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Alveolar macrophages and BDMCs undergo sequential biochemical changes during the chronic inflammatory response to chemically induced lung carcinogenesis in mice. Herein, we examine two chronic lung inflammation models-repeated exposure to BHT and infection with Mycobacterium tuberculosis-to establish whether similar macrophage phenotype changes occur in non-neoplastic pulmonary disease. Exposure to BHT or M. tuberculosis results in pulmonary inflammation characterized by an influx of macrophages, followed by systemic effects on the BM and other organs. In both models, pulmonary IFN-gamma and IL-4 production coincided with altered polarization of alveolar macrophages. Soon after BHT administration or M. tuberculosis infection, IFN-gamma content in BALF increased, and BAL macrophages became classically (M1) polarized, as characterized by increased expression of iNOS. As inflammation progressed in both models, the amount of BALF IFN-gamma content and BAL macrophage iNOS expression decreased, and BALF IL-4 content and macrophage arginase I expression rose, indicating alternative/M2 polarization. Macrophages present in M. tuberculosis-induced granulomas remained M1-polarized, implying that these two pulmonary macrophage populations, alveolar and granuloma-associated, are exposed to different activating cytokines. BDMCs from BHT-treated mice displayed polarization profiles similar to alveolar macrophages, but BDMCs in M. tuberculosis-infected mice did not become polarized. Thus, only alveolar macrophages in these two models of chronic lung disease exhibit a similar progression of polarization changes; polarization of BDMCs was specific to BHT-induced pulmonary inflammation, and polarization of granuloma macrophages was specific to the M. tuberculosis infection.

Background: Silibinin, a flavanone from milk thistle, inhibits the growth of tumors in several rodent models. We examined the effects of dietary silibinin on the growth, progression, and angiogenesis of urethane-induced lung tumors in mice., Methods: A/J mice (15 per group) were injected with urethane (1 mg/g body weight) or saline alone and fed normal diets for 2 weeks, after which they were fed diets containing different doses of silibinin (0%-1% [wt/wt] silibinin) for 18 or 27 weeks. Immunohistochemistry and Western blot analysis were used to examine angiogenesis and enzymatic markers of inflammation, proliferation, and apoptosis. All statistical tests were two-sided., Results: Urethane-injected mice exposed to silibinin had statistically significantly lower lung tumor multiplicities than urethane-injected mice fed the control diet lacking silibinin (i.e., control mice). Mice that received urethane and 1% (wt/wt) dietary silibinin for 18 weeks had 93% fewer large (i.e., 1.5-2.5-mm-diameter) lung tumors than control mice (mean number of tumors/mouse: 27 in the urethane group versus 2 in the urethane + 1% silibinin group, difference = 25 tumors/mouse, 95% confidence interval [CI] = 13 to 37 tumors/mouse, P = .005). Lung tumors of silibinin-fed mice had 41%-74% fewer cells positive for the cell proliferation markers proliferating cell nuclear antigen and cyclin D1 than lung tumors of control mice. Tumor microvessel density was reduced by up to 89% with silibinin treatment (e.g., 56 microvessels/400x field in tumors from control mice versus 6 microvessels/400x field in tumors from urethane + 1% silibinin-treated mice [difference = 50 microvessels/400x field, 95% CI = 46 to 54 microvessels/400x field; P<.001]). Silibinin decreased lung tumor expression of vascular endothelial growth factor (VEGF) and of inducible nitric oxide synthase and cyclooxygenase-2, two enzymes that promote lung tumor growth and progression by inducing VEGF expression., Conclusions: Silibinin inhibits lung tumor angiogenesis in an animal model and merits investigation as a chemopreventive agent for suppressing lung cancer progression.

Because chronic pulmonary diseases predispose to lung neoplasia, the identification of the molecular mechanisms involved could provide novel preventive, diagnostic, and therapeutic strategies. Toll-like receptors (TLRs) transduce exogenous and endogenous signals into the production of inflammatory cytokines to coordinate adaptive immune responses. To determine the role of Tlr4 in chronic lung inflammation, we compared lung permeability, leukocyte infiltration, and nuclear factor kappa B (NFkappaB) and activator protein 1 (AP-1) DNA binding in butylated hydroxytoluene (BHT)-treated (four weekly injections of 125-200 mg/kg each) inbred mouse strains with functional Tlr4 (OuJ and BALB) and mutated Tlr4 (HeJ and BALB(Lps-d)). We also measured primary tumor formation in these mice after single-carcinogen injection (3-methylcholanthrene; 10 microg/kg), followed by BHT treatment (six weekly injections of 125-200 mg/kg each). Mice with functional Tlr4 had reduced lung permeability, leukocyte inflammation, and primary tumor formation (BALB(Lps-d), mean = 22.3 tumors/mouse, versus BALB, mean = 13.9 tumors/mouse, difference = 8.4 tumors/mouse, 95% confidence interval = 4.6 to 12.1 tumors/mouse; P = .025) compared with mice with mutated Tlr4. NFkappaB DNA binding activity was higher in OuJ than in HeJ mice; however, AP-1 activity was elevated in HeJ mice. To our knowledge, this is the first model to demonstrate a modulatory role for Tlr4 in chronic lung inflammation and tumorigenesis.

Fewer lung adenomas were induced by urethan in BALB/cBy mice than in the A/J or SWR/J mouse strains. When BALB mice were crossed with either of these more sensitive strains the response of the progeny to urethan was most easily explained by a single gene which regulates susceptibility, with the more resistant phenotype behaving as a dominant trait. C56BL/6J mice were more resistant to adenoma induction than were BALB mice; progeny obtained when these two strains were crossed resembled the BALB susceptibility phenotype. As an approach to understanding the mechanism of action of this gene, agents that modulate adenoma initiation and tumor promotion were tested in BALB mice and other strains. The number of adenomas in BALB mice were increased severalfold by multiple urethan injections, which presumably affect initiation, and by the use of butylated o-hydroxytoulene as a promoting agent. Tumor incidence in A-mice was increased 50% by each treatment; neither procedure caused tumors to appear in the resistant DBA/2J, C3H/-21BG, or C57BL/6J strains. No relationship was observed between the strain dependency of the lethal effects of multiple injections of these agents and the relative susceptibilities of these strains to adenoma induction. The role of certain host factors in the regulation of tumor susceptibility was also tested. Homozygosity for the beige (bg) mutation had no effect on tumor numbers in C57 mice, suggesting that natural killer cells, deficient in bg/bg mice, played no major role in determining adenoma susceptibility in this strain. No correlation was found between the susceptibility of various sublines to urethan-induced lung adenoma and the reported relative tumoricidal capacities of the peritoneal macrophages from these sublines.

The effects of adrenalectomy (Ax) on urethan-induced lung tumors were determined in strains of mice that vary in their respective tumor susceptibilities: A/J (sensitive), BALB/cByJ (intermediate), and C57BL/6J (B6, resistant). Ax increased tumor number in both A/J (by 25%) and B6 mice (by 400%), but not in BALB/cByJ mice. The relative proportions of adenomas exhibiting the alveolar or papillary histological growth patterns were unchanged. Implantation of corticosterone-containing pellets into adrenalectomized B6 mice restored tumor multiplicity to that of sham-operated mice and into adrenalectomized A/J mice reduced multiplicity below that of sham-operated mice. Corticosterone, therefore, regulates neoplastic development of mouse lung epithelial cells.

For insight into the number of genes governing differential susceptibility such as in mice of the inbred strain A/J (A) highly susceptible to the induction of pulmonary adenomas by urethan in comparison to resistant strain C57BL/6J (B6) mice, tumor induction was studied in the AXB and BXA series of recombinant inbred (RI) strains derived respectively from A female X B6 male and B6 female X A male ancestral lines. Mice from 46 of these lines were given injections with 1 mg urethan/g (body wt), and the tumor number was assessed 4 months later. Lung tumor multiplicity for several RI lines was independently characterized in the Boulder, CO, and Montreal laboratories, and very similar numbers were obtained. Most lines had multiplicities intermediate to those of the progenitor strains, clearly indicating that more than a single gene accounted for the differences in lung tumor susceptibility between A and B6. The tumor incidence and multiplicity data fit very closely to what would be expected under a three-locus model, and we designated these genes "Pas" for pulmonary adenoma susceptibility. One locus called "Pas-1" had an effect on tumor multiplicity greater than that of the other loci.

Urethan-induced lung adenomas arising from alveolar type 2 cells and from bronchiolar Clara cells have distinct histologic patterns; and examination of all lung adenomas found in A/J, SWR/J, BALB/cByJ, 129/J, and RIIIS/J inbred mice, 14-16 weeks after a single urethan injection, demonstrated that both tumor types were present in all five strains. The relative numbers of each tumor type varied significantly among the different strains. No correlation was observed between the type of tumor formed and tumor multiplicity. Augmentation of tumor multiplicity by chronic butylated hydroxytoluene (BHT) treatment following urethan injection did not change the proportion of the 2 tumors from that observed with urethan alone. This finding demonstrated that BHT stimulated the development of both tumor types equally. The tumors found 14 weeks after a single urethan injection into neonatal BALB mice were larger than those found 14 weeks after urethan injection into adult BALB mice, but the proportion of alveolar and papillary tumors was the same for both groups. Lymphocytic infiltration into tumors was mainly associated with Clara cell-derived tumors. The proportion of type 2 cell- and Clara cell-derived lung adenomas appeared to be controlled primarily by the genetic background.

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.

Food additive butylated hydroxytoluene (BHT) (CAS: 128-37-0; 2,6-di-tert-butyl-p-cresol) can modulate the formation of lung adenomas in mice treated with the carcinogen urethan (CAS: 51-79-6; ethyl ester carbamic acid). An ip injection of BHT administered 6 hours before a single urethan injection decreased the number of tumors formed, whereas six weekly BHT injections following a single urethan dose increased tumor multiplicity. Biotransformation of BHT was apparently required for both prophylaxis and enhancement. Pretreatment of mice with cedrene, which perturbs drug metabolism, prevented both of these BHT activities. Analogues of BHT with different substitutions at position 6 of the phenol ring also affected tumor formation. 2-tert-Butyl-4-methylphenol inhibited, not enhanced, adenoma formation. 2-tert-Butyl-4,6-dimethylphenol (BDMP) (CAS: 1879-09-0; 6-tert-butyl-2,4-xylenol); increased tumor number in A/J but not in the BALB/cByJ mouse strain; its prophylactic activity could not be measured since urethan injection following BDMP treatment was lethal. Thus the presence of an alkyl group at this site on the phenol ring was not required for prophylaxis but was necessary for tumor enhancement. These results were consistent with the possibility that different BHT metabolites were responsible for each effect. Adenoma formation was enhanced by BHT in four strains of mice with a U+ phenotype (susceptible to urethan-induced lung adenomas); these strains were A, BALB/cBy, SWR/J, and RIIIS/J. BHT had no such effect, however, in the U+ strain 129/J. A U+ B- phenotype (urethan inducible but unresponsive to BHT enhancement) also was found among the recombinant inbred lines originally derived from a cross between U+B+ BALB/-cByJ and U-/B- C57BL/6ByJ progenitor strains. This finding showed that the genes determining sensitivity to urethan and to BHT had recombined independently of each other. An inability to metabolize BHT was probably not involved in the B- phenotype, since BHT caused reversible lung damage in the 2 U+B- recombinant inbred lines and strain 129, and biotransformation was required for this effect. These U+B- mice thus can be used to characterize the lung toxic effects of BHT in the absence of its tumor-enhancing activity and will serve as valuable controls in studies on mechanisms of tumor enhancement.