Abstract  This chapter focuses on the genetics of murine lung tumors. Some studies indicate that susceptibility to lung tumorigenesis is determined by a single gene, while other studies suggest the involvement of multiple genes. Most studies on the genetics of lung tumorigenesis in mice have considered tumor incidence and the number of tumors per animal as the phenotype, without considering the size of neoplastic lesions. There is no relationship between the susceptibility of any given mouse strain to lung tumors and its susceptibility to tumors of other organs. Susceptibility to spontaneous lung tumor development is paralleled by susceptibility to the induction of the same tumor type by chemical carcinogens. The study and identification of genetic factors affecting inherited predisposition to lung tumorigenesis in mice are of great interest as a model system for understanding pathogenetic mechanisms. Inbred mice represent good model systems for the identification of the number and chromosomal localization of genetic loci predisposing lung tumor development. The knowledge of the genetics of lung tumor susceptibility in mice is growing very quickly. Lung tumor is a relatively common type of cancer in humans, and the familial clustering of cases is rare compared to colon and breast cancer, where both nonhereditary and familial cases are recognized. The murine strains predisposed to lung tumor development may provide a unique experimental system for the analysis of the genetics of these tumors.

Abstract  About 10 years have elapsed since the first whole-genome scanning studies in the mouse to identify loci that affect susceptibility or resistance to tumorigenesis. In that time, >100 cancer modifiers have been mapped, and four strong candidate genes have been identified. Cancer modifier loci affect almost all types of mouse tumorigenesis, with some loci acting on the entire tumorigenic process, whereas others act on specific stages, e.g., tumor initiation or tumor growth/progression. Present evidence indicates that the effects of cancer modifier loci are tissue-specific and restricted to tumor cells. However, a subset of such loci may be involved in different types of tumors, and several chromosomal regions show significant clustering of cancer modifier loci. Human homologues of mouse cancer modifier loci most likely exist and play a role in the risk of sporadic cancer, although present experimental evidence for this possibility is sparse. Mouse cancer modifier loci might serve as the basis for understanding the genetic and biochemical mechanisms of polygenic inheritance of cancer predisposition/resistance. Identification of homologous cancer modifier loci in humans might, in turn, provide a step toward the development of diagnostic, preventive, and therapeutic strategies that target these loci.

Abstract  The cyclin-dependent kinase inhibitor 2a (Cdkn2a) locus encodes two distinct tumor suppressors, p16INK4a and p19ARF, whose functions interrelate in the regulation of cell proliferation as key components of the retinoblastoma and p53 pathways, respectively. In many types of cancer, alterations of Cdkn2a abrogate the functions of both suppressors, implying that both are integral to the genesis of certain cancer types. While this has been observed in mouse lung adenocarcinogenesis, recent observations also suggested that naturally occurring variation at the Cdkn2a locus is probably operative in the development of these tumors. Firstly, two common haplotypes of mouse Cdkn2a have been identified, each of which encodes cosegregating variants of p16INK4a and p19ARF. The p16INK4a variants differ at amino acids 18 (histidine or proline) and 51 (valine or isoleucine), whereas the p19ARF variants differ only at amino acid 72 (histidine or arginine). Secondly, genetic resistance to lung tumor formation appears to segregate with one particular haplotype, which also is deleted preferentially in lung adenocarcinomas of Cdkn2a heterozygous mice. Here we attempt to explain these observations and to characterize further the roles of p16INK4 and p19ARF in mouse lung tumorigenesis by examining the function and expression of each of the variants of Cdkn2a. Functional analysis showed that the proline 18/isoleucine 51 p16INK4a variant was diminished in cdk6 binding, cdk6 inhibition and NIH/3T3 fibroblast growth suppression compared with the histidine 18/valine 51 variant, whereas both of the p19ARF variants suppressed growth with similar potencies. Also, the different alleles for p16INK4a and p19ARF were transcribed equally in the normal lungs of Cdkn2a heterozygotes, as determined by comparative reverse transcription-polymerase chain reaction-single-stranded conformation polymorphism analysis. These results indicate that strain-specific variation in p16INK4a function is exploited in mouse lung tumorigenesis and strongly implicate a role for p16INK4a in lung cancer predisposition and development.

Abstract  Divinylbenzene-HP is used for producing vinyl polymers. Divinylbenzene-HP was nominated for study by the National Cancer Institute because of the potential for worker exposure and the structural similarity of divinylbenzene to styrene, a potential human carcinogen. Male and female F344/N rats and B6C3F1 mice were exposed to divinylbenzene-HP (80%) by inhalation for 2 weeks, 3 months, or 2 years. Genetic toxicology studies were conducted in Salmonella typhimurium, Escherichia coli, and mouse peripheral blood erythrocytes. 2-WEEK STUDY IN RATS Groups of five male and five female rats were exposed by whole body inhalation to divinylbenzene-HP at target concentrations of 0, 25, 50, 100, 200, or 400 ppm 6 hours plus T90 (12 minutes) per day, 5 days per week for 16 days. All rats survived to the end of the study. Significant decreases in mean body weights occurred in both male and female rats in the 400 ppm groups. Relative kidney weights of 50 ppm or greater males and relative liver weights of 200 and 400 ppm males were significantly greater than those of the chamber controls. A clear serous nasal/eye discharge was observed in groups of males exposed to 100 ppm or greater and females exposed to 50 ppm or greater. Minimal or mild rhinitis occurred in 400 ppm rats of both sexes. 2-WEEK STUDY IN MICE Groups of five male and five female mice were exposed by whole body inhalation to divinylbenzene-HP at target concentrations of 0, 25, 50, 100, 200, or 400 ppm for 6 hours plus T90 (12 minutes) per day, 5 days per week for 17 days. All 400 ppm males and females died on or before the second day of the study, and two male and two female 200 ppm mice died early. Mean body weights of 100 and 200 ppm males were significantly less than those of the chamber controls. Thymus weights of exposed groups of males were significantly less than those of the chamber controls, and relative liver weights of 100 and 200 ppm males were significantly increased. Kidney and liver weights of exposed groups of females were significantly greater than those of the chamber controls. Mice exposed to 200 and 400 ppm had liver lesions including degeneration, necrosis, hemorrhage or cytomegaly. Renal tubule necrosis and regeneration occurred at 200 ppm. Necrosis or metaplasia of nasal epithelium and glands occurred in the nose in all exposure groups. 3-MONTH STUDY IN RATS Groups of 10 male and 10 female rats were exposed to divinylbenzene-HP at concentrations of 0, 25, 50, 100, 200, or 400 ppm for 6 hours plus T90 (12 minutes) per day, 5 days per week for 14 weeks. All rats survived to the end of the study. There were no biologically significant changes in body weight in either sex. Nasal/eye discharge was noted in 400 ppm males and 100 ppm females. Kidney and liver weights of exposed groups of males and of 400 ppm females were generally greater than those of the chamber controls. In addition, the relative weights of the heart and testis were significantly increased in 200 and 400 ppm males. Incidences of degeneration of the olfactory epithelium in 200 and 400 ppm rats and basal cell hyperplasia of the olfactory epithelium in rats exposed to 100 ppm or greater were significantly increased. 3-MONTH STUDY IN MICE Groups of 10 male and 10 female mice were exposed to divinylbenzene-HP at concentrations of 0, 12.5, 25, 50, 100, or 200 ppm for 6 hours plus T90 (12 minutes) per day, 5 days per week for 14 weeks. All 200 ppm males and nine 200 ppm females died early. Final mean body weights were significantly lower in males and females exposed to 25, 50, or 100 ppm when compared with chamber controls. Lethargy or hypoactivity was observed in the higher exposure concentration groups. Exposure to divinylbenzene was associated with necrosis of the liver and kidney in 200 ppm males and females dying early. In all exposed groups of male and female mice, there was necrosis of nasal cavity lateral walls, olfactory epithelium, and glands with resultant atrophy of olfactory epithelium and glands in females. A lower number of animals had necrotic or degenerative changes of the upper respiratory tract. 2-YEAR STUDY IN RATS Groups of 50 male and 50 female rats were exposed to divinylbenzene-HP at concentrations of 0, 100, 200, or 400 ppm for 6 hours plus T90 (12 minutes) per day, 5 days per week for up to 105 weeks. Survival of 400 ppm females was significantly less than that of the chamber control group. Survival of all exposed groups of males was similar to that of the chamber control group. Mean body weights of 400 ppm males and females were significantly less than those of the controls during the second half of the study. Renal tubule carcinomas occurred in two of 50 males exposed to 400 ppm in the original kidney sections, an incidence that exceeded the historical control range. In 400 ppm males, the incidence of renal tubule hyperplasia was increased, and the incidence of nephropathy was significantly increased. Following combined analysis of single and step-section data, the incidences of renal tubule adenoma and adenoma or carcinoma (combined) were marginally higher in 200 and 400 ppm males, and the incidence of renal tubule hyperplasia was significantly increased in 400 ppm males. The incidences of malignant glial cell tumors (malignant astrocytoma and oligodendroglioma) in the brain were slightly increased in 100 and 200 ppm males, and the incidence in the 200 ppm group exceeded the historical range for chamber controls. There were increased incidences of degenerative and regenerative changes in the olfactory epithelium in the nose of all exposed groups of rats. The incidence of focal chronic inflammation in the lung of 400 ppm males was significantly greater than in the chamber control group. 2-YEAR STUDY IN MICE Groups of 50 male and 50 female mice were exposed to divinylbenzene-HP at concentrations of 0, 10, 30, or 100 ppm for 6 hours plus T90 (12 minutes) per day, 5 days per week for up to 105 weeks. Survival of all exposed groups of male and female mice was similar to that of the chamber controls. Mean body weights were lower relative to chamber controls in 100 ppm males and in 30 and 100 ppm females. The incidences of alveolar/bronchiolar adenoma and alveolar/bronchiolar adenoma or carcinoma (combined) in 100 ppm males were greater than chamber control incidences, but the incidences of adenoma or carcinoma (combined) were within the historical control range. The incidences of alveolar/bronchiolar adenoma and alveolar/bronchiolar adenoma or carcinoma (combined) in all exposed groups of females were generally greater than those of the chamber controls; the incidences were at the upper end or exceeded the historical control ranges. There was a greater incidence and severity of alveolar epithelial hyperplasia in 100 ppm females and a greater severity of this lesion in 30 ppm females, when compared to chamber controls. The incidences and/or severities of atypical bronchiole hyperplasia were significantly increased in all exposed groups of mice. Nonneoplastic nasal lesions occurred in most exposed mice. GENETIC TOXICOLOGY Divinylbenzene-HP was not mutagenic in any of three independent gene mutation assays using Salmonella typhimurium strains TA97, TA98, TA100, TA1535, or TA1537 or Escherichia coli tester strain WP2 uvrA with or without induced hamster or rat liver enzymes. No increases in the frequencies of micronucleated normochromatic erythrocytes or alterations in the percentages of polychromatic erythrocytes were seen in peripheral blood of male or female B6C3F1 mice exposed to divinylbenzene-HP by inhalation for 3 months. CONCLUSIONS Under the conditions of this 2-year inhalation study, there was equivocal evidence of carcinogenic activity of divinylbenzene-HP in male F344/N rats based upon the occurrence of carcinomas in the kidney and glial tumors in the brain. There was no evidence of carcinogenic activity in female F344/N rats exposed to 100, 200, or 400 ppm divinylbenzene-HP. There was no evidence of carcinogenic activity in male B6C3F1 mice exposed to 10, 30, or 100 ppm divinylbenzene-HP. There was equivocal evidence of carcinogenic activity of divinylbenzene-HP in female B6C3F1 mice based on the incidences of alveolar/bronchiolar adenoma or carcinoma (combined) in the lung. Exposure to divinylbenzene-HP caused nonneoplastic lesions of the nasal cavity in male and female rats and of the lung and nasal cavity in male and female mice.

Abstract  The pulmonary adenoma susceptibility 1 (Pas1) locus affects inherited predisposition and resistance to chemically induced lung tumorigenesis in mice. The A/J and C57BL/6J mouse strains carry the susceptibility and resistance allele, respectively. We identified and genotyped 65 polymorphisms in the Pas1 locus region in 29 mouse inbred strains, and delimited the Pas1 locus to a minimal region of 468 kb containing six genes. That region defined a core Pas1 haplotype with 42 tightly linked markers, including intragenic polymorphisms in five genes (Bcat1, Lrmp, Las1, Ghiso, and Kras2) and amino-acid changes in three genes (Lrmp, Las1, Lmna-rs1). In (A/J x C57BL/6J)F1 mouse lung tumors, the Lmna-rs1 gene was completely downregulated, whereas allele-specific downregulation of the C57BL/6J-derived allele was observed at the Las1 gene, suggesting the potential role of these genes in tumor suppression. These results indicate a complex multigenic nature of the Pas1 locus, and point to a functional role for both intronic and exonic polymorphisms of the six genes of the Pas1 haplotype in lung tumor susceptibility.

Abstract  Rodent species and strains show wide variations in susceptibility to lung tumorigenesis. In mice, hierarchical clustering of 29 inbred laboratory strains by pulmonary adenoma susceptibility 1 (Pas1) locus polymorphisms separated the strains into either an A/J- or a C57BL/6J-type Pas1 haplotype. A pooled analysis (including >8500 mice) of studies on spontaneous and chemically induced lung tumorigenesis in these strains revealed a significantly higher risk of spontaneous lung tumors [odds ratio (OR) 12.17; 95% confidence interval (CI) 9.00-16.45] as well as of chemically induced lung tumors (OR 15.14; 95% CI 12.51-18.31) in the A/J-type haplotype. Strain differences were observed with six different carcinogens, suggesting that Pas1 locus activity is carcinogen-independent. Thus, the present meta-analysis indicates a link between the genetic control of spontaneous and chemically induced lung tumor susceptibility in mice. The Pas1 susceptibility allele is frequent in the population of inbred mouse strains, whereas a counterpart appears to be absent or rare in rat and hamster strains. These findings might help in the interpretation of results of rodent carcinogenicity bioassays and assessing the risk of lung carcinogenesis from chemicals.

Abstract  Pulmonary adenoma susceptibility 1 (Pas1), the major locus affecting inherited predisposition to lung tumor development in mice, maps near the Kras2 gene. We previously reported a significant association between a KRAS2/RsaI polymorphism and the risk and prognosis of lung adenocarcinoma (ADCA) in the Italian population. In the present case-control study, we examined 269 lung ADCA patients, 121 squamous cell lung carcinoma patients, and 632 healthy individuals (general population controls) in the Japanese population with genetic markers spanning approximately 1200 kb in the KRAS2 region. Allele-specific oligonucleotide hybridization revealed the same KRAS2/RsaI polymorphism associated with risk and prognosis as in Italian lung ADCA patients; the polymorphism was significantly associated with clinical stage (P < 0.001) and survival rate (log rank = 0.0014), confirming the mapping of PAS1 and pointing to the role of this locus in human lung cancer.

Abstract  This report shows the tumor rates of control group animals from selected studies.

Abstract  Cumene is produced in a modified Friedel-Crafts reaction process that uses acidic catalysts to alkylate benzene with propylene. Cumene is the principal chemical used in the production of phenol and acetone. Cumene is used to produce acetophenone, α-methylstyrene, diisopropylbenzene, and dicumylperoxide; as a thinner; as a constituent of some petroleum-based solvents; in gasoline blending, diesel fuel, and high-octane aviation fuel; and as a raw material for peroxides and oxidation catalysts. Because cumene is a good solvent for fats and resins, it has been suggested as a replacement for benzene in many industrial applications. Cumene occurs naturally in petroleum and in a variety of foodstuffs. Cumene was nominated for study by the NIEHS because of its high production volume, presence in gasoline and other fuels, potential for human exposure, and lack of existing carcinogenicity test data. Male and female F344/N rats and B6C3F1 mice were exposed to cumene (greater than 99.9% pure) by inhalation for 2 weeks, 3 months, or 2 years. Genetic toxicology studies were conducted in Salmonella typhimurium, rat bone marrow, and mouse peripheral blood. 2-WEEK STUDY IN RATS Groups of five male and five female rats were exposed to cumene vapor at concentrations of 0, 250, 500, 1,000, 2,000, or 4,000 ppm, 6 hours plus T90 (12 minutes) per day, 5 days per week for 16 days. All rats exposed to 4,000 ppm died on day 1, and two male and three female rats exposed to 2,000 ppm died by day 4. Mean body weights of 2,000 ppm rats were significantly less than those of the chamber controls. Rats exposed to 2,000 ppm that died early were severely lethargic following daily exposure. Liver and kidney weights of all exposed groups were increased. Accumulation of minimal to mild hyaline droplets was observed in the renal tubular cortex of males exposed to concentrations of 250 to 2,000 ppm. 2-WEEK STUDY IN MICE Groups of five male and five female mice were exposed to cumene vapor at concentrations of 0, 250, 500, 1,000, 2,000, or 4,000 ppm, 6 hours plus T90 (12 minutes) per day, 5 days per week for 17 days. All mice exposed to 4,000 ppm died on day 1; all mice exposed to 2,000 ppm died on day 2, and four female mice exposed to 1,000 ppm died by day 4. Mean body weights of all exposed groups were similar to those of the chamber controls. Mice exposed to 2,000 ppm were severely lethargic after the first exposure. The four female mice exposed to 1,000 ppm that died early exhibited signs of lethargy and ataxia. Liver weights, both relative and absolute, were increased in all groups of surviving males and in 250 and 500 ppm female groups. 3-MONTH STUDY IN RATS Groups of 10 male and 10 female rats were exposed to cumene vapor at concentrations of 0, 62.5, 125, 250, 500, or 1,000 ppm, 6 hours plus T90 (12 minutes) per day, 5 days per week for 14 weeks. Additional clinical pathology groups of 10 male and 10 female rats were exposed to the same concentrations for 23 days. All rats survived to the end of the study, and mean body weights of all exposed groups were similar to those of the chamber controls. Kidney and liver weights of 250 ppm or greater males and liver weights of 1,000 ppm females were significantly greater than those of the chamber controls. There were significant differences between exposed and chamber control females in the relative length of time spent in the estrous stages. The amount of α2u-globulin in the right kidneys was significantly increased in male rats exposed to 125 ppm or greater. The incidences of medullary granular casts in males exposed to 250 ppm or greater were significantly increased. The severities of renal tubule cortex hyaline droplet accumulation and regeneration increased with increasing exposure concentration in male rats. 3-MONTH STUDY IN MICE Groups of 10 male and 10 female mice were exposed to cumene vapor at concentrations of 0, 62.5, 125, 250, 500, or 1,000 ppm, 6 hours plus T90 (12 minutes) per day, 5 days per week for 14 weeks. Eight 1,000 ppm females died during week 1 of the study. Mean body weights of males exposed to 500 or 1,000 ppm were significantly less than those of the chamber controls. The eight 1,000 ppm female mice that died during the first week of the study exhibited clinical signs of acute toxicity, including lethargy or ataxia. Liver weights of mice exposed to 500 or 1,000 ppm were significantly increased. The weight of the cauda epididymis and the spermatid count were significantly decreased in 1,000 ppm males. 2-YEAR STUDY IN RATS Groups of 50 male and 50 female rats were exposed to cumene vapor at concentrations of 0, 250, 500, or 1,000 ppm, 6 hours plus T90 (12 minutes) per day, 5 days per week for 105 weeks. Survival of all exposed groups of rats was similar to that of the chamber controls. Mean body weights of 1,000 ppm females were slightly less than those of the chamber controls during the second year of the study but were similar to the chamber controls at the end of the study. Incidences of adenoma of the respiratory epithelium in the nose occurred with a positive trend in males and were significantly increased in all exposed groups of males and in 250 ppm females. Incidences of hyperplasia of basal cells in the olfactory epithelium in the nose of all exposed groups and hyperplasia of the respiratory epithelium in the nose of all exposed groups of males and 1,000 ppm females were significantly increased. The incidences of renal tubule adenoma in all exposed groups of males, renal tubule carcinoma in 500 and 1,000 ppm males, and renal tubule adenoma or carcinoma (combined) in all exposed groups of males were increased; the difference from chamber controls for the combined incidence was significant at 500 ppm. The incidences of hyperplasia of the renal tubule and transitional epithelium of the renal pelvis in 500 and 1,000 ppm males and mineralization of the renal papilla in all exposed groups of males were significantly greater than those of the chamber controls. The incidence of interstitial cell adenoma (including bilateral) of the testis was significantly increased in 1,000 ppm male rats, and there was a positive trend in the incidences across all groups. 2-YEAR STUDY IN MICE Groups of 50 male and 50 female mice were exposed to cumene vapor at concentrations of 0, 125 (female mice only), 250, 500, or 1,000 (male mice only) ppm, 6 hours plus T90 (12 minutes) per day, 5 days per week for 105 weeks. An exposure concentration-related decrease in survival occurred in male mice, and the survival of 1,000 ppm males was significantly less than that of the chamber controls. Mean body weights of 1,000 ppm males were generally less than those of the chamber controls after week 8 of the study, and those of 500 ppm females were less from week 28 until week 76 of the study. The incidences of alveolar/bronchiolar adenoma, alveolar/bronchiolar carcinoma, and alveolar/bronchiolar adenoma or carcinoma (combined) in all exposed groups of mice occurred with positive trends and were significantly greater than those in the chamber controls. The incidences of alveolar epithelial bronchiole metaplasia and bronchiole hyperplasia were significantly increased in all exposed groups of mice. p53 and K-ras mutations were found in 52% and 87% of lung neoplasms in exposed mice compared to 0% and 14% in the chamber controls, respectively. In female mice, the incidences of hepatocellular adenoma and hepatocellular adenoma or carcinoma (combined) occurred with positive trends and were significantly increased in the 500 ppm group. In male mice, there were significant increases in the incidences of eosinophilic foci of the liver. The incidences of hemangiosarcoma in the spleen and of follicular cell adenoma in the thyroid gland were significantly increased in 1,000 ppm male mice. In the nose, the incidences of olfactory epithelium atrophy, basal cell hyperplasia of the olfactory epithelium, atypical hyperplasia of the olfactory epithelium, hyperplasia of olfactory epithelium glands, and suppurative inflammation were generally significantly increased in 500 and 1,000 ppm males and 500 ppm females. The incidence of squamous metaplasia of the respiratory epithelium was significantly increased in 500 ppm females. The incidence of basal cell hyperplasia was also significantly increased in 250 ppm females. The incidences of epithelial hyperplasia of the forestomach in the 500 and 1,000 ppm groups of males and the incidences of ulceration and inflammation of the forestomach in 1,000 ppm males were significantly increased. GENETIC TOXICOLOGY Cumene was not mutagenic in S. typhimurium strain TA97, TA98, TA100, or TA1535, when tested with and without liver S9 activation enzymes. Cumene induced small, but significant, increases in micronucleated polychromatic erythrocytes in bone marrow of male rats treated by intraperitoneal injection. In contrast, no increase in micronucleated erythrocytes was observed in peripheral blood of male or female mice exposed to cumene by inhalation for 3 months. CONCLUSIONS Under the conditions of these 2-year inhalation studies, there was clear evidence of carcinogenic activity of cumene in male F344/N rats based on increased incidences of respiratory epithelial adenoma in the nose and renal tubule adenoma or carcinoma (combined). Increased incidences of interstitial cell adenoma of the testis may have been related to exposure to cumene. There was some evidence of carcinogenic activity of cumene in female F344/N rats based on the incidences of respiratory epithelium adenoma in the nose. There was clear evidence of carcinogenic activity of cumene in male B6C3F1 mice based on increased incidences of alveolar/bronchiolar neoplasms. The increased incidences of hemangiosarcoma in the spleen and follicular cell adenoma in the thyroid gland in male mice may have been related to cumene exposure. There was clear evidence of carcinogenic activity of cumene in female B6C3F1 mice based on increased incidences of alveolar/bronchiolar neoplasms. Increased incidences of hepatocellular adenoma or carcinoma (combined) in female mice were also considered to be related to exposure to cumene. Exposure of male rats to cumene resulted in nonneoplastic lesions of the kidney characteristic of α2u-globulin accumulation. Exposure to cumene resulted in nonneoplastic lesions in the nose of male and female rats; the lung, nose, liver, and forestomach of male mice; and the lung and nose of female mice.

Abstract  Previous observation has shown that the wild-type Kras2 allele is a suppressor of lung cancer in mice. Here we report that loss of heterozygosity (LOH) of chromosome 12p was detected in approximately 50% of human lung adenocarcinomas and large cell carcinomas, and Kras2 mutations were detected at codon 12 in approximately 40% of adenocarcinomas and large cell carcinomas. Interestingly, all of the lung adenocarcinomas and large cell carcinomas containing a Kras2 mutation exhibited allelic loss of the wild-type Kras2 allele when a correlation between LOH of the region on chromosome 12p and Kras2 mutation was made. These results from human lung cancer tissues provide a strong evidence in support of our previous observation in mouse models that the wild-type Kras2 is a tumor suppressor of lung cancer.

Abstract  Our recent linkage study of urethane-induced pulmonary adenomas in SMXA RI strains of mouse revealed two host resistance genes, Par1 (chromosome 11) and Par3 (chromosome 12). The map positions of Par1 and Par3 correspond to human 17q11-23 and 14q11-24, based on synteny between mouse and human. In this study, we examined the loss of heterozygosity (LOH) in these two homologous human chromosomal regions in 30 primary lung adenocarcinoma samples with matched normal DNA. Using 15 highly polymorphic markers, two commonly deleted regions were identified on human chromosomes 14 and 17, respectively. At 17q21, nine (53%) of 17 informative tumors showed LOH between D17S588 and D17S518. On the other hand, at 14q11-12, seven (32%) of 22 informative tumors showed LOH at loci between D14S261 and D14S80. Subsequently, we examined 25 squamous cell carcinomas (SQ) and 24 small cell carcinomas (SCC). At 14q11-12, six (38%) of 16 informative SQ and five (42%) of 12 informative SCC showed LOH. In contrast, at 17q11-23, one (7%) of 15 informative SQ and two (14%) of 14 SCC showed LOH. Therefore, the gene on 17q seemed to affect selectively adenocarcinomas, whereas the other gene on 14q, all three types of lung carcinomas. These observations indicate that a comparative genetic analysis provides a promising approach to survey genes involved in multifactorial process of human lung carcinogenesis.

Abstract  This is the first in a series of review articles describing the current state of research on mouse lung tumorigenesis. The system is valuable as a biological model for studying stages of tumor development and the interaction of genetic and environmental factors which dispose towards neoplasia. Additionally, these tumors are analagous to bronchiolo-alveolar cancer in man. Three pulmonary adenoma susceptibility (Pas) genes regulate susceptibility; 1 of these is the proto-oncogene, K-ras2. Candidates for the other 2 genes include the H-2 histocompatibility locus and genes which regulate the basal proliferative rate of the cells from which these tumors arise. Tumor development is favored by a depressed immune system, immature age, and decreased levels of circulating corticosterone.

Abstract  Potential factors underlying the tumorigenic activity of ethylbenzene (EB) were examined in F344 rats and B6C3F1 mice inhaling 750 ppm EB vapor 6 h/day, 5 days/week, for one or four weeks. Target tissues (kidneys of rats and livers and lungs of mice) were evaluated for changes in organ weights, mixed function oxygenases (MFO), glucuronosyl transferase activities, S-phase DNA synthesis, apoptosis, alpha2u-globulin deposition, and histopathology. In male rats, kidney weight increases were accompanied by focal increases in hyaline droplets, alpha2u-globulin, degeneration, and S-phase synthesis in proximal tubules. In female rats, only decreased S-phase synthesis and MFO activities occurred. In mice, increased liver weights were accompanied by hepatocellular hypertrophy, mitotic figures, S-phase synthesis, and enzyme activities. S-phase synthesis rates in terminal bronchiolar epithelium were elevated and accompanied by loss of MFO activity. Exposure to a nontumorigenic level of 75 ppm for one week caused few changes. These data, considered with the general lack of EB genotoxicity, suggest a mode of tumorigenesis dependent upon increased cell proliferation and altered population dynamics in male rat kidney and mouse liver and lungs. A similar response in the kidneys of female rats appears to require a longer exposure period than was employed.

Abstract  Styrene, a widely used intermediate in the manufacture of plastics, elastomers, and resins, was selected for bioassay by the National Cancer Institute because of the widespread use of this compound and a lack of adequate carcinogenicity data. A bioassay for the possible carcinogenicity of styrene was conducted using Fischer 344 rats and B6C3F1 mice. Styrene was administered by gavage to groups of 50 male and 50 female animals of each species. Forty rats of each sex and twenty mice of each sex were placed on test as vehicle controls. The high, medium, and low dosages of styrene administered to rats were, respectively, 2,000, 1,000, and 500 mg/kg. The high and low dosages administered to mice were 300 and 150 mg/kg, respectively. The compound was administered for 78 weeks to high and medium dose rats, for 103 weeks to low dose rats, and for 78 weeks to mice. The period of compound administration was followed by an observation period of 27 weeks for high and medium dose rats, 1 week for low dose rats, and 13 weeks for mice. Mortality among male and female high dose rats was significantly higher than that among their respective vehicle controls. In response to this elevated and early mortality, an additional dosed group of each sex was included in the chronic bioassay. No significant positive association was apparent between dosage and mortality among any other dosed rat groups. For mice, there was a significant positive association between mortality and the dosages of styrene administered to males, but not to females. Adequate numbers of animals in all groups, except for the high dose male and female rats, survived sufficiently long to be at risk from late-developing tumors. Slight dose-related mean body weight depression was apparent when male rats and female mice were compared to their respective vehicle controls, indicating that the dosages administered to these animals during the chronic bioassay may have approximated the maximum tolerated dosages. There was no distinct depression in mean body weight when dosed female rats anddosed male mice were compared to their respective vehicle controls. However, since there was significant accelerated mortality among high dose female rats, it is possible that the dosage administered to the medium dose female rats may have exceeded the maximum tolerated dosage. In male mice, there was a significant positive association between styrene dosage and the incidences of a combination of adenomas and carcinomas of the lung. This finding was supported by the high dose to control Fisher exact comparison. However, the variation of the incidence of these neoplasms in historical control male mice at this laboratory does not permit a firm conclusion of carcinogenicity. There was no significant difference between tumor incidence at any other site in male mice, or at any site in rats or female mice, when dosed groups were compared to vehicle controls. The findings of an increased incidence of a combination of adenomas and carcinomas of the lung provided suggestive evidence for the carcinogenicity of styrene in male B6C3F1 mice. However, it is concluded that, under the conditions of this bioassay, no convincing evidence for the carcinogenicity of the compound was obtained in Fischer 344 rats or B6C3F1 mice of either sex.

Abstract  Benzofuran is used as an intermediate in the polymerization of coumarone-indene resins found in various corrosion-resistant coatings such as paints and varnishes, in water-resistant coatings for paper products and fabrics, and in adhesives approved for use in food containers. Toxicology and carcinogenesis studies were conducted by administering benzofuran (approximately 99% pure) in corn oil by gavage to groups of F344/N rats and B6C3F1 mice of each sex for 14 days, 13 weeks, or 2 years. Genetic toxicology tests were conducted in Salmonella typhimurium, mouse lymphoma cells, and Chinese hamster ovary (CHO) cells. Fourteen-Day Studies: Benzofuran doses for groups of five rats ranged from 63 to 1,000 mg/kg and from 16 to 250 mg/kg for mice. All male and female rats that received 1,000 mg/kg and one female rat that received 500 mg/kg died before the end of the studies. The final mean body weights of male rats that received 250 or 500 mg/kg were 13% or 21% lower than that of controls; the final mean body weight of female rats that received 500 mg/kg was 10% lower than that of controls. Final mean body weights of chemically exposed and control mice were similar. No compound-related histologic lesions were found in rats or mice. Thirteen-Week Studies: Doses for groups of 10 rats and 10 mice ranged from 31 to 500 mg/kg. One female rat that received 500 mg/kg and one that received 250 mg/kg died before the end of the study. Final mean body weights of male rats that received 125, 250, or 500 mg/kg were 11%, 17%, or 27% lower than that of vehicle controls; the final mean body weight of female rats that received 500 mg/kg was 11% lower than that of vehicle controls. Histologic lesions observed in chemically exposed rats included minimal hepatocellular necrosis, increased severity of nephropathy, and cytoplasmic vacuolization of the adrenal cortex. Seven male and three female mice that received 500 mg/kg and one male mouse that received 250 mg/kg died before the end of the 13-week studies. The final mean body weight of mice that received 500 mg/kg was 13% lower than that of vehicle controls. Nephrosis was observed in male mice that received 250 mg/kg. Based on reduced mean body weights, increased severity of nephropathy, and hepatocellular necrosis, benzofuran doses selected for the 2-year studies in rats were 30 or 60 mg/kg for males and 60 or 120 mg/kg for female. Based on increased mortality and nephrosis in male mice, doses selected for the 2-year studies in mice were 60 or 120 mg/kg for males and 120 or 240 mg/kg for females. Body Weights and Survival in the Two-Year Studies: Mean body weights of high dose rats and dosed male mice were 4%-11% lower than those of vehicle controls. Mean body weights of chemically exposed female mice were 8%-35% lower than those of vehicle controls. The survival of chemically exposed male rats was reduced after week 92 (survival at week 89: vehicle control, 47/50; low dose, 39/50; high dose, 38/50; final survival: vehicle control, 33/50; low dose, 12/50; high dose, 18/50). Survival of chemically exposed female rats and male mice was similar to that of vehicle controls after 2 years (female rats: 27/50; 23/50; 25/50; male mice: 33/50; 20/50; 28/50). Deaths of 10 low dose male mice at weeks 20-21 were caused by a dosing error; these animals were not included in survival and tumor analyses. Survival of chemically exposed female mice was reduced after week 89 (final survival: 37/50; 19/50; 21/50). Nonneoplastic and Neoplastic Effects in the Two-Year Studies: Nephropathy occurred with increased severity in chemically exposed male rats. The incidences of parathyroid hyperplasia, fibrous osteodystrophy, mineralization of the pulmonary artery, renal cortical cysts, and hyperplasia of the pelvic epithelium were increased in chemically exposed male rats. The incidence of nephropathy was increased in chemically exposed female rats (vehicle control, 29/50; low dose, 48/50; high dose, 39/50). Renal atypical tubular cell hyperplasia and renal tubular cell adenocarcinomas occurred in chemically exposed female rats (atypical tubular cell hyperplasia: 0/50; 1/50; 3/50; tubular cell adenocarcinomas: 0/50; 1/50; 4/50). No renal tubular cell adenocarcinomas have been observed in 2,094 female corn oil vehicle control F344/N rats in National Toxicology Program studies. Chronic inflammation, ulcers, and epithelial hyperplasia of the forestomach were observed at increased incidences in chemically exposed male rats (chronic inflammation: 1/50; 11/50; 6/49; ulcers: 1/50; 5/50; 8/49; epithelial hyperplasia: 9/50; 15/50; 18/49). Metaplastic hepatocytes arising within pancreatic islets occurred at an increased incidence in high dose female rats (0/50; 1/50; 11/49). The incidences of neurilemomas were markedly increased above the historical control incidences (0.1%-0.4%) in all groups of rats (male: 18/50; 13/50; 14/50; female: 7/50; 9/50; 3/50). Syncytial alteration of the liver occurred at increased incidences in male mice exposed to benzofuran. The incidences of hepatocellular adenomas, hepatoblastomas (high dose male mice) and hepatocellular adenomas, hepatocellular carcinomas, or hepatoblastomas (combined) were increased in chemically exposed mice (male--adenomas: 4/49; 24/39; 34/48; hepatoblastomas: 0/49; 3/39; 18/48; carcinomas, adenomas, or hepatoblastomas, combined: 12/49; 31/39; 40/48; female--adenomas: 1/50; 22/48; 21/47; hepatoblastomas: 0/50; 1/48; 2/47; carcinomas, adenomas, or hepatoblastomas, combined: 4/50; 25/48; 22/47). Squamous cell papillomas or carcinomas (combined) of the forestomach were increased in chemically exposed mice (male: 2/49; 11/39; 13/48; female: 2/50; 9/50; 5/50). The incidences of epithelial hyperplasia of the bronchioles were increased in chemically exposed mice. The incidences of alveolar/bronchiolar adenomas or carcinomas (combined) in high dose males and chemically exposed females were increased (adenomas or carcinomas, combined--male: 10/49; 9/39; 19/48; female: 2/50; 9/48; 14/47). Genetic Toxicology: Benzofuran was not mutagenic in S. typhimurium strains TA98, TA100, TA1535, or TA1537 in the presence or absence of exogenous metabolic activation. Benzofuran induced trifluorothymidine resistance in mouse L5178Y lymphoma cells treated in the absence of metabolic activation; this assay was not conducted with activation. Benzofuran induced sister chromatid exchanges but not chromosomal aberrations in CHO cells in the presence and absence of activation. Conclusions: Under the conditions of these 2-year gavage studies, there was no evidence of carcinogenic activity of benzofuran for male F344/N rats receiving doses of 30 or 60 mg/kg per day. There was some evidence of carcinogenic activity of benzofuran for female F344/N rats, based on increased incidences of tubular cell adenocarcinomas of the kidney. There was clear evidence of carcinogenic activity for male and female B6C3F1 mice, based on increased incidences of neoplasms of the liver, lung, and forestomach. Exposure to benzofuran increased the severity of nephropathy in male rats, increased the incidences of nephropathy in female rats, and induced hepatocellular metaplasia in the pancreas in female rats. Nonneoplastic lesions observed in mice exposed to benzofuran included syncytial alteration of the liver, bronchiolar epithelial hyperplasia, and epithelial hyperplasia of the forestomach.

Abstract  Coumarin is the basic structure of numerous naturally occurring compounds with important and diverse physiological activities. More than a thousand coumarin derivatives have been described, varying from simple coumarins containing alkyl and hydroxyl side chains to complex coumarins with benzoyl, furanoyl, pyranoyl, or alkylphosphorothionyl substituents. Coumarin and 3,4-dihydrocoumarin were nominated by the Food and Drug Administration and the National Cancer Institute for study because of the widespread use of coumarin in perfumes, cosmetics, and other products as a fragrance, continued interest in coumarin compounds as flavor-enhancing agents for foods, and the interest in structure-activity relationships of this important group of compounds. Coumarin is believed to be metabolized to a 3,4-epoxide intermediate, which may be responsible for its toxic effects, while 3,4-dihydrocoumarin, which lacks the 3,4-double bond, is not considered likely to form an epoxide intermediate. Toxicity and carcinogenicity studies were conducted by administering coumarin (97% pure) in corn oil by gavage to groups of male and female F344/N rats and B6C3F1 mice for 16 days, 13 weeks, and 2 years. Genetic toxicology studies were conducted in Salmonella typhimurium, cultured Chinese hamster ovary cells, Drosophila melanogaster, and B6C3F1 mice. 16-DAY STUDY IN RATS Groups of five male and five female rats received coumarin in corn oil by gavage at doses of 0, 25, 50, 100, 200, or 400 mg per kg body weight, 5 days a week for a total of 12 doses in a 16-day period. All female rats and four male rats receiving 400 mg/kg died. The mean body weight gains and final mean body weights of surviving dosed male and female rats were similar to those of the controls. There were no clinical signs of organ-specific toxicity, and there was no evidence of impaired blood coagulation from measurements of capillary clotting time or prothrombin and activated partial thromboplastin time. 16-DAY STUDY IN MICE Groups of five male and five female mice received coumarin in corn oil by gavage at doses of 0, 40, 75, 150, 300, or 600 mg per kg body weight, 5 days a week for a total of 12 doses in a 16-day period. All mice receiving 600 mg/kg, two male mice receiving 300 mg/kg, and one male mouse receiving 75 mg/kg died. The mean body weight gains and final mean body weights of surviving dosed male and female mice were similar to those of the controls. Clinical findings of inactivity, excessive lacrimation, piloerection, bradypnea, ptosis, or ataxia were observed in some mice from the 300 and 600 mg/kg groups within the first several hours after dosing. Capillary clotting time and platelet counts of dosed mice were similar to those of controls. 13-WEEK STUDY IN RATS Groups of 10 male and 10 female rats received coumarin in corn oil by gavage at doses of 0,19, 38, 75,150, or 300 mg per kg body weight. Three male and three female rats receiving 300 mg/kg died. The mean body weight gains and final mean body weights of male rats that received 150 and 300 mg/kg were significantly lower than those of the controls. There were no clinical signs related to specific organ toxicity. Male and female rats receiving coumarin exhibited dose-related decreases in mean erythrocyte volume and mean erythrocyte hemoglobin, and dose-related increases in erythrocyte counts. Serum levels of total bilirubin and one or more cytoplasmic enzymes including alanine aminotransferase, aspartate aminotransferase, ornithine carbamoyltransferase, and/or sorbitol dehydrogenase in males and females receiving 300 mg/kg were higher than those of controls. The absolute and relative liver weights of male and female rats that received 150 and 300 mg/kg were significantly greater than those of the controls. Centrilobular hepatocellular degeneration and necrosis, chronic active inflammation, and bile duct hyperplasia were observed in the liver of rats receiving 150 or 300 mg/kg. The high dose selected for the 2-year study was 100 mg/kg, which was just below the level at which mortality, lower final mean body weights, and treatment-related liver lesions were observed in the 13-week study. 13-WEEK STUDY IN MICE Groups of 10 male and 10 female mice received coumarin in corn oil by gavage at doses of 0, 19, 38, 75, 150, or 300 mg per kg body weight. Two male mice receiving 300 mg/kg died. The mean body weight gain and final mean body weight of surviving male mice that received 300 mg/kg were significantly lower than those of the controls. No clinical signs of toxicity were observed. Male and female mice receiving coumarin exhibited dose-related decreases in mean erythrocyte volume and mean erythrocyte hemoglobin. The absolute and relative liver weights of males and females that received 150 and 300 mg/kg were significantly greater than those of the controls. Centrilobular hepatocellular hypertrophy was observed in male and female mice receiving 300 mg/kg. The high dose selected for the 2-year study was 200 mg/kg, which was just below the level at which mortality and liver lesions were observed in the 13-week study. 2-YEAR STUDY IN RATS: Groups of 60 male and 60 female rats were administered coumarin in corn oil by gavage at doses of 0, 25, 50, or 100 mg per kg body weight. After 15 months, 10 animals from each group were evaluated. Survival, Body Weights, and Clinical Findings None of the male rats receiving 100 mg/kg and only two males receiving 50 mg/kg survived until the end of the study (vehicle control, 28/50; 25 mg/kg, 9/50; 50 mg/kg, 2/51; 100 mg/kg, 0/50). Survival of dosed female rats was similar to that of the controls (29/50, 38/50, 36/50, 30/50). The reduced survival in dosed male rats was primarily attributed to chemical-related exacerbation of spontaneously occurring renal disease. Final mean body weights of female rats that received 100 mg/kg and all dosed groups of male rats were lower than those of the controls. There were no clinical signs of toxicity in rats, other than nonspecific signs relating to debilitation as a result of renal or other spontaneous disease. Hematology and Clinical Chemistry At the 15-month interim evaluation, the values for one or more hematologic parameters including mean erythrocyte volume, mean erythrocyte hemoglobin in 50 and 100 mg/kg rats, and hematocrit or hemoglobin in 100 mg/kg rats were significantly lower than those of controls. Activated partial thromboplastin times were also significantly lower in 50 and 100 mg/kg males, while platelet counts were significantly higher. Activities of alanine aminotransferase, sorbitol dehydrogenase, or g-glutamyltransferase in 50 and 100 mg/kg male and 100 mg/kg female rats were significantly higher than those of the controls at the 15-month interim evaluation. Pathology Findings The principal lesions associated with the administration of coumarin to rats for up to 2 years occurred in the liver, kidney, and forestomach. While the hepatic lesions were seen in all groups of males, they occurred only in the 50 and 100 mg/kg females. The lesions consisted of a spectrum of changes including hepatocellular necrosis, fibrosis, cytologic alteration, and increased severity of bile duct hyperplasia. The incidences of hepatocellular neoplasms were not increased in dosed rats. There was a chemical-related increase in the average severity of nephropathy in all groups of dosed male and female rats. There were corresponding increased incidences of parathyroid gland hyperplasia in all groups of dosed males, probably as a result of compromised renal function. In the standard evaluation of single kidney sections, a low incidence of renal adenomas was seen in all groups of males and in 100 mg/kg females (males: vehicle control, 1/49; 25 mg/kg, 2/50; 50 mg/kg, 2/51; 100 mg/kg, 1/50; females: 0/49, 0/50, 0/50, 2/49). An evaluation of step sections identified additional individuals with renal tubule focal hyperplasia (males: 2/49, 12/50, 10/51, 6/50; females: 1/49, 0/50, 4/50, 2/49) and adenoma (males: 0/49, 4/50, 5/51, 4/50; females: 0/49, 0/50, 1/50,1/49) in the dosed groups. The incidences of forestomach ulcers in all groups of dosed male rats and in 100 mg/kg female rats were significantly greater than those of the controls (males: 7/48, 24/50, 35/51, 34/50; females: 1/48, 1/49, 6/50, 9/48). STOP-EXPOSURE EVALUATION A group of 40 male rats received 100 mg/kg coumarin in corn oil by gavage for 9 months, when 20 of the animals were necropsied and evaluated. The remainder of the male rats received only the corn oil vehicle during the 15-month recovery period. Similarly, a group of 30 male rats received 100 mg/kg coumarin in corn oil by gavage for 15 months, when 10 of the rats were necropsied and evaluated. The remaining 20 rats received only corn oil during the 9-month recovery period. A group of 20 vehicle control male rats were necropsied at 9 months, and another 10 vehicle control male rats were necropsied at 15 months. While chemical-related hepatic lesions were seen at both the 9- and 15-month interim evaluations, the incidences and severities of these lesions following the recovery period were generally similar to controls. Thus, the hepatic lesions produced by 9 or 15 months of exposure were reversible. In contrast to the liver lesions, the severity of nephropathy in male rats following the recovery period was significantly greater than that of males examined at the 9- and 15-month interim evaluations. This is not unexpected, since nephropathy is a progressive degenerative disease that naturally increases in severity with age. The incidence of renal tubule hyperplasia in the 15-month stop-exposure group (dosed for 15 months followed by the recovery period) and the incidence of renal tubule adenoma in the 9-month stop-exposure group were significantly greater than those of the control group. 2-YEAR STUDY IN MICE Groups of 70 male and 70 female mice were administered coumarin in corn oil by gavage at doses of 0, 50, 100, or 200 mg per kg body weight for up to 2 years. After 15 months, 19 or 20 mice from each group were evaluated. Survival, Body Weights, and Clinical Findings Survival of dosed male and female mice was similar to that of the controls (males: vehicle control, 43/50; 50 mg/kg, 47/50; 100 mg/kg, 42/50; 200 mg/kg, 37/51; females: 33/50, 40/50, 42/51, 28/51). The mean body weights of 200 mg/kg male and female mice were lower than those of controls throughout much of the study. There were no clinical findings related to chemical administration. Hematology and Clinical Chemistry Mean erythrocyte volume, mean erythrocyte hemoglobin, and hematocrit of 200 mg/kg males and mean erythrocyte volume of 200 mg/kg females were significantly lower than those of the controls. Blood platelet counts of 200 mg/kg males and females were significantly higher than those of controls. There were no biologically significant differences in enzyme activities between dosed and control mice. Pathology Findings The principal toxic lesions associated with the administration of coumarin to mice occurred in the liver. The incidences of centrilobular hypertrophy in 100 and 200 mg/kg males and 200 mg/kg females were significantly greater than those of controls. The incidences of syncytial alteration in all male dose groups and in 200 mg/kg females were also significantly greater than controls. The incidences of eosinophilic foci, a putative preneoplastic lesion, and of hepatocellular adenoma were significantly greater in the 50 and 100 mg/kg females. Hepatocellular carcinomas occurred with low incidences in the dosed females, but none occurred in the controls. The overall incidence of hepatocellular neoplasms (benign and malignant combined) in the 50 and 100 mg/kg females (control, 8/50; 50 mg/kg, 27/49; 100 mg/kg, 31/51; 200 mg/kg, 13/50) exceeds the range in historical controls (range 2%-34%; 129/898, 14.4%) from recent NTP studies. The reason for a lack of liver response in 200 mg/kg female mice is not known, but may be due in part to the decrease in body weight. While the incidences of eosinophilic foci were marginally greater in dosed male mice, the incidences of hepatocellular neoplasms were similar among the dosed and control groups. The incidences of alveolar/bronchiolar adenomas were significantly greater in 200 mg/kg male and female mice than in the controls. Further, the incidence of alveolar/bronchiolar carcinoma in 200 mg/kg females was also significantly greater than in controls. The overall incidence of pulmonary neoplasms (benign and malignant combined) in the 200 mg/kg groups (males: 14/50, 9/50,15/50, 25/51; females: 2/51, 5/49, 7/49, 27/51) exceeds the range in historical controls (males: range 6%-28%; 166/900, 18.4%; females: range 0%-14%; 58/899, 6.5%) from recent NTP studies. The incidence of squamous cell papilloma of the forestomach in 50 mg/kg males was greater than that of the controls (2/50, 8/50, 2/50, 0/51) and also exceeds the range of this neoplasm in control male mice from recent NTP studies (range 0%-14%; 27/902, 3.0%). The incidence of squamous cell papilloma of the forestomach in 50 mg/kg female mice was also slightly increased (1/52, 5/50, 2/51, 2/51); however, the incidence did not exceed the NTP historical range (27/901, 3%; range, 0%-10%). GENETIC TOXICOLOGY Coumarin induced gene mutations in Salmonella typhimurium strain TA100 in the presence, but not in the absence, of exogenous metabolic activation (S9); no mutations were induced in strains TA98, TA1535, or TA1537, with or without S9. In Chinese hamster ovary cells, coumarin induced sister chromatid exchanges in the absence of S9, and chromosomal aberrations in the presence of S9. Coumarin did not induce sex-linked recessive lethal mutations in germ cells of male Drosophila melanogaster treated either as adults by feeding or injection, or as larvae by feeding. No increase in the frequency of micronucleated erythrocytes was observed in peripheral blood of male and female B6C3F1 mice administered coumarin by gavage for 13 weeks. CONCLUSIONS Under the conditions of these 2-year gavage studies there was some evidence of carcinogenic activity of coumarin in male F344/N rats based on increased incidences of renal tubule adenomas. There was equivocal evidence of carcinogenic activity of coumarin in female F344/N rats based on a marginally increased incidence of renal tubule adenomas. There was some evidence of carcinogenic activity of coumarin in male B6C3F1 mice based on the increased incidence of alveolar/bronchiolar adenomas. There was clear evidence of carcinogenic activity of coumarin in female B6C3F1 mice based on increased incidences of alveolar/bronchiolar adenomas, alveolar/bronchiolar carcinomas, and hepatocellular adenomas. The marginally increased incidences of squamous cell papillomas of the forestomach in male and female mice receiving 50 mg/kg may have been related to coumarin administration. The administration of coumarin to rats was also associated with an increased severity of nephropathy in the kidney and of bile duct hyperplasia in the liver, increased incidences of ulcers of the forestomach, and necrosis, fibrosis, and cytologic alteration of the liver. Administration of coumarin to mice was also associated with centrilobular hypertrophy, syncytial alteration, and eosinophilic focus in the liver.

Abstract  The C3H/HeJ (C3H), A/J and BALB/cByJ (BALB) mouse strains are respectively resistant, sensitive and intermediate regarding the induction of lung tumors by urethane. The phenotypic difference between C3H and A/J is largely determined by the Pas1 (Pulmonary adenoma susceptibility 1) gene on chromosome 6, the A/J allele of which dominantly increases the tumor burden. We recently found that BALB mice possess a unique lung tumor resistance gene on chromosome 18, designated Par2 (Pulmonary adenoma resistance 2), which partially, but dominantly suppresses the sensitive phenotype of A/J mice (Oncogene 13: 1599-1604, 1996). It has, however, remained unclear why BALB mice carrying the Par2 gene are significantly more sensitive to urethane-induced lung carcinogenesis than C3H mice that have no dominant lung tumor resistance genes. In the present study, using (C3H x BALB)F1 x C3H backcross mice treated with urethane, we demonstrated that BALB mice possess the disease allele of the Pas1 gene despite their 15-fold more resistance relative to A/J mice (LOD = 22.6). The BALB Par2 allele only significantly reduced the mean lung tumor multiplicity (LOD = 4.4) in the backcross population carrying the BALB allele of Pas1, indicating that the intermediate BALB phenotype may at least in part be the result of interactions between these two dominant genes. While the BALB Pas1 allele increased both the mean multiplicity and size of lung tumors, the BALB Par2 allele affected only the mean tumor multiplicity, implying that they are involved in different stages of multi-step lung carcinogenesis. In addition, we found that 68% of lung tumors from the BALB Pas1-positive backcross mice contained activating point mutations of the Kras2 oncogene, tightly linked to the Pas1 locus, whereas these genetic alterations were absent in tumors from BALB Pas1-negative mice. The Par2 genotype exhibited no effect on this parameter. Since the activating point mutations were observed exclusively in the BALB allele as already reported with lung tumors in (C57BL/6J x BALB/cJ)F1 mice, BALB Pas1 or possibly Kras2 itself may confer selective growth advantage on the affected urethane-initiated lung lesions.

Abstract  Ethylbenzene is mainly used in the manufacture of styrene. Ethylbenzene is also a major component of mixed xylenes used as solvents in agricultural and home insecticide sprays, rubber and chemical manufacturing, and household degreasers, paints, adhesives, and rust preventives. Ethylbenzene is also used as an antiknock agent in aviation and motor fuels. Ethylbenzene was nominated for study by the National Institute for Occupational Safety and Health (NIOSH) and the Occupational Safety and Health Administration (OSHA) because of its potential for widespread human exposure and because of its structural similarity to benzene and toluene. Male and female F344/N rats and B6C3F1 mice were exposed to ethylbenzene (greater than 99% pure) by inhalation for 2 years. Genetic toxicology studies were conducted in Salmonella typhimurium, mouse lymphoma cells, cultured Chinese hamster ovary cells, and mouse peripheral blood erythrocytes. In previously reported 13-week toxicity studies in which F344/N rats and B6C3F1 mice were exposed to ethylbenzene by whole body inhalation exposure, no histopathologic changes were observed (NTP, 1992). 2-YEAR STUDY IN RATS: Groups of 50 male and 50 female F344/N rats were exposed to 0, 75, 250, or 750 ppm ethylbenzene by inhalation, 6 hours per day, 5 days per week, for 104 weeks. Survival, Body Weights, and Clinical Findings Survival of male rats in the 750 ppm group was significantly less than that of the chamber controls. Mean body weights of 250 and 750 ppm males were generally less than those of the chamber controls beginning at week 20. Mean body weights of exposed groups of females were generally less than those of chamber controls during the second year of the study. Pathology Findings In male rats exposed to 750 ppm, the incidences of renal tubule adenoma and adenoma or carcinoma (combined) were significantly greater than the chamber control incidences. In addition, the incidence of renal tubule hyperplasia in 750 ppm males was significantly greater than that in the chamber controls. The findings from an extended evaluation (step section) of the kidneys showed a significant increase in the incidences of renal tubule adenoma and hyperplasia in 750 ppm males and females; the incidence of renal tubule adenoma or carcinoma (combined) was significantly increased in 750 ppm males. The severities of nephropathy in 750 ppm male and all exposed female rats were significantly increased relative to the chamber controls. The incidence of interstitial cell adenoma in the testis of 750 ppm males was significantly greater than that in the chamber control group and slightly exceeded the historical control range for inhalation studies. 2-YEAR STUDY IN MICE: Groups of 50 male and 50 female B6C3F1 mice were exposed to 0, 75, 250, or 750 ppm ethylbenzene by inhalation, 6 hours per day, 5 days per week, for 103 weeks. Survival, Body Weights, and Clinical Findings Survival of exposed groups of male and female mice was similar to that of the chamber controls. Mean body weights of female mice exposed to 75 ppm were greater than those of the chamber controls from week 72 until the end of the study. Pathology Findings In 750 ppm males, the incidences of alveolar/ bronchiolar adenoma and alveolar/bronchiolar adenoma or carcinoma (combined) were significantly greater than those in the chamber control group but were within the NTP historical control ranges. The incidence of alveolar epithelial metaplasia in 750 ppm males was significantly greater than that in the chamber controls. In 750 ppm females, the incidences of hepatocellular adenoma and hepatocellular adenoma or carcinoma (combined) were significantly greater than those in the chamber control group but were within the historical control ranges. The incidence of eosinophilic foci in 750 ppm females was significantly increased compared to that in the chamber controls. There was a spectrum of nonneoplastic liver changes related to ethylbenzene exposure in male mice, including syncytial alteration of hepatocytes, hepatocellular hypertrophy, and hepatocyte necrosis. rosis. The incidences of hyperplasia of the pituitary gland pars distalis in 250 and 750 ppm females and the incidences of thyroid gland follicular cell hyperplasia in 750 ppm males and females were significantly increased compared to those in the chamber control groups. GENETIC TOXICOLOGY: Ethylbenzene gave little indication of mutagenicity, in vitro or in vivo. No induction of mutations was noted in Salmonella typhimurium strain TA97, TA98, TA100, or TA1535 with or without S9 metabolic activation, and no increases in sister chromatid exchanges or chromosomal aberrations were observed in cultured Chinese hamster ovary cells treated with ethylbenzene, with or without S9. In the mouse lymphoma assay, a significant mutagenic response was noted in the absence of S9, but only at the highest nonlethal dose tested and with accompanying cytotoxicity; the test was not performed with S9. No increases in the frequency of micronucleated erythrocytes were observed in vivo in peripheral blood samples from male and female mice exposed to ethylbenzene for 13 weeks. CONCLUSIONS: Under the conditions of these 2-year inhalation studies, there was clear evidence of carcinogenic activity of ethylbenzene in male F344/N rats based on increased incidences of renal tubule neoplasms. The incidences of testicular adenoma were also increased. There was some evidence of carcinogenic activity of ethylbenzene in female F344/N rats based on increased incidences of renal tubule adenomas. There was some evidence of carcinogenic activity of ethylbenzene in male B6C3F1 mice based on increased incidences of alveolar/bronchiolar neoplasms. There was some evidence of carcinogenic activity of ethylbenzene in female B6C3F1 mice based on increased incidences of hepatocellular neoplasms. Exposure of male and female rats to ethylbenzene resulted in increased incidences of renal tubule hyperplasia and increased severities of nephropathy. Exposure of male mice to ethylbenzene resulted in increased incidences of alveolar epithelial metaplasia, syncytial alteration of hepatocytes, hepatocellular hypertrophy, hepatocyte necrosis, and thyroid gland follicular cell hyperplasia. In female mice, ethylbenzene exposure resulted in increased incidences of eosinophilic foci of the liver, pituitary gland pars distalis hyperplasia, and thyroid gland follicular cell hyperplasia. Synonyms: EB; ethylbenzol; phenylethane

Abstract  Naphthalene, a white, crystalline powder, is used as a moth repellent and in the manufacture of phthalic and anthranilic acids, naphthylamines, and synthetic resins. The 2-year studies were conducted by exposing groups of male and female B6C3F1 mice to naphthalene (>99% pure) vapor for 6 hours daily, 5 days per week, for 104 weeks. Genetic toxicology studies were conducted in Salmonella typhimurium and Chinese hamster ovary cells. 2-Year Studies: Groups of male and female mice were exposed to atmospheres containing 0 (75 mice per group), 10 (75 mice per group), or 30 ppm (150 mice per group) naphthalene. Mice from each group were included for 14-day hematology evaluations (male: 0 ppm, 5 animals; 10 ppm, 4; 30 ppm, 10; female: 0 ppm, 4; 10 ppm, 5; 30 ppm, 10). Mean body weights of exposed mice were slightly lower than those of controls throughout the studies. Survival of male control mice was significantly less than that of exposed mice; the lower survival was the result of wound trauma and secondary infections related to fighting among the group-housed mice (0 ppm, 26/70, 37%; 10 ppm, 52/69, 75%; 30 ppm, 118/133, 89%). Survival of exposed female mice was similar to that of controls (59/69, 86%; 57/65, 88%; 102/135, 76%). Neoplastic and Nonneoplastic Effects in the 2-Year Studies: No increase in tumor incidence related to naphthalene administration was observed in male mice. In females, the incidence of pulmonary alveolar/bronchiolar adenomas was significantly greater in the high-dose group than in the controls (5/69, 7%; 2/65, 3%; 28/135, 21%). One other high-dose female had an alveolar/bronchiolar carcinoma. The combined incidence of alveolar/ bronchiolar adenomas and carcinomas in the high-dose females was above those for control female B6C3F1 mice from NTP feed, water, and inhalation studies (91/1,166, 7.8%, range 0%-16%). These lung tumors were attributed to naphthalene exposure. Nonneoplastic lesions attributed to naphthalene exposure were observed in the nose and lungs of mice of both sexes. In the nose, naphthalene exposure was associated with an increase in the incidence and severity of chronic inflammation, metaplasia of the olfactory epithelium, and hyperplasia of respiratory epithelium. Chronic inflammation in the lung was associated with chemical exposure. Genetic Toxicology: Naphthalene was negative for the induction of gene mutations in Salmonella typhimurium strains TA100, TA1535, TA1537, and TA98 with and without exogenous metabolic activation (S9). In cytogenetic tests with Chinese hamster ovary cells, naphthalene induced sister chromatid exchanges with and without S9 activation. Exposure to naphthalene induced a significant increase in chromosomal aberrations in Chinese hamster ovary cells in the presence of S9. Conclusions: Under the conditions of these 2-year inhalation studies, there was no evidence of carcinogenic activity of naphthalene in male B6C3F1 mice exposed to 10 or 30 ppm. There was some evidence of carcinogenic activity of naphthalene in female B6C3F1 mice, based on increased incidences of pulmonary alveolar/ bronchiolar adenomas. In both male and female mice, naphthalene caused increased incidences and severity of chronic inflammation, metaplasia of the olfactory epithelium, and hyperplasia of the respiratory epithelium in the nose and chronic inflammation in the lungs. Synonyms: Naphthalin, Naphthene, Tar Camphor

Abstract  The mapping near Kras2 of pulmonary adenoma susceptibility 1 (Pas1), a major locus affecting inherited predisposition to lung cancer in mice prompted us to test the homologous human region (12p12) for association with lung adenocarcinoma, by a population-based study. We genotyped 213 lung adenocarcinoma patients and 219 healthy blood donor subjects for five polymorphic markers mapping in the putative region of interest. Three marker polymorphisms, located in a region spanning approximately 700 kb, were significantly associated with lung adenocarcinoma risk. Furthermore, polymorphisms in KRAS2 and PTHLH loci were also associated with tumor prognosis. These results suggest the existence of a human Pas1 homologous locus on chromosome 12p12.

Abstract  Pulmonary adenoma susceptibility 1 (Pas1) is a major locus affecting inherited predisposition to the development of lung adenocarcinoma in mice, and is mapped to chromosome 6q near the Kras2 gene. However, it is still unclear whether the PAS1 locus on human chromosome 12p11.2-p12.1, the region showing synteny to the mouse Pas1 region, is involved in susceptibility to human lung adenocarcinoma development. Thus, we conducted a case-control study of 100 lung adenocarcinoma cases and 100 controls using 20 highly polymorphic microsatellite markers dispersed in a 13 cM region covering a putative PAS1 locus. The differences in the allele and genotype distributions were observed at several loci, and the difference was at a maximum at the D12S1034 locus (P = 0.034 and P = 0.036, respectively). The differences in the allele and genotype distributions at D12S1034 remained significant in the analysis in which 239 lung adenocarcinoma cases and 63 controls were added to the 100 cases and 100 controls used for the initial screening (P = 0.031 and P = 0.027, respectively). The D12S1034 locus was located 800-1350 kb proximal to the KRAS2 locus, and in the region syntenic to the core Pas1 region of approximately 1.5 Mb in size where a single haplotype is shared by several mouse-inbred strains susceptible to lung adenocarcinoma development. These results indicate that the PAS1 locus is located in the vicinity of D12S1034 and a genetic variation(s) at this locus is involved in susceptibility to human lung adenocarcinoma.