboratory animals. Protein carbonyls, the product of oxidative modification of amino acid residues, may result from macrophage and neutrophil inflammatory responses to inhaled particles. We hypothesized that increased levels of protein carbonyl groups in the bronchoalveolar lavage fluid (BALF) may serve as a biomarker of oxidative stress in rodents exposed to extremely high airborne concentrations of poorly soluble particles (PSP) of low toxicity. The objective of the present study was to compare the BALF protein carbonyl levels in three rodent species following a subchronic PSP exposure known to result in pulmonary pathology in chronically exposed rats under similar conditions. Female Fischer 344 rats, B6C3F1 mice, and Syrian golden hamsters were identically exposed by whole-body inhalation to concentrations of aerosolized pigmentary titanium dioxide (TiO2)(MMAD and GSD, 1.42 and 1.3 µm, respectively) for 6 h/day and 5 days/wk for 13 wk. Groups of animals were exposed to 0, 10, 50, or 250 mg/m(3) of pigmentary TiO2. Levels of protein carbonyl groups in BALF were measured at the termination of the 13-wk exposure with an ELISA assay utilizing a 2,4-dinitrophenylhydrazine fluorescent probe. Protein carbonyl levels were elevated in rats at both the mid and high dose (50 and 250 mg/m(3)), while in mice and hamster the levels were elevated only at the high dose (250 mg/m(3)). The elevations in protein carbonyl levels paralleled changes in BALF-associated cytologic and biochemical inflammatory indices, including total protein levels and neutrophil counts. Inflammatory changes in all three species were limited to animals exposed to the highest concentrations of particles. Rats were the only species tested that had coincidental elevation of both protein carbonyls and a high inflammatory response measured in BALF following the 50-mg/m(3) exposure. These results suggest that the measurement of protein carbonyl groups in BALF may be a useful biomarker of particle-induced oxidant change, although this endpoint should be used in conjunction with other oxidative endpoints as a total assessment of oxidant stress.

Comparison of Selected Pulmonary Responses of Rats, Mice, and Syrian Golden Hamsters to Inhaled Pigmentary Titanium Dioxide.

We present a preliminary report of a bioassay designed to compare and contrast selected pulmonary responses of female B6C3F1 mice, Fischer 344 rats, and Syrian golden hamsters to inhaled pigmentary titanium dioxide (TiO2). Animals were administered 10, 50, or 250 mg/m(3) TiO2 for 6 h/day and 5 days/wk, for 13 wk. Recovery groups were held for an additional 4-, 13-, or 26-wk period. Following exposure and at each recovery time, TiO2 burdens in the lung and lung-associated lymph nodes were determined. A separate group of animals was used at each time point to assess the inflammatory response of the lung by assaying total protein in bronchoalveolar lavage fluid (BALF) and cytologic examination of cells recovered in BALF. Burdens (mg/mg dry weight) of TiO2 in the lung following exposure to 10, 50, or 250 mg/m(3) TiO2 were 5.2, 53.5, and 170.2 for the mouse; 7.1, 45.1, and 120.4 for the rat; and 2.6, 14.9, and 120.3 for the hamster. With time after exposure, lung burdens of TiO2 particles were decreased and lymph-node burdens increased. Changes in the hamsters' burdens were more rapid than those in mice and rats. Increases in BALF cell numbers (macrophages and neutrophils) and in total protein were observed in all 3 species following exposure to 50 and 250 mg/m(3) TiO2, with the magnitude of response being the grea test in the rat. These responses remained elevated relative to control levels at 26 wk postexposure. Histopathologic examination of lungs showed a concentration-dependent retention pattern of particles that varied by species. Hypertrophy and hyperplasia of alveolar epithelium along with alveolar metaplastic and fibrotic changes were observed in rats exposed to 250 mg/m(3) TiO2. Alveolar epithelial proliferative changes were associated with inflammation in mice and hamsters, but the metaplastic and fibrotic changes noted in rats were not present in similarly exposed mice or hamsters. These data suggest that rats exposed subchronically to extremely high concentrations of pigmentary TiO2 differ from mice and hamsters in their cellular responses in the lung as well as in the way they clear and sequester particles. These differences may partly explain the differential outcome of pulmonary responses in various rodent species following chronic inhalation exposure to poorly soluble particles.

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.

Comparison of pleural responses of rats and hamsters to subchronic inhalation of refractory ceramic fibers.

In the present subchronic study, we compared pleural inflammation, visceral pleural collagen deposition, and visceral and parietal pleural mesothelial cell proliferation in rats and hamsters identically exposed to a kaolin-based refractory ceramic fiber, (RCF)-1 by nose-only inhalation exposure, and correlated the results to translocation of fibers to the pleural cavity. Fischer 344 rats and Syrian golden hamsters were exposed to 650 fibers/cc of RCF-1, for 4 hr/day, 5 days/week for 12 weeks. Following 4 and 12 weeks of exposure, and after a 12-week recovery period, pleural lavage fluid was analyzed for cytologic and biochemical evidence of inflammation. Visceral and parietal pleural mesothelial cell proliferation was assessed by immunocytochemical detection of bromodeoxyuridine incorporation. Pleural collagen was quantitated using morphometric analysis of lung sections stained with Sirius Red. Fiber-exposed rats and hamsters had qualitatively similar pleural inflammation at each time point. Mesothelial cell proliferation was more pronounced in hamsters than in rats at each time point and at each site. In both species, the mesothelial cell labeling index was highest in the parietal pleural mesothelial cells lining the surface of the diaphragm at each time point. Hamsters but not rats had significantly elevated collagen in the visceral pleura at the 12-week postexposure time point. Fibers were found in the pleural cavities of both species at each time point. These fibers were generally short and thin. These results suggest that mesothelial cell proliferation and fibroproliferative changes in the pleura of rodents following short-term inhalation exposure are associated with fiber translocation to the pleura and may be predictive of chronic pleural disease outcomes following long-term exposure.

Effects of immobilization restraint on Syrian golden hamsters.

Rodent nose-only inhalation toxicology systems comprise whole-body immobilization in plastic restraint tubes. This method of restraint is known to have a variety of effects on animals. In the studies reported here, two independent toxicology laboratories examined the effects of inhalation tube restraint in Syrian golden hamsters, a species that has recently gained importance in inhalation studies of fibrous particulates. Body weight, food and water consumption, core body temperature, and plasma cortisol and corticosterone concentrations were assessed in animals immobilized in nose-only inhalation tubes, and the results were compared with those from unrestrained cage-control animals. Animals were immobilized for either 6 h/ day, 5 days/week for 13 weeks (subchronic), or 4 h/day for 14 consecutive days (subacute), mimicking exposure conditions commonly used in nose-only inhalation studies. Tube restraint was found to induce a marked decrease in body weight, which increased in response to cessation of restraint. The body weight decrement was associated with significant differences in food and water consumption between the restrained and control groups in the subacute study and only food consumption in the subchronic study. During the restraint period, core body temperature in the immobilized animals increased slightly but not above the normal range for this species. Plasma cortisol and corticosterone concentrations were not significantly increased with use of restraint, compared with values in controls. Immobilization-associated body weight depression in Syrian golden hamsters is important for the evaluation of nose-only inhalation study results because many normal physiologic parameters, as well as toxicant-induced effects, are associated with body weight status.

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.

Pharmacokinetics of methanol and formate in female cynomolgus monkeys exposed to methanol vapors.

The 1990 Clean Air Act Amendments contain mandates for reduced automotive emissions and add new requirements for the use of alternative fuels such as methanol to reduce certain automotive pollutants. Methanol is acutely toxic in humans at relatively low doses, and the potential for exposure to methanol will be increased if it is used in automotive fuel. Formate is the metabolite responsible for neurotoxic effects of acute methanol exposure. Since formate metabolism is dependent on folate, potentially sensitive folate-deficient subpopulations, such as pregnant women, may accumulate formate and be at higher risk from low-level methanol exposure. Our objective was to determine the pharmacokinetics of 14C-methanol and 14C-formate in normal and folate-deficient monkeys after exposure to 14C-methanol vapors at environmentally relevant concentrations: below the threshold limit value (TLV), at the TLV of 200 parts per million (ppm), and above the TLV. Four normal adult female cynomolgus monkeys were individually anesthetized with isoflurane, and each was exposed by endotracheal intubation to 10, 45, 200, or 900 ppm 14C-methanol for 2 hours. Concentrations of the inhaled and exhaled 14C-methanol, blood concentrations of 14C-methanol and 14C-formate, exhaled 14C-carbon dioxide (14CO2), and respiratory parameters were measured during exposure. After exposure, 14C-methanol and 14CO2 exhaled, 14C-methanol and 14C-formate excreted in urine, and 14C-methanol and 14C-formate in blood were quantified. The amounts of exhaled 14C-methanol and 14CO2, blood concentrations of 14C-methanol and 14C-formate, and 14C-methanol and 14C-formate excreted in urine were linearly related to methanol exposure concentration. For all exposures, blood concentrations of 14C-methanol-derived formate were 10 to 1000 times lower than endogenous blood formate concentrations (100 to 200 mM) reported for monkeys and were several orders of magnitude lower than levels of formate known to be toxic. Since the metabolism of formate in primates depends on the availability of tetrahydrofolate, the same four monkeys were next placed on a folate-deficient diet until folate concentrations in red blood cells consistent with moderate folate deficiency (29 to 107 ng/mL) were achieved. Monkeys were then reexposed to the highest exposure concentration, 900 ppm 14C-methanol, for a similar 2-hour period, and again the pharmacokinetic data described above were obtained. Even with a reduced folate status, monkeys exposed to 900 ppm methanol for 2 hours had peak concentrations of methanol-derived formate that were well below the endogenous levels of formate. Although these results represent only a single exposure and therefore preclude broad generalizations, they do suggest the body contains sufficient folate stores to effectively detoxify small doses of methanol-derived formate from exogenous sources, such as those that might occur during normal use of automotive fuel.