The objective of this study was to characterize the renal toxicity and carcinogenicity of p-nitrobenzoic acid in F344 rats. Dose levels in 13-week and 2-year studies ranged from 630-10,000 ppm and 1,250-5,000 ppm, respectively. At 13 weeks, renal lesions included minimal to mild hyaline droplet accumulation in male rats and karyomegaly in male and female rats. At 2 years, renal lesions included proximal tubule epithelial cell hyperplasia in male rats and oncocytic hyperplasia in high-dose male and female rats, and a decreased severity of nephropathy in males and females. The hvaline droplets in renal tubular epithelial cells of male rats at 13 weeks were morphologically similar to those described in alpha2u-globulin nephropathy. Using immunohistochemical methods, alpha2u-globulin accumulation was associated with the hyaline droplets. In addition, at 13 weeks, cell proliferation as detected by PCNA immunohistochemistry was significantly increased in males exposed to 5,000 and 10,000 ppm when compared to controls. Cytotoxicity associated with alpha2U-globulin nephropathy such as single-cell necrosis of the P2 segment epithelium or accumulation of granular casts in the outer medulla did not occur in the 13-week study. In addition, chronic treatment related nephrotoxic lesions attributed to accumulation of alpha2u-globulin such as linear foci of mineralization within the renal papilla, hyperplasia of the renal pelvis urothelium and kidney tumors were not observed. Although there was histologic evidence of alpha2u-globulin accumulation in male rats at 13 weeks, the minimal severity of nephropathy suggests that the degree of cytotoxicity was below the threshold, which would contribute to the development of renal tumors at 2 years.

Isoprene is the 2-methyl analog of 1,3-butadiene, a genotoxic and carcinogenic compound in rats and mice. Male B6C3F1 mice were exposed to 0, 2200 or 7000 ppm isoprene by inhalation (6 h/day; 5 days/week) for 26 weeks. Following a 26-week recovery period, an increased incidence of Harderian gland (HG) neoplasms was observed at both concentrations. The present study was designed to characterize genetic alterations in the K-ras and H-ras protooncogenes in HG neoplasms. Mutations in K-ras and H-ras were identified by single-strand conformational analysis and direct sequencing of polymerase chain reaction (PCR) amplified DNA, isolated from paraffin-embedded sections of HG neoplasms. A higher frequency of ras mutations, in particular K-ras mutations, was detected in isoprene-induced neoplasms than in 1,3-butadiene-induced or control HG neoplasms. All of the isoprene-induced HG neoplasms exhibited activated K-ras (60%) or H-ras (40%) mutations. In contrast, ras mutations were detected in 69% of HG neoplasms from 1,3-butadiene exposed mice (14% K-ras and 55% H-ras) and in 56% of HG neoplasms obtained from control B6C3F1 mice (8% K-ras and 48% H-ras). The predominant mutations in isoprene-induced HG neoplasms, but not in previously or newly analysed 1,3-butadiene-induced HG neoplasms, consisted of A-->T transversions (CAA-->CTA) at K-ras codon 61 (15/30) and C-->A transversions (CAA-->AAA) at H-ras codon 61 (8/30). Two-thirds of the K-ras CTA mutations were detected in HG neoplasms from the 2200 ppm exposure group while one-third was present in the 7000 ppm group. Isoprene-induced HG neoplasms with K-ras or H-ras mutations had an elevated proliferating cell nuclear antigen (PCNA) index, compared to spontaneous HG neoplasms without ras mutations. The high frequency and specificity of the ras mutation profile suggest that ras protooncogene activation contributes to isoprene-induced HG tumorigenesis.

The Predictive-Toxicology Evaluation (PTE) project conducts collaborative experiments that subject the performance of predictive-toxicology (PT) methods to rigorous, objective evaluation in a uniquely informative manner. Sponsored by the National Institute of Environmental Health Sciences, it takes advantage of the ongoing testing conducted by the U.S. National Toxicology Program (NTP) to estimate the true error of models that have been applied to make prospective predictions on previously untested, noncongeneric-chemical substances. The PTE project first identifies a group of standardized NTP chemical bioassays either scheduled to be conducted or are ongoing, but not yet complete. The project then announces and advertises the evaluation experiment, disseminates information about the chemical bioassays, and encourages researchers from a wide variety of disciplines to publish their predictions in peer-reviewed journals, using whatever approaches and methods they feel are best. A collection of such papers is published in this Environmental Health Perspectives Supplement, providing readers the opportunity to compare and contrast PT approaches and models, within the context of their prospective application to an actual-use situation. This introduction to this collection of papers on predictive toxicology summarizes the predictions made and the final results obtained for the 44 chemical carcinogenesis bioassays of the first PTE experiment (PTE-1) and presents information that identifies the 30 chemical carcinogenesis bioassays of PTE-2, along with a table of prediction sets that have been published to date. It also provides background about the origin and goals of the PTE project, outlines the special challenge associated with estimating the true error of models that aspire to predict open-system behavior, and summarizes what has been learned to date.

Large granular lymphocyte (LGL) leukemia commonly occurs in the Fischer-344/N rat. The high spontaneous incidence complicates the interpretation of results from chronic carcinogenicity studies that use this rat strain. As a result, a comprehensive characterization of LGL leukemia is necessary to help understand the leukemogenic process and the applicability of staging for assessing the progression of this disease. In the current study, the proliferation rate of LGL leukemia cells from untreated control Fischer-344 (F-344) rats in 3 stages of leukemia compared to nonleukemic age-matched rats was determined by immunohistochemical staining for proliferating cell nuclear antigen (PCNA). In histologic sections of spleen from aged F-344/N rats affected by LGL leukemia, there was a significant increase of both PCNA labeling and mitotic indices that was most advanced in the spleen of rats with more severe LGL leukemia. These results support biological significance for the morphologic staging system currently in use.

The National Toxicology Program recently completed longterm ozone inhalation studies in B6C3F1 mice and F344/N rats. Mice and rats were exposed to 0, 0.5 or 1.0 p.p.m. ozone by inhalation for 24 or 30 months. There was an increased incidence of lung neoplasms in B6C3F1 mice. However, there was no evidence of carcinogenicity in F344/N rats. The objectives of this study were to (i) evaluate benign and malignant lung neoplasms from B6C3F1 mice for mutations in the K-ras gene at codons 12, 13 and 61, (ii) determine if the frequency and spectra of K-ras mutations were unique for ozone-induced lung neoplasms, (iii) determine if specific K-ras mutations were associated with the size and morphological patterns of lung neoplasms or ozone exposure concentrations and (iv) screen lung neoplasms by immunohistochemical methods for the p53 protein. K-ras mutations were detected by single-strand conformation analysis and identified by direct sequencing of polymerase chain reaction-amplified DNA isolated from formalin-fixed, paraffin-embedded neoplasms. K-ras mutations were detected in 73% of ozone-induced neoplasms, as compared with 33% of lung neoplasms from controls. The predominant mutations consisted of A-->T transversions at codon 61 (8/19) and G-->T transversions at codon 12 (7/19). Specific K-ras mutations in lung neoplasms were not associated with various morphological patterns. Our data suggests that ozone may cause direct and/or indirect DNA damage in the K-ras proto-oncogene of B6C3F1 mice.

The role of O6‐methylguanine (O6MG) DNA adduct formation and persistence in the formation of 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanone (NNK)—induced lung tumors from resistant C57BL/6 and susceptible A/J mice was investigated. In addition, the frequencies of pulmonary tumor formation and Ki‐ras activation were defined in C57BL/6 mice treated with NNK or vinyl carbamate (VC), and the role of the p53 gene in pulmonary carcinogenesis in these resistant mice was examined. One day after treatment with 100 mg/kg NNK, O6MG adduct concentrations were twofold to eightfold higher in Clara cells and type II cells than in small cells or whole lungs from both mouse strains. The concentrations of O6MG in isolated cells decreased at a similar rate in the two strains of mice. Lung tumors were detected by 27 mo of age in 18% of the C57BL/6 mice after a single 100 mg/kg dose of NNK and in 46% of these mice after a single 60 mg/kg dose of VC. In contrast, the tumor incidence in untreated C57BL/6 mice was 4%. Only one of 22 lung tumors from C57BL/6 mice treated with NNK contained an activated Ki‐ras gene that was associated with an O6MG DNA adduct, whereas previous studies detected activated Ki‐ras oncogenes in most of the NNK‐induced lung tumors analyzed from susceptible A/J and resistant C3H mice. The small differences in formation and persistence of the O6MG adduct in whole lung or isolated lung cells from A/J and C57BL/6 strains do not account for the differences in either susceptibility for tumor formation or activation of the Ki‐ras gene between these strains. In contrast to the low number of NNK‐induced tumors with Ki‐ras mutations in the resistant mice, 11 of 20 lung tumors from VC‐treated mice contained activated Ki‐ras genes. Neither p53 tumor suppressor gene mutations nor overexpression of the p53 protein were detected in spontaneous or chemically induced lung tumors in C57BL/6 mice. Thus, although Ki‐ras activation was detected in some tumors, pathways independent of ras activation and p53 inactivation also appear to be involved in lung tumorigenesis in this resistant mouse strain.

An enhanced immunohistochemical procedure to detect proliferating cell nuclear antigen (PCNA), an endogenous cell replication marker, has been successfully applied to formalin-fixed, paraffin-embedded archival rat and mouse tissues. The procedure involves microwave oven heating of tissue sections in a commercially available antigen retrieval solution of heavy metal salts. Successful immunohistochemical staining of PCNA can be consistently obtained in tissues fixed for over 24 months in formalin and in sections made from paraffin blocks stored in our tissue archives for up to 19 months. Use of this technique will allow retrospective staining of rodent tissues for identification of S phase cells as an indication of DNA replicative activity in previously conducted toxicity and carcinogenicity studies.

An automated method for lectin staining using the Fisher Code-On™ series, with a modification of its general program, “Immunocode,” is described. The lectin wheat germ agglutinin (WGA) was used for staining procedures. Comparisons were made between the lectin staining quality of formalin- vs modified B5-fixed lung tissue, using manual and automated staining techniques. Although automated and manual methods for lectin staining were comparable, the results obtained by automated staining were highly reproducible and less subject to inconsistencies due to human error. Automated staining also decreased consumption of costly reagents and saved laboratory personnel time (~3.0 hr, automated vs 4.8 hr, manually). The intensity and discreteness of WGA staining was superior in the modified B5- vs formalin-fixed lung tissues with both automated and manual staining methods. (The J Histotechnol 14:149, 1991)

The microscopic and ultrastructural characteristics of spontaneous pulmonary neoplasms in strain A (strA) mice are described. Fifty-one spontaneous lung tumors were identified in 34 out of 57, 11-23-month-old male strA/Hen mice. Grossly, all tumors appeared as yellow-white, discrete nodules ranging in size from 1.0-10 mm. Tumor types were randomly distributed throughout the lung; however, the right lung lobes were most frequently involved. Histologically, tumors were classified as adenoma (34/51) or carcinoma (17/51) as defined by standard histopathologic criteria. Adenomas were usually less than 4 mm in diameter and had solid (16/34), papillary (10/34), or mixed (8/34) histologic growth patterns. Carcinomas were usually greater than 4 mm in diameter and had papillary (13/17) or mixed (4/17) histologic growth patterns. Ultrastructurally, benign tumors consisted of solid or papillary areas of neoplastic type II-like cells. Cells comprising malignant tumors had varying ultrastructural characteristics ranging from well-differentiated alveolar cell types to undifferentiated cells having intracytoplasmic osmiophilic dense bodies, vacuoles, or few specialized organelles commonly observed in mature nonneoplastic pulmonary epithelial cells.

The application of the collagenase portal vein perfusion technique for the isolation of intestinal cells resulted in the preparation of highly viable enterocytes. Cell viability was found to be greater than 90% as tested by LDH release and Trypan blue exclusion techniques. According to the results of marker enzyme determinations, collected cells were mostly of matured villus type, characterized by high disaccharidase and very low thymidine kinase activity. In vitro treatment of the isolated cells with the anticancer agent cis-diamminedichloroplatinum (II) caused decrease of the metabolic processes, i.e. glucose oxidation and protein synthesis, demonstrating that beyond the production of DNA-crosslinks other mechanisms may play a role in the cytotoxic effect of the drug. It should be stressed, however, that prolonged incubation of the cell suspension over 30 min at physiological temperature may itself lead to gradual decrease of the viability and to disturbance of the metabolic activity of the cells.

Intestinal cells were isolated from male Fischer 344 rats by the collagenase portal vein perfusion procedure and evaluated for direct effects of cis-diamminedichloroplatinum (CDDP) and ethylacrylate (EtAc) on metabolic activities. Specific activities of marker enzymes of intestinal crypt and villus cells indicated that the preparations contained predominantly villus cells. Cell viability was generally greater than 90%, and was maintained longest when the cells were suspended in M-199 medium supplemented with 1% BSA. EtAc, an industrial intermediate which is toxic to tissues which are directly exposed to this chemical, had no apparent effect on rates of glucose metabolism or protein synthesis in suspensions of the isolated intestinal cells. These metabolic processes, however, were inhibited by the anticancer agent, CDDP; the mechanism of cytotoxicity of CDDP may therefore be due to interference with intermediary metabolism. The present studies indicate that isolated intestinal cell suspensions may be useful in examining direct and immediate effects of chemicals which are toxic to the intestinal epithelium, and in evaluating potential cytotoxic effects of CDDP analogs which have been developed.

Gelatin-sorbitol microcapsules containing 44.1% trichloroethylene (TCE) were prepared and mixed in NIH-07 rodent meal diet and provided at microcapsule concentrations of 0 (untreated control group), 1.25, 2.5, 5.0, or 10% (equivalent to 0, 0.55, 1.10, 2.21, or 4.41% TCE, respectively) to groups of 10 male F344 rats for 14 days. An additional control group received diets containing 5% empty capsules. For comparisons, TCE dissolved in corn oil was administered by gavage to different groups of 10 male rats for 14 consecutive days at dose levels adjusted to correspond to those in the feed study. Treatment-related deaths occurred only in the highest dose group of the gavage study. Body weight gain and feed consumption were reduced in high-dose groups of both the feed and gavage studies. There was no measurable loss of TCE in feed sampled from the cages during the study. Dose-related increases in organ (liver and kidney) weight/body weight ratios, individual cell necrosis in the liver, and hepatic microsomal NADPH cytochrome c reductase and peroxisomal palmitoyl-CoA oxidase and catalase activities were found in both the dosed-fed and gavage groups. Induction of cytochrome P-450 occurred only in the dosed-feed study. There were no significant compound-related pathologic lesions observed in the kidneys, the only other organ examined microscopically. Differences in lethality, cytochrome P-450 levels, and induction of microsomal or peroxisomal enzyme activities were attributed to differences in the method of dosing (gavage versus dosed-feed). The demonstration of no significant loss of TCE from the feed and of similar toxic effects produced by microencapsulated TCE via feed and TCE in corn oil via gavage indicate that microencapsulation can provide an excellent alternative exposure route for studying the oral toxicological properties of volatile chemicals, such as TCE, in rats.

Application of Microencapsulation for Toxicology Studies. II. Toxicity of Microencapsulated Trichloroethylene in Fischer 344 Rats. MELNICK, R. L., JAMESON, C. W., GOEHL, T. J., MARONPOT, R. R., COLLINS, B. J., GREENWELL, A., HARRINGTON, F. W., WILSON, R. E., TOMAS-ZEWSKI, K. E., AND AGARWAL, D. K. (1987). Fundam. Appl. Toxicol. 8, 432–442. Gelatin–sorbitol microcapsules containing 44.1% trichloroethylene (TCE) were prepared and mixed in NIH-07 rodent meal diet and provided at microcapsule concentrations of 0 (untreated control group), 1.25,2.5, 5.0, or 10% (equivalent to 0, 0.55, 1.10, 2.21, or 4.41% TCE, respectively) to groups of 10 male F344 rats for 14 days. An additional control group received diets containing 5% empty capsules. For comparisons, TCE dissolved in corn oil was administered by gavage to different groups of 10 male rats for 14 consecutive days at dose levels adjusted to correspond to those in the feed study. Treatment-related deaths occurred only in the highest dose group of the gavage study. Body weight gain and feed consumption were reduced in high-dose groups of both the feed and gavage studies. There was no measurable loss of TCE in feed sampled from the cages during the study. Dose-related increases in organ (liver and kidney) weight/body weight ratios, individual cell necrosis in the liver, and hepatic microsomal NADPH cytochrome c re-ductase and peroxisomal palmitoyl-CoA oxidase and catalase activities were found in both the dosed-fed and gavage groups. Induction of cytochrome P-450 occurred only in the dosed-feed study. There were no significant compound-related pathologic lesions observed in the kidneys, the only other organ examined microscopically. Differences in lethality, cytochrome P-450 levels, and induction of microsomal or peroxisomal enzyme activities were attributed to differences in the method of dosing (gavage versus dosed-feed). The demonstration of no significant loss of TCE from the feed and of similar toxic effects produced by microencapsulated TCE via feed and TCE in corn oil via gavage indicate that microencapsulation can provide an excellent alternative exposure route for studying the oral toxicological properties of volatile chemicals, such as TCE, in rats.

A rapid decrease in male fertility in laboratory animals exposed to 1,2-dibromo-3-chloropropane (DBCP) has been suggested to be due, in part, to a postglycolytic inhibition of sperm carbohydrate metabolism. The present studies were performed to identify the specific site of DBCP-induced inhibition of intermediary metabolism. 14CO2 generation by epididymal sperm, isolated from Fischer 344 rats, was measured using radiolabeled tricarboxylic acid (TCA) cycle intermediates: acetyl CoA, citrate, alpha-ketoglutarate, and succinate. There was 0-28% inhibition of CO2 generation after addition of 0.5 mM DBCP and 81-98% inhibition with 3 mM DBCP, with all four substrates. The activities of alpha-ketoglutarate dehydrogenase, pyruvate dehydrogenase, malate dehydrogenase, and lactate dehydrogenase were not inhibited by DBCP. Since the DBCP-induced inhibition of metabolism of different substrates to CO2 was similar, and since DBCP did not inhibit enzyme activities of glycolysis or the TCA cycle, a common site of inhibition was suspected. In evaluations of mitochondrial electron transport chain activity, DBCP (3 mM) inhibited oxygen consumption resulting from metabolism of endogenous substrates plus alpha-ketoglutarate or malate by about 80%. When succinate, an FAD-dependent oxidation, was used as a substrate, oxygen consumption was not inhibited by DBCP. It is concluded that DBCP inhibits sperm carbohydrate metabolism at the NADH dehydrogenase step in the mitochondrial electron transport chain.

Male Fischer 344 rats were given a single, sc injection of 1,2-dibromo-3-chloropropane (DBCP) at 6 or 25 days of age. One group of treated animals was killed 1 to 3 days afterward to compare the dose and time relationships of the acute toxic response of neonatal and weanling male rats to DBCP and another group at approximate sexual maturity (approximately 120 days of age) to detect residual toxic effects resulting from acute exposure. The 6-day-old rats were more susceptible than the 25-day-old rats to the acute toxic effects of DBCP, as characterized by reduced 48-hr survival, renal dysfunction, and renal and hepatic necrosis over the dose range of 80 to 320 mg/kg. The lowest dose tested, 20 mg/kg, and all higher doses reduced subsequent body and gonadal weight gains, and caused hypospermatogenesis or seminiferous tubular atrophy in animals exposed at 6 days of age and killed at sexual maturity. Similar effects were observed in animals exposed at 25 days of age, except that doses of 160 mg/kg or greater were required to produce residual toxic effects. These data indicate enhanced susceptibility of neonatal male rats to the gonadotoxic effects of dibromochloropropane, including the possibility of apparent irreversible injury caused by acute exposure.

A single oral dose of 4.0 mmol/kg bromobenzene transiently depleted hepatic and renal reduced nonprotein sulfhydryl group (NPS) concentrations, caused hepatocellular necrosis, and increased serum glutamic-pyruvic transaminase activity in male Fischer 344 rats. The depletion of NPS had partially reversed by 24 hr, and NPS concentrations were approximately twice normal values by 48 hr post-treatment. When the effects of single and repeated (once daily for 2, 4, or 10 days) treatments with 4.0 mmol/kg were compared, it was apparent that the severity of hepatotoxicity lessened and the percentage depletions of hepatic and renal NPS concentrations decreased with increasing length of bromobenzene treatment. There were essentially no signs of toxicity following the tenth treatment with 4.0 mmol/kg. Single-treatment studies indicated the following dose-response: 2.0 mmol/kg bromobenzene depleted liver NPS and was hepatotoxic, 0.5 mmol/kg caused a lesser depletion of liver NPS and was not (overtly) hepatotoxic, and 0.0625 mmol/kg was the maximum dose that did not deplete liver NPS. The responses to single and multiple (ten) treatments with these representative doses were compared. Liver injury was observed after a single but not after the tenth daily treatment with 2.0 mmol/kg. Both the single and the tenth administrations of 2.0 mmol/kg depleted hepatic NPS, but the percentage of depletion was greater after the first than after the tenth dose. Liver injury was not detected with lower dose regimens. The patterns of NPS depletion in liver and kidney were similar after single or multiple (ten) treatments. The minimum NPS concentrations produced, however, were lower after single than after multiple treatments. The molar amounts of liver NPS depleted after the tenth treatment appeared to be equivalent to or greater than those after the first, but prior bromobenzene exposure resulted in a higher concentration of tissue NPS being present at the time of the final treatment. Thus, the minimum tissue concentrations of NPS were greater after multiple treatments than after single treatments, despite the loss of equivalent amounts of NPS. It is concluded from these studies that repeated treatment produces resistance to bromobenzene hepatotoxicity. This protective adaptation may be due to a chemically induced increase in liver glutathione concentration.