Abstract  Styrene (ST) is an important industrial chemical. In long-term inhalation studies, ST-induced lung tumors in mice but not in rats. To test the hypothesis that the lung burden by the reactive metabolite styrene-7,8-oxide (SO) would be most relevant for the species-specific tumorigenicity, we investigated the SO burden in isolated lungs of male Sprague-Dawley rats and in-situ prepared lungs of male B6C3F1 mice ventilated with air containing vaporous ST and perfused with a modified Krebs-Henseleit buffer (37°C). Styrene vapor concentrations were determined in air samples collected in the immediate vicinity of the trachea. They were almost constant during each experiment. Styrene exposures ranged from 50 to 980 ppm (rats) and from 40 to 410 ppm (mice). SO was quantified from the effluent perfusate. Lungs of both species metabolized ST to SO. After a mathematical translation of the ex-vivo data to ventilation and perfusion conditions as they are occurring in vivo, a species comparison was carried out. At ST concentrations of up to 410 ppm, mean SO levels in mouse lungs ranged up to 0.45 nmol/g lung, about 2 times higher than in rat lungs at equal conditions of ST exposure. We conclude that the species difference in the SO lung burden is too small to consider the genotoxicity of SO as sufficient for explaining the fact that only mice developed lung tumors when exposed to ST. Another cause is considered as driving force for lung tumor development in the mouse.

Abstract  The lung, which is in intimate contact with the external environment, is exposed to a number of toxicants both by virtue of its large surface area and because it receives 100% of the cardiac output. Lung diseases are a major disease entity in the U.S. population ranking third in terms of morbidity and mortality. Despite the importance of these diseases, key issues remain to be resolved regarding the interactions of chemicals with lung tissue and the factors that are critical determinants of chemical-induced lung injury. The importance of cytochrome P450 monooxygenase dependent metabolism in chemical-induced lung injury in animal models was established over 25 years ago with the furan, 4-ipomeanol. Since then, the significance of biotransformation and the reasons for the high degree of pulmonary selectivity for a myriad of different chemicals has been well documented, mainly in rodent models. However, with many of these chemicals there are substantial differences in the susceptibility of rats vs. mice. Even within the same species, varied levels of the respiratory tract respond differently. Thus, key pieces of data are still missing when evaluating the applicability of data generated in rodents to primates, and as a result of this, there are substantial uncertainties within the regulatory community with regards to assessing the risks to humans for exposure to some of these chemicals. For example, all of the available data suggest that the levels of cytochrome P450 monooxygenases in rodent lungs are 10-100 times greater than those measured in the lungs of nonhuman primates or in man. At first glance, this suggests that a significant margin of safety exists when evaluating the applicability of rodent studies in the human, but the issues are more complex. The intent of this review is to outline some of the work conducted on the site and species selective toxicity and metabolism of the volatile lung toxic aromatic hydrocarbon, naphthalene. We argue that a complete understanding of the cellular and biochemical mechanisms by which this and other lung toxic compounds generate their effects in rodent models with subsequent measurement of these cellular and biochemical events in primate and human tissues in vitro will provide a far better basis for judging whether the results of studies done in rodent models are applicable to humans.

Abstract  In the mouse, the histocompatibility-2 (H-2) haplotype influences induction of lung and intestinal tumors by N-ethyl-N-nitrosourea (ENU) treatment of fetuses or infant mice. The differentiation of lung and intestinal epithelium is known to be regulated by glucocorticoids. We show that glucocorticoid-induced development of alveolar lung volume is H-2 influenced and that glucocorticoid treatment of fetuses also influences prenatal ENU induction of lung and intestinal tumors. These glucocorticoid effects on tumorigenesis are also H-2 influenced. The number of papillary lung tumors increased in B10 (H-2b) and decreased in B10.A (H-2a) mice. In the intestine, the number of tumors increased in H-2b females and decreased in H-2b males. In H-2a mice, the number of intestinal tumors was unchanged but their location was altered. We propose that the H-2 complex influences tumorigenesis in lung and small intestine by affecting the hormonal regulation of differentiation of target epithelial cells.

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  Injury models have suggested that the lung contains anatomically and functionally distinct epithelial stem cell populations. We have isolated such a regional pulmonary stem cell population, termed bronchioalveolar stem cells (BASCs). Identified at the bronchioalveolar duct junction, BASCs were resistant to bronchiolar and alveolar damage and proliferated during epithelial cell renewal in vivo. BASCs exhibited self-renewal and were multipotent in clonal assays, highlighting their stem cell properties. Furthermore, BASCs expanded in response to oncogenic K-ras in culture and in precursors of lung tumors in vivo. These data support the hypothesis that BASCs are a stem cell population that maintains the bronchiolar Clara cells and alveolar cells of the distal lung and that their transformed counterparts give rise to adenocarcinoma. Although bronchiolar cells and alveolar cells are proposed to be the precursor cells of adenocarcinoma, this work points to BASCs as the putative cells of origin for this subtype of lung cancer.

Abstract  Styrene is widely used with significant human exposure, particularly in the reinforced plastics industry. In mice it is both hepatotoxic and pneumotoxic, and this toxicity is generally thought to be associated with its metabolism to styrene oxide. Styrene causes lung tumors in mice but not in rats. The question is how the tumorigenic effect in mouse lung may relate to the human. This review examines the comparison of the metabolic activation rates (1) between the liver and lung and (2) for the lung, between the rodent and human. Emphasis is placed on the specific cytochromes P450 present in the lungs of humans and what role they might play in the bioactivation of styrene and other compounds. In general, pulmonary metabolism is very slow compared to hepatic metabolism. Furthermore, metabolic rates in humans are slow compared to those in rats and mice. There is a wide difference in what specific cytochromes P450 investigators have reported as being present in human lung which makes comparisons, both inter-species and inter-organ, difficult. The general low activity for cytochrome P450 activity in the lung, especially for CYP2F1, the human homolog for CYP2F2 which has been identified in mice as being primarily responsible for styrene metabolism, argues against the hypothesis that human lung would produce enough styrene oxide to damage pulmonary epithelial cells leading to cell death, increased cell replication and ultimately tumorigenicity, the presumed mode of action for styrene in the production of the mouse lung tumors.

Abstract  A decrease in the intracellular concentrations of the transcripts for some tumor suppressor genes has been found during murine lung tumorigenesis; for p15INK4b and p16INK4a, this was due to homozygous deletions. We report here a decrease in the mRNA levels of the mutated in colorectal cancer (Mcc) and adenomatous polyposis coli (Apc) genes in mouse lung tumors and some neoplastic cell lines. This was assessed both by northern blotting and reverse transcriptase-polymerase chain reaction of RNA isolated from lung tumors that had been induced by urethane, N-nitrosodiethylamine, or 3-methylcholanthrene in (A/J x C57BL/6) F1 or A/J mice. A reduced amount of both Mcc and Apc messages was also seen when two neoplastic cell lines, a spontaneous transformant (E9) and a line derived from a chemically induced solid tumor (82-132), were compared with two independently derived nontumorigenic cell lines (E10 and C10); E9 was derived from E10, and all of these lines are probably of alveolar type 2 cell origin. A cell line derived from a chemically induced papillary lung tumor probably of bronchiolar Clara cell origin (LM2) had Mcc mRNA levels similar to those of C10 and E10 but reduced Apc mRNA levels. A line (p53-823) derived from a papillary tumor that arose in a mouse with a mutated p53 transgene had a reduced amount of the Mcc gene product only. These differential changes in the relative amounts of Apc and Mcc messages in LM2 and p53-823) cells may serve as useful models for studying the regulation of their expression. Both messages had half-lives of 6-9 h in normal E10 and neoplastic E9 cells, so decreased message stability does not account for these reductions. This is the first report of estimated degradation rates of these mRNAs. Apc and Mcc message content did not vary as a function of growth status of the cell lines. Single-strand conformation polymorphism analysis did not reveal mutations in Apc coding regions known to have a high mutation frequency in human colon tumors. Loss of heterozygosity of Apc and Mcc was not found in tumors that developed in the F1 mice, implying a lack of allelic deletions. These changes in tumor suppressor gene expression may contribute to the development and maintenance of neoplasia in lung epithelium.

Abstract  A tabulation of carcinogenic potency (TD50) by species for 492 chemicals that induce tumors in rats or mice is presented. With the use of the Carcinogenic Potency Database, experimental results are summarized by indicating in which sex-species groups the chemical was tested and the respective evaluations of carcinogenicity. A comparison of three summary measures of TD50 for chemicals with more than one positive experiment per species shows that the most potent TD50 value is similar to measures that average values or functions of values. This tabulation can be used to investigate associations between rodent potency and other factors such as mutagenicity, teratogenicity, chemical structure, and human exposure.

Abstract  Metabolic activation and DNA binding of aflatoxin B1 (AFB1), N-nitrosodimethylamine (DMN) and benzo[a]pyrene (B[a]P) were compared in human, rat and mouse hepatocytes and human pulmonary alveolar macrophages (PAM). The degree of carcinogen activation by hepatocytes and PAM was measured by cell-mediated mutagenesis assays in which co-cultivated Chinese hamster V79 cells were used to monitor mutagenic metabolites. Hepatocytes from human, mouse and rat metabolized DMN and released the active metabolites to induce either ouabain- or 6-thioguanine-resistant mutation. The mutation frequencies mediated by hepatocytes of the 3 animal species were approximately 3-9 mutants/10(5) survivors at a concentration of 0.2 mM DMN. The variations of radioactivity bound to liver cell DNA were relatively small in cultured mouse, rat, and human hepatocytes exposed to 14C label DMN (0.5 mM) and the binding values were in a range of 6-12 X 10(3) pmoles/mg DNA. However, rat hepatocytes were at least 10-fold more effective than either human or mouse hepatocytes in generating mutagenic metabolites of AFB1 and also had a much higher AFB1 metabolite DNA-binding value. The AFB1 DNA-binding levels were 4.1, 12-27 (range), 120 pmoles/mg DNA respectively in mouse, human, and rat liver cells following AFB1 (3.3 microM) exposure for 20 h. Hepatocytes from the 3 animal species were unable to mediate mutation in the presence of 4 microM B[a]P; PAM activated B[a]P and effectively mediated mutation in the co-cultivated V79 cells. In contrast to results with hepatocytes, PAM failed to generate enough mutagenic metabolites of AFB1 (3.3 microM) and the mediation of mutations was seen only at very high concentration of DMN (80 mM). The genotoxic effects of the 3 carcinogens on hepatocytes from different species in vitro were in agreement with the in vivo animal experiments in that mice are relatively resistant to AFB1 carcinogenesis whereas rats are sensitive; B[a]P is not effective as a complete liver carcinogen in adult rat and mouse whereas DMN induces liver cancer.

Abstract  Lung cancer is the leading cause of cancer-related death in people and is mainly due to environmental factors such as smoking and radon. The National Toxicology Program (NTP) tests various chemicals and mixtures for their carcinogenic hazard potential. In the NTP chronic bioassay using B6C3F1 mice, the incidence of lung tumors in treated and control animals is second only to the liver tumors. In order to study the molecular mechanisms of chemically induced lung tumors, an understanding of the genetic changes that occur in spontaneous lung (SL) tumors from untreated control animals is needed. The authors have evaluated the differential transcriptomic changes within SL tumors compared to normal lungs from untreated age-matched animals. Within SL tumors, several canonical pathways associated with cancer (eukaryotic initiation factor 2 signaling, RhoA signaling, PTEN signaling, and mammalian target of rapamycin signaling), metabolism (Inositol phosphate metabolism, mitochondrial dysfunction, and purine and pyramidine metabolism), and immune responses (FcγR-mediated phagocytosis, clathrin-mediated endocytosis, interleukin 8 signaling, and CXCR4 signaling) were altered. Meta-analysis of murine SL tumors and human non-small cell lung cancer transcriptomic data sets revealed a high concordance. These data provide important information on the differential transcriptomic changes in murine SL tumors that will be critical to our understanding of chemically induced lung tumors and will aid in hazard analysis in the NTP 2-year carcinogenicity bioassays.

Abstract  This study was conducted to determine species differences in the metabolism of ethylbenzene (EB) in liver and lung. EB (0.22-7.0mM) was incubated with mouse, rat and human liver and lung microsomes and the formation of 1-phenylethanol (1PE), acetophenone (AcPh), 2-ethylphenol (2EP), 4-ethylphenol (4EP), 2,5-ethylquinone, and 3,4-ethylquinone were measured. Reactive metabolites (2,5-dihydroxyethylbenzene-GSH [2EP-GSH] and 3,4-dihydroxyethylbenzene-GSH [4EP-GSH]) were monitored via glutathione (GSH) trapping technique. None of the metabolites were formed at detectable levels in incubations with human lung microsomes. Percent conversion of EB to 1PE ranged from 1% (rat lung; 7.0mM EB) to 58% (mouse lung; 0.22mM EB). More 1PE was formed in mouse lung than in mouse liver microsomes, although formation of 1PE by rat liver and lung microsomes was similar. Metabolism of EB to 1PE was in the order of mouse > rat > human. Formation of AcPh was roughly an order of magnitude lower than 1PE. Conversion of EB to ring-hydroxylated metabolites was much lower (0.0001% [4EP-GSH; rat lung] to 0.6% [2EP-GSH; mouse lung]); 2EP-GSH was typically 10-fold higher than 4EP-GSH. Formation of 2EP-GSH was higher by lung (highest by mouse lung) than liver microsomes and the formation of 2EP-GSH by mouse liver microsomes was higher than rat and human liver microsomes. Increasing concentrations of EB did lead to a decrease in amount of some formed metabolites. This may indicate some level of substrate- or metabolite-mediated inhibition. High concentrations of 2EP and 4EP were incubated with microsomes to further investigate their oxidation to ethylcatechol (ECat) and ethylhydroquinone (EHQ). Conversion of 2EP to EHQ ranged from 6% to 9% by liver (mouse > human > rat) and from 0.1% to 18% by lung microsomes (mouse >> rat >> human). Conversion of 4EP to ECat ranged from 2% to 4% by liver (mouse > human similar to rat) and from 0.3% to 7% by lung microsomes (mouse >> rat >> human). Although ring-oxidized metabolites accounted for a relatively small fraction of overall EB metabolism, its selective elevation in mouse lung microsomes is nonetheless consistent with the hypothesized mode of action for observed preferential toxicity of EB to the lung in this species.