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  A >20-fold increase in X-ray computed tomography (CT) use during the last 30 years has caused considerable concern because of the potential carcinogenic risk from these CT exposures. Estimating the carcinogenic risk from high-energy, single high-dose exposures obtained from atomic bomb survivors and extrapolating these data to multiple low-energy, low-dose CT exposures using the Linear No-Threshold (LNT) model may not give an accurate assessment of actual cancer risk. Recently, the National Lung Cancer Screening Trial (NLST) reported that annual CT scans of current and former heavy smokers reduced lung cancer mortality by 20%, highlighting the need to better define the carcinogenic risk associated with these annual CT screening exposures. In this study, we used the bitransgenic CCSP-rtTA/Ki-ras mouse model that conditionally expresses the human mutant Ki-ras(G12C) gene in a doxycycline-inducible and lung-specific manner to measure the carcinogenic risk of exposure to multiple whole-body CT doses that approximate the annual NLST screening protocol. Irradiated mice expressing the Ki-ras(G12C) gene in their lungs had a significant (P = 0.01) 43% increase in the number of tumors/mouse (24.1 ± 1.9) compared to unirradiated mice (16.8 ± 1.3). Irradiated females had significantly (P < 0.005) more excess tumors than irradiated males. No tumor size difference or dose response was observed over the total dose range of 80-160 mGy for either sex. Irradiated bitransgenic mice that did not express the Ki-ras(G12C) gene had a low tumor incidence (≤ 0.1/mouse) that was not affected by exposure to CT radiation. These results suggest that (i) estimating the carcinogenic risk of multiple CT exposures from high-dose carcinogenesis data using the LNT model may be inappropriate for current and former smokers and (ii) any increased carcinogenic risk after exposure to fractionated low-dose CT-radiation may be restricted to only those individuals expressing cancer susceptibility genes in their tissues at the time of exposure.

Abstract  Mutation in RAS proteins is one of the most common genetic alterations observed in human and experimentally induced rodent cancers. In vivo, oncogenic mutations have been shown to occur at exons 12, 13, and 61, resulting in any 1 of 19 possible point mutations in a given tumor for a specific RAS isoform. While some studies have suggested a possible role of different mutant alleles in determining tumor severity and phenotype, no general consensus has emerged on the oncogenicity of different mutant alleles in tumor formation and progression. Part of this may be due to a lack of a single, signature pathway that shows significant alterations between different mutations. Rather, it is likely that subtle differences in the activation, or lack thereof, of downstream effectors by different RAS mutant alleles may determine the eventual outcome in terms of tumor phenotype. This paper reviews our current understanding of the potential role of different RAS mutations on tumorigenesis, highlights studies in model cell culture and in vivo systems, and discusses the potential of expression array and computational network modeling to dissect out differences in activated RAS genes in conferring a transforming phenotype.

Abstract  Styrene is one of the most important industrial intermediates consumed in the world. Human exposure to styrene occurs mainly in the reinforced plastics industry, particularly in developing countries. Styrene has been found to be hepatotoxic and pneumotoxic in humans and animals. The biochemical mechanisms of styrene-induced toxicities remain unknown. Albumin and hemoglobin adduction derived from styrene oxide, a major reactive metabolite of styrene, has been reported in blood samples obtained from styrene-exposed workers. The objectives of the current study focused on cellular protein covalent binding of styrene metabolite and its correlation with cytotoxicity induced by styrene. We found that radioactivity was bound to cellular proteins obtained from mouse airway trees after incubation with (14)C-styrene. Microsomal incubation studies showed that the observed protein covalent binding required the metabolic activation of styrene. The observed radioactivity binding in protein samples obtained from the cultured airways and microsomal incubations was significantly suppressed by co-incubation with disulfiram, a CYP2E1 inhibitor, although disulfiram apparently did not show a protective effect against the cytotoxicity of styrene. A 2-fold increase in radioactivity bound to cellular proteins was detected in cells stably transfected with CYP2E1 compared to the wild-type cells after (14)C-styrene exposure. With the polyclonal antibody developed in our lab, we detected cellular protein adduction derived from styrene oxide at cysteinyl residues in cells treated with styrene. Competitive immunoblot studies confirmed the modification of cysteine residues by styrene oxide. Cell culture studies showed that the styrene-induced protein modification and cell death increased with the increasing concentration of styrene exposure. In conclusion, we detected cellular protein covalent modification by styrene oxide in microsomal incubations, cultured cells, and mouse airways after exposure to styrene and found a good correlation between styrene-induced cytotoxicity and styrene oxide-derived cellular protein adduction.

Abstract  National Toxicology Program (NTP) inhalation studies demonstrated that cumene significantly increased the incidence of alveolar/bronchiolar adenomas and carcinomas in B6C3F1 mice. Cumene or isopropylbenzene is a component of crude oil used primarily in the production of phenol and acetone. The authors performed global gene expression analysis to distinguish patterns of gene regulation between cumene-induced tumors and normal lung tissue and to look for patterns based on the presence or absence of K-ras and p53 mutations in the tumors. Principal component analysis segregated the carcinomas into groups with and without K-ras mutations, but failed to separate the tumors based on p53 mutation status. Expression of genes associated with the Erk MAP kinase signaling pathway was significantly altered in carcinomas with K-ras mutations compared to tumors without K-ras mutations or normal lung. Gene expression analysis also suggested that cumene-induced carcinomas with K-ras mutations have greater malignant potential than those without mutations. In addition, significance analysis of function and expression (SAFE) demonstrated expression changes of genes regulated by histone modification in carcinomas with K-ras mutations. The gene expression analysis suggested the formation of alveolar/bronchiolar carcinomas in cumene-exposed mice typically involves mutation of K-ras, which results in increased Erk MAP kinase signaling and modification of histones.

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  Pregnant C3H/HeNCr MTV- mice were given a single intraperitoneal injection of 0.5 mmol N-nitrosoethylurea/kg on days 14, 16, or 18 of gestation. Six of the male offspring were sacrificed for study at the ages of 2, 4, 8, 16, 32, and 52 weeks. Grossly visible lung tumors were counted and all lungs were sectioned completely, saving every tenth section for histologic evaluation. All N-nitrosoethylurea-induced mouse lung tumors have previously been shown to originate from alveolar type II cells. Lung tumors were diagnosed as solid, papillary, or mixed solid/papillary types, and at the largest area of each tumor, the perimeter was measured and compared with the number of sections per tumor. The fraction of tumors detected grossly depended on size and, on average, only 51% of neoplasms present were detected macroscopically. A significant correlation was seen between the mean number of histological sections and perimeter length per tumor, in particular for small and medium sized papillary neoplasms. The growth of solid tumors was limited to a maximum size, after which they progressed towards papillary types. The numbers of transplacentally induced mouse lung tumors were distributed in direct proportion to the weight of the individual lung lobes, unrelated to day of treatment of type or tumor. Tumor biology depended on the day of treatment reflecting numbers of degree of differentiation of fetal alveolar type II cells, i.e., the target cell: most tumors developed in offspring treated on day 16, tumor size was greater and progression from solid to papillary neoplasms faster at earlier treatments, increase in tumor multiplicity postnatally was only seen in mice treated late in gestation, and mice treated on day 14 or day 16 showed a consistent ratio of solid to papillary tumors.

Abstract  Urethan (CAS: 51-79-6)-induced pulmonary adenomas that arise from either alveolar type II pneumocytes of bronchiolar Clara cells have distinct histologic growth patterns and can thus be distinguished from each other. Strain differences were reported in the relative proportions of tumors derived from each cell type when these tumors were examined 14 weeks after urethan treatment. For determination as to whether these proportions changed at later stages of growth, tumors in A/J, SWR/J, RIIIS/J, BALB/cByJ, 129/J, and C57BL/6J mice were examined at 28 and 56 weeks after urethan treatment. Tumor multiplicity increased with time in all strains. Small tumors were predominantly type II cell derived in most strains, whereas medium and large tumors were derived mainly from Clara cells. This suggests that type II tumors are restricted in growth while Clara tumors may continue to grow. Medium and large Clara-derived tumors made up a larger proportion of the total tumor population at 28 and 56 weeks than at 14 weeks post urethan, even in A/J mice that typically display 85% type II cell-derived tumors at the earlier time. Several large Clara cell-derived tumors exhibited characteristics of cancer, whereas no type II cell-derived tumor was observed to do so. These results implicate the bronchiolar Clara cell as the predominant cell of origin of pulmonary adenocarcinomas in mice.

Abstract  The International Programme on Chemical Safety (IPCS) is leading an activity to harmonize approaches to cancer risk assessment as a part of its larger project on the Harmonization of Approaches to the Assessment of Risk from Exposure to Chemicals. Through a series of workshops and the evaluation of case studies, a number of key components of risk assessments relating to harmonization were identified: transparency, terminology, weight of evidence, flexibility, and accessibility/communication. A major impediment to harmonization identified in the consideration of weight of evidence was the evaluation of mode of action. To address this need, a conceptual framework was developed, based on the general principles involved in considering the chemical induction of a specific tumor in animals. This is based partly on the Bradford Hill criteria for causality as modified by Faustman et al. (1997) for developmental toxicity. The framework is described in this paper followed by a worked example. It is recognized that the framework addresses only one stage in the overall characterization of hazard to humans of chemical carcinogens. Another important but separate step is the assessment of relevance to humans. This is a priority area for future work in this project.

Abstract  The continued progress of modelling lung cancer in mice has led not only to new means of understanding the molecular pathways governing human lung cancer, but it has also created a vast reservoir of alternative tools to test treatments against this malignancy. More sophisticated somatic mouse models for nonsmall cell lung cancer, small cell lung cancer and pulmonary squamous cell carcinoma have been generated that closely mimic human lung cancer. These models enable us to identify the cells of origin and the role of stem cells in the maintenance of the various types of lung cancer. Moreover, results of lung cancer intervention studies are now starting to reveal the full potential of these somatic mouse models as powerful pre-clinical models.

Abstract  Identifying the cells of origin of lung cancer may lead to new therapeutic strategies. Previous work has focused upon the putative bronchoalveolar stem cell at the bronchioalveolar duct junction as a cancer cell of origin when a codon 12 K-Ras mutant is induced via adenoviral Cre inhalation. In the present study, we use two "knock-in" Cre-estrogen receptor alleles to inducibly express K-RasG12D in CC10(+) epithelial cells and Sftpc(+) type II alveolar cells of the adult mouse lung. Analysis of these mice identifies type II cells, Clara cells in the terminal bronchioles, and putative bronchoalveolar stem cells as cells of origin for K-Ras-induced lung hyperplasia. However, only type II cells appear to progress to adenocarcinoma.

Abstract  To investigate the role of an activated K-Ras gene in the initiation and maintenance of lung adenocarcinomas, we developed transgenic mice that express murine K-Ras4b(G12D) under the control of doxycycline in type II pneumocytes. Focal proliferative lesions of alveolar type II pneumocytes were observed as early as seven days after induction with doxycycline; after two months of induction, the lungs contained adenomas and adenocarcinomas, with focal invasion of the pleura at later stages. Removal of doxycycline caused a rapid fall in levels of mutant K-Ras RNA and concomitant apoptotic regression of both the early proliferative lesions and the tumors. Tumor burden was dramatically decreased by three days after withdrawal, and tumors were undetectable after one month. When similar experiments were performed with animals deficient in either the p53 gene or the Ink4A/Arf locus, tumors arose more quickly (within one month of exposure to doxycycline) and displayed more obvious histological features of malignancy; nevertheless, these tumors also regressed rapidly when the inducer was removed, implying that continued production of mutant K-Ras is necessary to maintain the viability of tumor cells in the absence as well as the presence of tumor suppressor genes. We also show that the appearance and regression of these pulmonary tumors can be readily monitored in anesthetized transgenic animals by magnetic resonance imaging.

Abstract  In the A/J strain of mice, urethane (ethyl carbamate) induces lung hyperplasia, adenoma, and adenocarcinoma in a time-dependent manner. These distinct morphological stages may correlate with sequential molecular genetic changes in this mouse model. To test this hypothesis, we investigated the presence of mutations involving Ki-ras and p53 in urethane-induced lung lesions in A/J mice at early and late stages of tumorigenesis. We precisely microdissected 40 lung lesions from paraffin-embedded sections. Ki-ras mutations around codon 61 and p53 mutations in exons 5-8 were identified by polymerase chain reaction-single-strand conformation polymorphism and DNA sequencing techniques. In 29 early-stage lung lesions classified as hyperplasias (seven) or adenomas (22), we observed 19 Ki-ras mutations (66%), including three silent mutations and one double mutation at different codons, and one silent p53 mutation (3.5%). In 11 late-stage adenomas, we identified nine activating Ki-ras mutations (82%) and four missense p53 mutations (36%). These results indicate that Ki-ras mutations arise early, whereas p53 mutations occur relatively late during the benign stages of urethane-induced lung carcinogenesis in A/J mice.

Abstract  About 30% of human tumours carry ras gene mutations. Of the three genes in this family (composed of K-ras, N-ras and H-ras), K-ras is the most frequently mutated member in human tumours, including adenocarcinomas of the pancreas ( approximately 70-90% incidence), colon ( approximately 50%) and lung ( approximately 25-50%). To construct mouse tumour models involving K-ras, we used a new gene targeting procedure to create mouse strains carrying oncogenic alleles of K-ras that can be activated only on a spontaneous recombination event in the whole animal. Here we show that mice carrying these mutations were highly predisposed to a range of tumour types, predominantly early onset lung cancer. This model was further characterized by examining the effects of germline mutations in the tumour suppressor gene p53, which is known to be mutated along with K-ras in human tumours. This approach has several advantages over traditional transgenic strategies, including that it more closely recapitulates spontaneous oncogene activation as seen in human cancers.

Abstract  Lung cancer kills more Americans yearly than any other neoplastic process. Mortality rates have changed little over the past several decades, despite improvements in surgical techniques, radiation therapy and chemotherapy. The identification of mutations in oncogenes and tumor suppressor genes in human lung tumor specimens, including K-ras, p53, p16INK4a and Rb, offers molecular explanations for tumor development and resistance to therapy. Mouse models of human lung cancer may advance our understanding of this disease. The examination of mice which develop lung cancer either spontaneously or due to carcinogen exposure, and the creation of mouse strains harboring the specific genetic mutations found in human lung cancer are among strategies being pursued.

Abstract  Immunohistochemistry can serve as a valuable adjunct to rodent tumor pathology. Specific antigens may be localized to cells and tissues in normal organs, preneoplastic lesions, and benign and malignant tumors. The immunoreactivity of polyclonal and monoclonal antibodies to these antigens provide a more accurate basis for tumor diagnosis and aid in understanding pathogenesis. Ultimately, the application of more precise understanding of tumor histogenesis and diagnosis will lead to more accurate interpretations of tumor incidence data for safety assessment in toxicology.

Abstract  Lung cancer rates have peaked among men in many areas of the world, but rates among women continue to rise. Most lung cancers are squamous cell carcinoma, small cell carcinoma, or adenocarcinoma; trends vary according to type. We compiled population-based morphology-specific incidence data from registries contributing to the International Agency for Research on Cancer (IARC) databases. Unspecified cancers and carcinomas were reallocated based on a registry, time period, sex and age group-specific basis. Where available, data from several registries within a country were pooled for analysis. Rates per 100,000 person-years for 1980-1982 to 1995-1997 were age-adjusted by the direct method using the world standard. Squamous cell carcinoma rates among males declined 30% or more in North America and some European countries while changing less dramatically in other areas; small cell carcinoma rates decreased less rapidly. Squamous and small cell carcinoma rates among females generally rose, with the increases especially pronounced in the Netherlands and Norway. In contrast, adenocarcinoma rates rose among males and females in virtually all areas, with the increases among males exceeding 50% in many areas of Europe; among females, rates also rose rapidly and more than doubled in Norway, Italy and France. Rates of all lung cancer types among women and adenocarcinoma among men continue to rise despite declining cigarette use in many Western countries and shifts to filtered/low-tar cigarettes. Renewed efforts toward cessation and prevention are mandatory to curb the prevalence of cigarette smoking and to reduce lung cancer rates eventually.

Abstract  Male A/J mice were injected i.p. with a single dose of urethan and fed 0.75% butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA) or ethoxyquin in the diet. All animals were killed 4 months after urethan and the number of lung tumors counted. Exposure to BHT, but not to BHA or ethoxyquin significantly enhanced formation of lung tumors if animals were given the BHT-containing diet once a week for 8 consecutive weeks or were kept on it continuously for 8 weeks. Prefeeding mice with BHT had no effect on tumor formation but prefeeding BHA reduced the number of tumors formed by urethan. It is concluded from this and previous work that in mouse lung BHT enhances tumor formation regardless of route of administration and over a 100-fold dose range.

Abstract  The use of average qualitative concordance between two bioassay endpoints is considered, with emphasis directed at agreement between rats and mice from results of long-term carcinogenicity studies. It is noted that concordance varies as a function of the underlying potency or toxicity of the chemicals over which the averaging is performed. Thus, the averaging process dilutes large observed concordances from potent chemicals, and possibly inflates lower observed concordances from weakly active chemicals. Stratification over some measure of potency is suggested as a method for taking these effects into account. Statistical simulations of concordance analyses limited to low-potency ranges are employed to examine the concordance measure in greater detail. It is seen that at low potencies, observed concordance is consistently underestimated, reaching maximum levels of only about 80%.

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.