Proposal of a 2-tier histologic grading system for canine cutaneous mast cell tumors to more accurately predict biological behavior.

Currently, prognostic and therapeutic determinations for canine cutaneous mast cell tumors (MCTs) are primarily based on histologic grade. However, the use of different grading systems by veterinary pathologists and institutional modifications make the prognostic value of histologic grading highly questionable. To evaluate the consistency of microscopic grading among veterinary pathologists and the prognostic significance of the Patnaik grading system, 95 cutaneous MCTs from 95 dogs were graded in a blinded study by 28 veterinary pathologists from 16 institutions. Concordance among veterinary pathologists was 75% for the diagnosis of grade 3 MCTs and less than 64% for the diagnosis of grade 1 and 2 MCTs. To improve concordance among pathologists and to provide better prognostic significance, a 2-tier histologic grading system was devised. The diagnosis of high-grade MCTs is based on the presence of any one of the following criteria: at least 7 mitotic figures in 10 high-power fields (hpf); at least 3 multinucleated (3 or more nuclei) cells in 10 hpf; at least 3 bizarre nuclei in 10 hpf; karyomegaly (ie, nuclear diameters of at least 10% of neoplastic cells vary by at least two-fold). Fields with the highest mitotic activity or with the highest degree of anisokaryosis were selected to assess the different parameters. According to the novel grading system, high-grade MCTs were significantly associated with shorter time to metastasis or new tumor development, and with shorter survival time. The median survival time was less than 4 months for high-grade MCTs but more than 2 years for low-grade MCTs.

Tissue culture of human renal epithelial cells using a defined serum-free growth formulation.

BACKGROUND: Development of the culture of renal epithelial cells in a serum-free growth medium was driven by the need to examine the effects of hormones and other effector molecules on differentiated cell function without interference from the complex mixture of substances in serum. The present report details this laboratory's cumulative experience in the use of a defined growth medium for the propagation of epithelial cells from adult, fetal, and malignant human renal tissue. METHODS: Routine cell culture technology was used to determine the capability of a defined growth medium to support the growth of renal epithelial cells isolated by collagenase dissociation of tissue from adult and fetal kidneys, renal cell carcinoma, and Wilms' tumors. RESULTS: The defined growth medium formulation consistently allows the isolation and growth of transporting renal epithelial cells from both normal adult and fetal kidneys. This growth medium only rarely supports the growth of epithelial cells from renal cell carcinomas and Wilms' tumors. CONCLUSIONS: The method developed for the culture of human proximal tubule cells requires minimal cell culture expertise and equipment, and results in the repeatable isolation of transporting epithelial cell cultures that retain features of differentiated proximal tubule cells.

Copper associated acute hepatic failure in a dog.

A 1.5-year-old Dalmatian was examined because of vomiting, weight loss, and high serum activities of alanine aminotransferase and aspartate aminotransferase. Abdominal ultrasonography revealed normal appearing hepatic structure with echogenicity, but histologic examination of hepatic biopsy specimens revealed extensive necrosis of hepatocytes involving the centrilobular areas. Macrophages and remaining hepatocytes contained pigments that were positive for copper by rubeanic acid-staining and hepatic copper concentration was high. The dog was treated with crystalloid fluids, antibiotics, and a low copper diet; its condition deteriorated, and the dog was euthanatized. Primary copper storage disease was suspected on the basis of histologic findings and high copper concentration in the liver.

Differential activity of aspirin, ketoprofen and sulindac as cancer chemopreventive agents in the mouse urinary bladder.

In vivo studies were conducted to compare the activity of three non-steroidal anti-inflammatory drugs as inhibitors of urinary bladder carcinogenesis induced in B6D2F1 (BDF) mice by N-butyl-N-(4-hydroxybutyl)nitrosamine (OH-BBN). Mice received continuous dietary exposure to non-toxic doses of aspirin, sulindac or ketoprofen beginning 1 week prior to the first of eight weekly doses of 7.5 mg OH-BBN; studies were terminated at 24 weeks after the first carcinogen dose. Both dose levels of sulindac (200 and 400 mg/kg diet) and both dose levels of ketoprofen (40 and 80 mg/kg diet) reduced the incidence of transitional cell carcinoma of the urinary bladder by >70% from that seen in dietary controls. The high dose of sulindac conferred the greatest protection against bladder cancer induction. In contrast, when administered at 400 and 800 mg/kg diet aspirin was inactive as a chemopreventive agent in the OH-BBN/BDF bladder cancer model. The significant potency of sulindac and ketoprofen as inhibitors of urinary bladder carcinogenesis, when considered with their history of safe human use, suggests that these agents merit further study as drugs for cancer chemoprevention in this target tissue.

Chemoprevention of MNU-induced mammary tumorigenesis by hormone response modifiers: toremifene, RU 16117, tamoxifen, aminoglutethimide and progesterone.

The effects of structurally different antiestrogens, progesterone and the aromatase inhibitor aminoglutethimide, were evaluated for chemopreventive activity in the N-methyl-N-nitrosourea (MNU)-induced mammary carcinogenesis model. Treatment with either RU 16117, progesterone or aminoglutethimide resulted in a significant decrease in cancer multiplicity [> or = 50%; P < .05] when administered individually at doses 80% of the maximally tolerated dose [MID]. Toremifene was also remarkably effective in inhibiting MNU-induced mammary tumorigenesis although this inhibition was achieved at a dose which caused a significant decrease in body weight gain. Aminoglutethimide, RU 16117 and toremifene citrate, in addition to their effects on tumor multiplicity, caused significant increases in the latency period for tumor development. Combinations of aminoglutethimide, progesterone and/or a suboptimal dose of tamoxifen citrate also proved to be effective in inhibiting the development of MNU-induced mammary cancers; however, the combination regimen was no more effective than either aminoglutethimide or progesterone administered alone. These results suggested that agents altering the hormonal environment, regardless of their mechanism of action, may provide protection against the development of hormone responsive mammary cancer.

Chemoprevention of OH-BBN-induced bladder cancer in mice by oltipraz, alone and in combination with 4-HPR and DFMO.

The chemopreventive efficacy of the schistosomicidal drug oltipraz (5-(2-pyrazinyl)-4-methyl-1,2-dithiol-3-thione) was evaluated against urinary bladder transitional cell carcinoma (TCC) induced in male C57BL/6 x DBA/2FI (BDF) mice by N-butyl-N(4-hydroxybutyl)nitrosamine (OH-BBN). Oltipraz was fed in the diet from one week prior to OH-BBN dosing until sacrifice, six months later. The agent at 250 mg/kg diet significantly reduced the incidence of TCC compared with that in carcinogen controls. Oltipraz also significantly reduced TCC incidence when fed at 500 mg/kg diet for 76 days, then at 125 mg/kg diet until the end of the test period. Treatment with this higher dose level of oltipraz also appeared to decreases the depth of tumor invasion. At lower dose levels of 100 and 200 mg/kg diet, oltipraz alone had no effect on tumor incidence. It also was tested at these dose levels in combinations with 2-difluoromethylornithine (DFMO) and with all-trans-N-(4-hydroxyphenyl)retinamide (4-HPR). Treatment with the combination of 640 mg DFMO/kg and 100 mg oltipraz/kg diet was efficacious, although DFMO alone at 640 mg/kg diet was inactive. The combination of 1280 mg DFMO/kg and 200 mg oltipraz/kg diet reduced TCC incidence significantly compared with carcinogen controls, but the effect was no greater than that of DFMO alone at 1280 mg/kg, and weight gain was suppressed compared with carcinogen controls. The depth of tumor invasion was decreased with this combination treatment. Combinations of oltipraz at 100 and 200 mg/kg diet, 4-HPR at 156 and 313 mg/kg diet, and DFMO at 640 and 1280 mg/kg diet were efficacious and without apparent toxicity. Nonetheless, the three agent combinations cannot be considered more effective than DFMO alone at 1280 mg/kg diet or the lower dose combination of oltipraz and DFMO.

Expression patterns of loricrin in various species and tissues.

In this study we analyzed the expression patterns of loricrin in various species and tissues using immunohistochemistry, immunoblotting and Northern blots. Loricrin is a glycine-, serine- and cysteine-rich protein expressed very late in epidermal differentiation in the granular layers of normal mouse and human epidermis. Later on in differentiation, loricrin becomes crosslinked as a major component into the cornified cell envelope by the formation of N epsilon-(gamma-glutamyl)lysine isopeptide bonds. This process either occurs directly or by the intermediate accumulation in L-keratohyaline granules of mouse epidermis and human acrosyringia. Loricrin was identified in all mammalian species analyzed by virtue of its highly conserved carboxy-terminal sequences revealing an electric mobility of approximately 60 kDa in rodents, rabbit and cow and of approximately 35 kDa in lamb and human on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Loricrin is expressed in the granular layer of all mammalian orthokeratinizing epithelia tested including oral, esophageal and fore-stomach mucosa of rodents, tracheal squamous metaplasia of vitamin A deficient hamster and estrogen induced squamous vaginal epithelium of ovary ectomized rats. Loricrin is also expressed in a few parakeratinizing epithelia such as BBN [N-butyl-N-(4-hydroxybutyl)nitrosamine]-induced murine bladder carcinoma and a restricted subset of oral and single vaginal epithelial cells in higher mammals. Our results provide further evidence that the program of squamous differentiation in internal epithelia of the upper alimentary tract in rodents and higher mammals differ remarkably. In addition, we also have noted the distinct distribution patterns of human loricrin and involucrin, another major precursor protein of the cornified cell envelope.

Chemoprevention of OH-BBN-induced bladder cancer in mice by piroxicam.

Piroxicam inhibited induction of transitional cell carcinoma in mouse urinary bladder by N-butyl-N-(4-hydroxybutyl)-nitrosamine. At 15 mg piroxicam/kg diet, tumor incidence was reduced 82% (P < 0.0001) compared with carcinogen controls. At 30 mg piroxicam/kg diet, tumor incidence was reduced 70% (P < 0.001). Results at the higher dose level suggested that piroxicam also may have inhibited invasion slightly. Combination treatment with 2-difluoromethyl-ornithine (DFMO) or all-trans-N-(4-hydroxyphenyl)retinamide (4-HPR) or both agents did not improve the chemopreventive potential of piroxicam. However, the three-agent combination of 30 mg piroxicam/kg, 1200 mg DFMO/kg and 313 mg 4-HPR/kg diet was highly effective. Tumor incidence was reduced 91% (P < 0.0001) compared with carcinogen controls. Unfortunately, the high efficacy was somewhat compromised by a significant decrease in survival and body weight gain in mice receiving the combination of agents compared with the carcinogen control.

Chemoprevention of MNU-induced mammary tumors in the mature rat by 4-HPR and tamoxifen.

The chemopreventive efficacies of the retinoid all-trans-N-(4-hydroxyphenyl)-retinamide (4-HPR) and the anti-estrogen tamoxifen citrate were evaluated against N-methyl-N'-nitrosourea (MNU) induced mammary cancer in 120-day old female Sprague-Dawley rats. The agents were tested alone and in combination. They were administered in a modified AIN-76A diet, beginning 60 days prior to a single i.v. dose of 50 mg MNU/kg-bw and continuing until the end of the study, 180 days post-carcinogen treatment. At 782 mg/kg diet, 4-HPR alone significantly inhibited the induction of mammary adenocarcinomas compared with carcinogen controls. At 0.250 mg/kg diet, tamoxifen alone reduced tumor incidence compared with carcinogen controls. At 0.125 mg/kg diet, tamoxifen was ineffective. Combinations of 782 mg 4-HPR/kg diet with either 0.250 or 0.125 mg tamoxifen/kg diet were effective in inhibiting MNU-induced adenocarcinomas. The reductions in tumor incidence were greater for these combinations than for either agent alone. 4-HPR and 0.250 mg tamoxifen/kg diet decreased tumor incidence 81% (p less than 0.005), whereas 4-HPR and 0.125 mg tamoxifen/kg diet decreased tumor incidence 72% (p less than 0.005) compared with carcinogen controls. The combination of 391 mg 4-HPR/kg diet and 0.500 mg tamoxifen/kg diet was also tested and was effective in reducing tumor incidence.

Animal models for chemoprevention of respiratory cancer.

Of the several models for lung carcinogenesis, two appear appropriate for chemoprevention studies based upon dose response, tumor type, and tumor localization. One model utilizes the direct-acting carcinogen methylnitrosourea (MNU), and the other utilizes a carcinogen (diethylnitrosamine) requiring metabolic activation. Tumors appear rapidly in both models (within 6 months), and the model systems are responsive to modulation by several classes of potential chemopreventive agents. For example, the retinoid N-(4-hydroxyphenyl) retinamide reduces the incidence of lung adenosquamous carcinoma, but retinol or beta-carotene are ineffective when administered alone. However, concomitant administration of these compounds reduces the incidence of non-neoplastic dysplasias as well as adenosquamous carcinomas of the lung. In the MNU system, retinoids in general have been ineffective in reducing the incidence of tracheobronchial squamous-cell carcinomas.

Retinoids as chemopreventive agents for breast cancer.

Although several retinoids have been evaluated for prevention of mammary carcinogenesis in rats and mice, retinyl acetate (RA) and N-(4-hydroxyphenyl)retinamide (4-HPR) proved most effective. In rats, dietary administration of the retinoids reduced the incidence and number, and increased the latency of N-methyl-N-nitrosourea (MNU)-induced mammary cancers. 4-HPR reduced the number of hyperplastic alveolar nodules (HAN) in MTV- mice and the number of tumors in MTV+ mice. Other studies indicate that the synergistic effect of retinoid administration and hormonal deprivation is more efficacious in prevention of MNU-induced mammary cancer than either modality alone. Furthermore, retinoids alone and the combination of retinoid and tamoxifen inhibit the appearance of mammary cancers following the surgical removal of the first cancer as well as inhibit the growth of established cancers. Again, the combined modality was the most effective. Retinoids also exert an antiproliferative effect upon the mammary epithelium in vivo, which is represented morphologically by a bare duct system with little branching, end buds, and few, if any, alveoli. In organ culture, retinoids inhibit mammary end bud differentiation and proliferation induced by insulin and prolactin or carcinogens.

Chemoprevention of experimental bladder cancer.

The chemopreventive efficacy of several compounds was evaluated in the N-butyl-N-(4-hydroxybutyl)nitrosamine (OH-BBN)-induced urinary bladder cancer model using C57BL/6 x DBA/2F1 (BDF) male mice. Compounds were administered in a defined semipurified diet (AIN-76-A) either as single agents or in combination. As single agents and at the doses employed, 2-alpha-difluoromethylornithine (DFMO), piroxicam, oltipraz, and sodium molybdate effectively inhibited the incidence of transitional cell carcinoma (TCC). 4-Hydroxyphenyl retinamide (4-HPR) was ineffective. Body weight gain and survival was not affected by the doses of agents used. Combinations of two agents which increased efficacy were 4-HPR+DFMO, DFMO+piroxicam, 4-HPR+oltipraz, and DFMO+oltipraz. Three-agent combinations which showed enhanced efficacy against TCC induction were 4-HPR+Na molybdate+DFMO, 4-HPR+DFMO+piroxicam, and 4-HPR+DFMO+oltipraz. Although the three-agent combinations were, for the most part, no more effective than the two-agent combinations at the doses employed, all combination regimens significantly reduced bladder cancer incidence even when single agent administration did not.

Inhibition of rat mammary gland chemical carcinogenesis by dietary dehydroepiandrosterone or a fluorinated analogue of dehydroepiandrosterone.

The chemopreventive efficacy of p.o. administered dehydroepiandrosterone (DHEA), DHEA plus N-(4-hydroxyphenyl)retinamide (4-HPR), or 16 alpha-fluoro-5-androsten-17-one (DHEA analogue 8354) was examined in rats treated with N-methyl-N-nitrosourea (MNU; 50 mg/kg body weight, i.v.) at 50 days of age. Semipurified diet (AIN-76A) containing each steroid alone, or DHEA plus 4-HPR, was administered during initiation (-1 week to +1 week post-MNU), promotion/progression (+1 week post-MNU to termination), or both phases (-1 week post-MNU to termination) of the carcinogenic process. Neither DHEA nor DHEA analogue 8354 (0.2%, w/w) significantly affected the initiation of mammary cancer when administered alone; however, DHEA (0.2%, w/w) plus 4-HPR (1 mmol/kg diet) significantly reduced cancer multiplicity (26%) when given during initiation. All three treatments were strongly effective when given during promotion/progression, significantly reducing mammary cancer multiplicity by 77% (DHEA), 84% (DHEA/4-HPR), and 66% (DHEA analogue 8354), relative to carcinogen controls. Cancer incidence was significantly inhibited by DHEA (33% inhibition) and DHEA/4-HPR (24% reduction) during promotion/progression. However, the most effective chemopreventive treatment encompassed both phases of carcinogenesis. Thus, under these conditions, DHEA (0.2% or 0.1%, w/w) reduced cancer incidence (52% and 32% reductions, respectively) and multiplicity (91% and 86% reductions, respectively). Further reduction in mammary cancer incidence was observed in animals that received DHEA (both doses) plus 4-HPR (1 and 0.5 mmol/kg diet, respectively). DHEA analogue 8354 (0.2% or 0.1%, w/w) given for the duration of the study reduced only cancer multiplicity (61% and 56% reductions, respectively). Tumor-related mortality was significantly lower in rats that received long-term treatment with DHEA or DHEA/4-HPR, when compared with carcinogen controls. Except for a slight, but significant, postcarcinogen decrease in the mean body weights of rats treated concomitantly with DHEA (plus or minus 4-HPR) and MNU, additional gross manifestations of steroid-induced toxicity were not observed.

Respiratory carcinogenesis of nitroaromatics.

The carcinogenic potential of 1-nitropyrene, a major mutagenic constituent of diesel-exhaust particles, was investigated using a hamster respiratory-carcinogenesis model. The specific aims of the investigation were to assess the activity of 1-nitropyrene as a complete carcinogen (Study 1) and as a cocarcinogen (Study 2) when administered in combination with the known environmental carcinogen benzo[a]pyrene. Preparations of 1-nitropyrene and benzo[a]pyrene adsorbed onto an equal mass of carbon carrier particles (Stokes diameter 2 to 5 microns, greater than 70 percent) were used for the intratracheal administration. Evaluation of 1-nitropyrene as a complete carcinogen involved exposing male Syrian golden hamsters to 1 or 2 mg of 1-nitropyrene either once or twice each week. A once-per-week instillation of 2 mg of benzo[a]pyrene served as a positive control. Two groups of animals received sterile saline only (saline controls) or carbon particles suspended in saline (particle controls). In addition, a group of untreated hamsters served as shelf controls. Evaluation of 1-nitropyrene as a cocarcinogen involved treating animals once each week with either 1 or 2 mg of 1-nitropyrene with or without concomitant exposure to 0.25 mg of benzo[a]pyrene. The studies were terminated after 92 weeks of treatment. In both studies, hamsters receiving 1-nitropyrene showed a dose-related decrease in survival and body-weight gain. In general, animals in Study 2 showed better survival than those in Study 1. A high intercurrent mortality was observed in the control groups of Study 1. In order to adjust for intercurrent mortality, tumor incidences were analyzed after modeling (using Cox regression) the effect of treatment in all animals for the period they were alive. A broad spectrum of neoplastic and nonneoplastic lesions was observed in the lungs and tracheas of all hamsters except shelf controls. Because of the histologic complexity of these lesions, the slides were coded, and the tissues were evaluated by an unbiased pathologist. The tumor types included papillomas, adenomas, adenocarcinomas, and squamous cell carcinomas, the latter being the most prevalent type in benzo[a]pyrene-treated animals. In view of the low incidence of tumors in 1-nitropyrene-treated hamsters, the tumor incidences in various experimental groups were compared regardless of the tumor type, location, and multiplicity. A small, but significant, increase in tumor incidence, with a dose-response trend, was observed only in Study 2 hamsters receiving 1-nitropyrene once weekly. In contrast, treatment with benzo[a]pyrene adsorbed onto carbon particles induced benign and malignant tumors virtually in all animals that survived more than 50 weeks of treatment.(ABSTRACT TRUNCATED AT 400 WORDS)

Interspecies analysis of the chemopreventive efficacy of dietary alpha-difluoromethylornithine.

The anticarcinogenic efficacy of the polyamine biosynthesis inhibitor, alpha-difluoromethylornithine (DFMO), was assessed in three rodent models of human epithelial cancer. In DMBA-induced female, Sprague-Dawley rats, DMFO treatment (3.2 or 6.4 g/kg diet) for 180 days significantly inhibited mammary carcinogenesis and reduced tumor-related intercurrent mortality compared to untreated controls. In male, C57BL/6x DBA/2F1 mice induced with N-butyl-N(4-hydroxybutyl)nitrosamine (OH-BBN), DFMO treatment (2 or 4 g/kg diet) concurrent with the period of carcinogen administration significantly reduced the incidence and severity of urinary bladder carcinomas. In methylnitrosourea (MNU)-induced male Syrian golden hamsters, DFMO (3.2 g/kg diet) numerically reduced the incidence and size of tracheal carcinoma relative to untreated controls. DFMO-mediated toxicity was not evident in any of the animals on study, although a slight reduction in mean body weight gain was evident in rats and mice.

Chemopreventive efficacy of combined retinoid and tamoxifen treatment following surgical excision of a primary mammary cancer in female rats.

Dietary N-(4-hydroxyphenyl)retinamide (4-HPR; 3 mmol/kg diet) and s.c. injections of the antiestrogen, tamoxifen (Tx; 10 micrograms or 20 micrograms per rat, thrice weekly) were used together as adjunct chemopreventive therapy in groups of 39-40 female, Sprague-Dawley rats that each received an i.v. injection (50 mg/kg b.w.) of the mammary gland carcinogen N-methyl-N-nitrosourea (MNU). Treatment was started immediately following the surgical excision of the first (primary) mammary carcinoma from each MNU-treated rat and was continued for 180 days. When compared to the effect of treatment with 4-HPR or Tx (30 micrograms/wk) alone, the combination treatments significantly enhanced terminal survival and reduced nonrecurrent mammary cancer incidence and multiplicity. Data showing the incidence of rats bearing the first through fifth additional cancers to appear following surgical resection of a primary lesion demonstrate that combined treatment with 4-HPR/Tx was immediately and consistently more efficacious than either agent per se in suppressing subsequent tumor appearance. This effect was apparently related to the dose of Tx. These results suggest that combined treatment with 4-HPR/Tx is superior to that of either agent alone in blocking progression of incipient neoplastic lesions at both early and later stages of the process.

Elevated glucose alters paracellular transport of cultured human proximal tubule cells.

Cultures of human proximal tubule cells were exposed to elevated concentrations of glucose and dome formation was assessed over a 22 day period of growth. Cultures grown on 5.5 mM glucose formed five domes per microscopic field while those exposed to elevated glucose concentrations (11.0 mM to 27.5 mM) formed only two to three domes per field. The areas of the domes formed by the cells grown on elevated glucose concentrations were reduced as compared to those formed on 5.5 mM glucose. An analysis of the electrical properties of cells grown on elevated glucose concentrations by Ussing chamber technique disclosed a marked reduction in potential difference, short circuit current, and resistance compared to cells grown on 5.5 mM glucose. Routine ultrastructural analysis disclosed that cells grown on elevated glucose concentrations appeared to have fewer tight junction complexes. Further examination utilizing freeze fracture methodology demonstrated that cells grown on elevated glucose concentrations averaged two to three sealing strands per junction as compared to an average of five sealing strands for cells grown on 5.5 mM glucose. The cells grown on elevated glucose concentrations were also noted to possess a greater number of gap junctions. These results demonstrate that elevated glucose concentrations can alter the paracellular route, and possibly the transcellular route, of transport regulation in cultured human proximal tubule cells.

Electrophysiology and ultrastructure of cultured human proximal tubule cells.

The present study was undertaken to determine if cultures of human proximal tubule cells would retain in vivo properties inherent to this segment in the intact nephron. Ussing chamber studies demonstrated that these cultured cells generated transepithelial potential differences of approximately -2.0 mV and resistances of 0.310 K omega.cm2, supporting the concept that the proximal tubule is a "leaky" epithelium. The electrical properties did not change when the cells were exposed to amiloride (10(-4) M), but did respond to acetazolamide (10(-4) M), consistent with responses known to occur in proximal tubules. Ultrastructural analysis of these cells demonstrated features indicative of proximal tubule cells. When grown on permeable supports, where apical and basolateral growth medium compartments were maintained separate from each other, the cells were noted to undergo increased differentiation with morphological evidence of cell polarity. Freeze fracture analysis demonstrated well-formed tight junction strands and segregation of unique numbers of intramembranous particles in apical, lateral, and basal membranes. The replicas also demonstrated the presence of aggregates though to represent gap junctions, structures which occur exclusively in the proximal segment of the human nephron. These observations provide evidence that this human cell culture model originates from the human proximal tubule and retains, in culture, many of the properties associated with proximal tubule cell function and structure in vivo.

Potentiation of vitamin A hepatotoxicity by butylated hydroxytoluene.

The interaction between the natural vitamin A ester retinyl acetate (RA) and the phenolic antioxidant butylated hydroxytoluene (BHT) in the induction of biliary hyperplasia and hepatic fibrosis in female Sprague-Dawley rats was characterized. Using a 3 X 3 matrix design, rats were fed diets supplemented with (per kilogram diet) 0, 125, or 250 mg RA and/or 0, 2500, or 5000 mg BHT. The 125-mg dose of RA induced no gross hepatotoxicity, while the 250-mg dose of RA induced a low incidence of hepatic fibrosis in rats examined after 120 and 180 days of exposure. Exposure to BHT alone induced hepatocellular hypertrophy and dose-related increases in liver weight, but no hepatocellular pathology. Simultaneous administration of RA plus BHT resulted in significant increases in the incidence of biliary hyperplasia and hepatic fibrosis compared to that induced by RA alone. BHT reduced total hepatic vitamin A content at all RA dose levels. Thus, mechanisms other than increases in liver vitamin A levels must underlie the potentiation by BHT of RA hepatotoxicity.