Annexin A1 subcellular expression in laryngeal squamous cell carcinoma.

AIMS: Annexin A1 (ANXA1) is a soluble cytoplasmic protein, moving to membranes when calcium levels are elevated. ANXA1 has also been shown to move to the nucleus or outside the cells, depending on tyrosine-kinase signalling, thus interfering in cytoskeletal organization and cell differentiation, mostly in inflammatory and neoplastic processes. The aim was to investigate subcellular patterns of immunohistochemical expression of ANXA1 in neoplastic and non-neoplastic samples from patients with laryngeal squamous cell carcinomas (LSCC), to elucidate the role of ANXA1 in laryngeal carcinogenesis. METHODS AND RESULTS: Serial analysis of gene expression experiments detected reduced expression of ANXA1 gene in LSCC compared with the corresponding non-neoplastic margins. Quantitative polymerase chain reaction confirmed ANXA1 low expression in 15 LSCC and eight matched normal samples. Thus, we investigated subcellular patterns of immunohistochemical expression of ANXA1 in 241 paraffin-embedded samples from 95 patients with LSCC. The results showed ANXA1 down-regulation in dysplastic, tumourous and metastatic lesions and provided evidence for the progressive migration of ANXA1 from the nucleus towards the membrane during laryngeal tumorigenesis. CONCLUSIONS: ANXA1 dysregulation was observed early in laryngeal carcinogenesis, in intra-epithelial neoplasms; it was not found related to prognostic parameters, such as nodal metastases.

Skin allograft survival and analysis of renal parameters after FTY720 + tacrolimus treatment in mice.

Calcineurin inhibitors such as cyclosporine (CsA) and tacrolimus (FK506) show similar efficacy to prevent rejection within the first year after organ transplantation. However, their use is limited by side effects, such as kidney damage, hypertension, new-onset diabetes, and hyperlipidemia. The consensus opinion suggests that compared with CsA, FK506 has fewer negative effects on blood pressure, serum lipids, and renal function. Nevertheless, FK506 use is associated with a higher incidence of posttransplantation diabetes mellitus. FTY720 is a new compound that has shown beneficial effects in animal models of rejection in transplantation, ischemia/reperfusion injury, autoimmune diseases, and tumor development. Our aim was to investigate whether FTY720 + tacrolimus association could provide additional immunosuppression without causing renal toxicity. FTY720 as a monotherapy or in association with FK506 was administered to C57BL/6 mice for 21 days to prevent skin graft rejection and to evaluate renal function and structure. Increased skin allograft survival in the FTY720 + FK506 group was associated with decreased cell numbers in the spleen, blood, and axillary lymph nodes. Changes in major histocompatibility complex (MHC) class II and intercellular adhesion molecule-1 (ICAM-1) expressions in splenocytes were also found in this group. The major effects already described for FK506 (diabetes) or FTY720 (lymphopenia) were observed after 21 days administration even when the drugs were associated. FTY720 associated with FK506 caused fewer changes in kidney structure, and blood glucose levels were lower than in FK506 monotherapy.

Apoptosis in different gastric lesions and gastric cancer: relationship with Helicobacter pylori, overexpression of p53 and aneuploidy.

Apoptosis has an essential function in maintaining the integrity of the gastrointestinal mucosa. Its deregulation is associated with the occurrence of lesions such as in atrophic gastritis, peptic ulcers, intestinal metaplasia, and stomach tumorigenesis. Thus, the aim of the present study was to investigate the frequency of apoptotic cells (apoptotic index, AI) by using two different immunohistochemical techniques, TUNEL and anti-activated caspase-3 antibody (CPP32), in gastric dyspepsia [chronic gastritis (CG, N = 34), chronic atrophic gastritis (CAG, N = 11), gastric ulcer (GU, N = 17), and intestinal metaplasia (IM, N = 15)], normal gastric mucosae (NM, N = 8), and gastric adenocarcinoma (GC, N = 12). The relationship was investigated between the AI and Helicobacter pylori infection, diagnosed by PCR, overexpression of p53 protein determined by immunohistochemistry, and aneuploidy by fluorescence in situ hybridization, as performed by our laboratory in previous studies. No significant differences were observed in AI between the different groups, whether by the TUNEL technique (F = 1.60; p = 0.1670) or by CPP32 antibody (F = 1.70; p = 0.1420). Nonetheless, CAG and CG groups had AI statistically higher than those of normal mucosae. These two groups (CAG and CG) also showed a higher frequency of apoptosis-positive cases (TUNEL+ or CPP32+). Generally, there was no correlation between the AI detected by the TUNEL and CPP32 techniques in the groups studied, except in the GC group (r = 0.70). Moreover, there was no significant association between apoptosis and H. pylori infection, overexpression of p53 protein and aneuploidy, but the H. pylori-positive cases only of GU (p = 0.0233) and IM (p = 0.0253) groups displayed a statistically higher AI compared to H. pylori-negative NM, when the CPP32 antibody technique was used. Thus, CG and CAG have increased apoptosis, which may occur independent of an association with H. pylori infection, aneuploidy and overexpression of p53 protein.

FTY720 prevents renal T-cell infiltration after ischemia/reperfusion injury.

Ischemia/reperfusion (I/R) injury, a common early feature in renal transplantation, results from both free radical species generation and local inflammatory responses that attract different types of cells. The interaction with infiltrating leukocytes could promote damage and death of resident renal cells contributing to worsening of renal function. It has been shown that depletion of host T cells protects against kidney damage after I/R injury, although the mechanism is not fully understood. FTY720, a synthetic analog of a natural product extracted from Isaria sincclairii has shown modulatory properties in experimental models of autoimmune disease, transplantation, and I/R injury. FTY720 alters lymphocyte responses to chemokine homing signals, thereby decreasing the number of lymphocytes in inflammatory sites. We evaluated renal function in mice at 3, 5, and 7 days after I/R injury in the presence or absence of FTY720 treatment. FTY720 treatment promoted earlier recovery of renal function associated with a lower number of renal-infiltrating lymphocytes. These findings confirm previous results showing a protective effect of FTY720 in I/R injury models.

Rhabdomyolysis, acute renal failure, and death after monensin ingestion.

We report a case of human monensin intoxication; to our knowledge, this is the first reported case in the medical literature. The patient took a dose of monensin three times higher than a dose considered lethal for cattle and developed a clinical picture similar to that reported in veterinary medicine. There was an early and extremely severe rhabdomyolysis followed by acute renal failure, heart failure, and death. The main changes observed at autopsy were extensive skeletal muscle necrosis, complement deposition at the myocardial level, pulmonary edema, and acute tubular damage.

Silver nitrate is superior to talc slurry in producing pleurodesis in rabbits.

STUDY OBJECTIVE: The ideal agent for producing pleurodesis has not been identified. Although talc is the agent most commonly used at the present time, there are concerns about its safety. Silver nitrate is a possible alternative agent. The purpose of the present study was to compare the effectiveness of intrapleural silver nitrate and talc slurry in producing pleurodesis in rabbits. Additionally, the total amount of pleural collagen and the distribution of thick and thin collagen fibers were studied. DESIGN: Two groups of 10 rabbits received either 0.50% silver nitrate or 400 mg/kg talc in a total volume of 2 mL intrapleurally. The animals were killed 28 days after injection, and the pleural spaces were assessed grossly for evidence of pleurodesis and microscopically for evidence of inflammation and fibrosis. Collagen was assessed with the van Gieson's and picrosirius stains. RESULTS: The macroscopic pleurodesis (scale, 0 to 4; mean +/- SEM) resulting from the intrapleural injection of silver nitrate (3.4+/-0.2) was significantly better (p<0.001) than that resulting from talc (1.6+/- 0.1). The mean degree of microscopic pleural fibrosis induced by silver nitrate (3.3+/-0.3) was significantly higher (p = 0.003) than that induced by talc (1.8+/-0.1). The mean amount of microscopic pleural collagen (van Gieson's) was significantly greater (p<0.001) in the rabbits that received silver nitrate (3.0+/-0.2) than in those that received talc (1.6+/-0.2). The distribution of thick and thin collagen fibers did not differ between the groups. CONCLUSIONS: We conclude that, in our rabbit model, intrapleural silver nitrate was more effective than talc in producing a pleurodesis.

Autopsy findings in early and late postoperative death after partial left ventriculectomy.

BACKGROUND: Partial left ventriculectomy (PLV) is an alternative to heart transplantation for patients with severe heart failure. However, this procedure is accompanied by high morbidity and mortality. Therefore, we studied the hearts of 12 patients who underwent this procedure to increase our understanding of the causes of bad outcome. METHODS: We analyzed the autopsy hearts of 11 of 16 patients who died after PLV, and one heart from a patient who underwent heart transplantation. RESULTS: Six patients died less than 30 days postoperatively, 4 of cardiogenic shock, 1 of arrhythmia, and 1 of coagulopathy. Five patients died from 36 to 120 days after the procedure, 4 of cardiogenic shock and 1 of arrhythmia. The patient who underwent heart transplantation had a cardiogenic shock 230 days after PLV. Ten hearts weighed more than 500 g and nine had myocardial infarction that extended to the papillary muscles. Four patients had infarction of both papillary muscles and 3 of them had episodes of arrhythmia, suggesting some relation between these events. CONCLUSIONS: We found several important morphologic clues for bad outcome: infarction of both papillary muscles, which may be associated with the development of arrhythmia, and myocardial infarction and pericardial hemorrhage, which may contribute to the outcome of heart failure.

Urban levels of air pollution modifies the progression of urethane-induced lung tumours in mice.

This paper investigates the effects of air pollution in urethane-induced lung tumours in mice by means of histological, morphometrical, and DNA ploidy. The experimental exposure was done in locations with different air pollution profiles: a polluted area (downtown São Paulo) and a "clean" environment. Swiss mice were employed and urethane (3 g/kg) was used as a carcinogenic substance. All the animals, whether exposed or not to air pollution, were sacrificed after 6 months, and the lung lesions were analysed. The results showed a significant effect of air pollution on tumour progression, observed by changes in the phenotype of the tumour cells as demonstrated by morphometry and DNA ploidy. We observed more atypical adenomas in the air pollution-exposed group (p = 0.02). Coherently, morphometric differences were also detected between the two groups. Neoplasms of exposed mice exhibited an increase in the nuclear fraction (p = 0.002) and in the nucleus/cytoplasm ratio (p = 0.011), as a decrease in the stromal fraction (p < 0.001). There was a higher risk of aneuploidy in the 6-months-of-air-pollution-exposure group (relative risk: 1.58; 95% of confidence interval: 1.007 to 2.403). These results indicate that urban air pollution accelerates the process of progression towards malignancy.

Functional hypotheses for aberrant glycosylation in tumor cells.

Aberrant glycosylation is a common feature of neoplastic cells. Although described for many years, the role of aberrant patterns of glycosylation is not fully understood. Our group has been focusing on the role of glycosylation in cell:matrix interactions, such as adhesion, spreading and migration on defined substrata (e.g., laminin and fibronectin). Animal lectins, such as galaptins, also seem to be involved in these processes.

Perioperative myocardial infarction in a patient with tuberculous constrictive pericarditis in the absence of coronary artery disease.

A 34-year-old man developed severe heart failure due to constrictive pericarditis. Pericardiectomy was carried on and the patient died 12 hours after surgery. Necropsy revealed an extensive hemorrhagic myocardial infarction involving the lateral free wall of the left ventricle in the absence of coronary artery disease. In addition, necropsy revealed tuberculosis as the etiology of constrictive pericarditis. Thus, myocardial infarction may occur in constrictive pericarditis in the setting of pericardiectomy and absence of coronary artery disease.

Leishmania (Leishmania) amazonensis infection in mice treated with FTY720.

In transplantation, parasite diseases are transmitted from the donor, or appear as de novo infections, or activate from a dormant insource as a consequence of immunosuppression. Clinical findings have shown that an intact immune system is crucial to prevent recurrence of Leishmania infection. We used BALB/c and C57BL/6 mice to evaluate the role of FTY720 in leishmaniasis. Mice inoculated with Leishmania (Leishmania) amazonensis were followed over 7 weeks for foot thickness measurements after initiation of FTY720 treatment. After 10 days of treatment, spleen, blood, and the foot were harvested for evaluation. BALB/c showed greater evident foot thickness than C57BL/6 mice. Oral treatment with FTY720 (1 mg/kg/d) over 10 days produced the same outcome. Increases in CD4(+) and CD8(+) T cells were observed after infection; FTY720 treatment was associated with a decrease in CD4(+) T cells only in BALB/c mice, whereas CD8(+) T cells were decreased in both mice strains. CD11b(+) expression decreased after infection with a discrete increase after FTY720 treatment. Lymphopenia was observed among all FTY720-treated mice. In conclusion, we observed that FTY720 produced no worse an outcome as monotherapy in established infections with L (L) amazonensis.

Genomics and proteomics approaches to the study of cancer-stroma interactions.

BACKGROUND: The development and progression of cancer depend on its genetic characteristics as well as on the interactions with its microenvironment. Understanding these interactions may contribute to diagnostic and prognostic evaluations and to the development of new cancer therapies. Aiming to investigate potential mechanisms by which the tumor microenvironment might contribute to a cancer phenotype, we evaluated soluble paracrine factors produced by stromal and neoplastic cells which may influence proliferation and gene and protein expression. METHODS: The study was carried out on the epithelial cancer cell line (Hep-2) and fibroblasts isolated from a primary oral cancer. We combined a conditioned-medium technique with subtraction hybridization approach, quantitative PCR and proteomics, in order to evaluate gene and protein expression influenced by soluble paracrine factors produced by stromal and neoplastic cells. RESULTS: We observed that conditioned medium from fibroblast cultures (FCM) inhibited proliferation and induced apoptosis in Hep-2 cells. In neoplastic cells, 41 genes and 5 proteins exhibited changes in expression levels in response to FCM and, in fibroblasts, 17 genes and 2 proteins showed down-regulation in response to conditioned medium from Hep-2 cells (HCM). Nine genes were selected and the expression results of 6 down-regulated genes (ARID4A, CALR, GNB2L1, RNF10, SQSTM1, USP9X) were validated by real time PCR. CONCLUSIONS: A significant and common denominator in the results was the potential induction of signaling changes associated with immune or inflammatory response in the absence of a specific protein.

Association between apolipoprotein E genotype, serum lipids, and colorectal cancer in Brazilian individuals.

We evaluated genetic variants of apolipoprotein E (APOE HhaI) and their association with serum lipids in colorectal cancer (CRC), together with eating habits and personal history. Eight-seven adults with CRC and 73 controls were studied. APOE*2 (rs7412) and APOE*4 (rs429358) were identified by polymerase chain reaction-restriction fragment length polymorphism. APOE gene polymorphisms were similar in both groups, but the epsilon4/epsilon4 genotype (6%) was present only in controls. The patients had reduced levels (mean +/- SD) of total cholesterol and low-density lipoprotein cholesterol fraction (180.4 +/- 49.5 and 116.1 +/- 43.1 mg/dL, respectively) compared to controls (204.2 +/- 55.6, P = 0.135 and 134.7 +/- 50.8 mg/dL; P = 0.330, respectively) indicating that they were not statistically significant after the Bonferroni correction. The APOE*4 allele was associated with lower levels of total cholesterol, low- and high-density lipoprotein cholesterol fraction and increased levels of very low-density lipoprotein cholesterol fraction and triglycerides only among patients (P = 0.014). There was a positive correlation between the altered lipid profile and increased body mass indexes in both groups (P < 0.010). Moreover, a higher rate of hypertension and overweight was observed in controls (P < 0.002). In conclusion, the presence of the epsilon4/epsilon4 genotype only in controls may be due to a protective effect against CRC. Lower lipid profile values among patients, even those on lipid-rich diets associated with the APOE*4 allele, suggest alterations in the lipid synthesis and metabolism pathways in CRC.

Comparison of the effects of lactated Ringer solution with and without hydroxyethyl starch fluid resuscitation on gut edema during severe splanchnic ischemia.

The type of fluid used during resuscitation may have an important impact on tissue edema. We evaluated the impact of two different regimens of fluid resuscitation on hemodynamics and on lung and intestinal edema during splanchnic hypoperfusion in rabbits. The study included 16 female New Zealand rabbits (2.9 to 3.3 kg body weight, aged 8 to 12 months) with splanchnic ischemia induced by ligation of the superior mesenteric artery. The animals were randomized into two experimental groups: group I (N = 9) received 12 mL x kg-1 x h-1 lactated Ringer solution and 20 mL/kg 6% hydroxyethyl starch solution; group II (N = 7) received 36 mL x kg-1 x h-1 lactated Ringer solution and 20 mL/kg 0.9% saline. A segment from the ileum was isolated to be perfused. A tonometric catheter was placed in a second gut segment. Superior mesenteric artery (Q SMA) and aortic (Qaorta) flows were measured using ultrasonic flow probes. After 4 h of fluid resuscitation, tissue specimens were immediately removed for estimations of gut and lung edema. There were no differences in global and regional perfusion variables, lung wet-to-dry weight ratios and oxygenation indices between groups. Gut wet-to-dry weight ratio was significantly lower in the crystalloid/colloid-treated group (4.9 +/- 1.5) than in the crystalloid-treated group (7.3 +/- 2.4) (P < 0.05). In this model of intestinal ischemia, fluid resuscitation with crystalloids caused more gut edema than a combination of crystalloids and colloids.

Spatial expression of two anti-inflammatory mediators, annexin 1 and galectin-1, in nasal polyposis.

BACKGROUND: There is renewed interest in the role played by specific counter-regulatory mechanisms to control the inflammatory host response, poorly investigated in human pathology. Here, we monitored the expression of two anti-inflammatory mediators, annexin 1 and galectin-1, and assessed their potential link to glucocorticoids' (GCs) effective control of nasal polyposis (NP). METHODS: Total patterns of mRNA and protein expression were analysed by quantitative real-time PCR (qPCR) and Western blotting analyses, whereas ultrastructural immunocytochemistry was used for spatial localization and quantification of each mediator, focusing on mast cells, eosinophils and epithelial cells. RESULTS: Up-regulation of the annexin 1 gene, and down-regulation of galectin-1 gene, was detected in polypoid tissue compared with nasal mucosa. Patient treatment with betamethasone augmented galectin-1 protein expression in polyps. At the cellular level, control mast cells and eosinophils displayed higher annexin 1 expression, whereas marked galectin-1 immunolabelling was detected in the granule matrix of mast cells. Cells of glandular duct epithelium also displayed expression of both annexin 1 and galectin-1, augmented after treatment. CONCLUSION: Mast cells and epithelial cells appeared to be pivotal cell types involved in the expression of both annexin 1 and galectin-1. It is possible that annexin 1 and galectin-1 could be functionally associated with a specific mechanism in NP and that GC exert at least part of their beneficial effects on the airway mucosa by up-regulating, in a specific cell target fashion, these anti-inflammatory agonists.

Value of the mesothelium-associated antibodies thrombomodulin, cytokeratin 5/6, calretinin, and CD44H in distinguishing epithelioid pleural mesothelioma from adenocarcinoma metastatic to the pleura.

Until recently, the standard approach of most laboratories in distinguishing epithelioid pleural mesothelioma from metastatic adenocarcinoma has been a negative result from a panel of adenocarcinoma-associated antibodies. However, several "mesothelium-associated" antibodies have been proposed as useful in this situation, and we have applied four of these putative mesothelioma markers--thrombomodulin, cytokeratin 5/6, calretinin, and CD44H--to a series of 61 epithelioid pleural mesotheliomas and 63 metastatic adenocarcinomas with known primary sites (lung = 19; breast = 21; ovary = 6; colon = 10; kidney = 4; uterus, epididymis, pancreas = 1 case each). Of the mesotheliomas, 55 of 61 (90%) stained for thrombomodulin, 56 of 61 (92%) for cytokeratin 5/6, 47 of 51 cases (92%) were positive for calretinin, and 39 of 43 (91%) were positive for CD44H. Of the metastatic adenocarcinomas, 12 of 63 (19%) cases were positive for thrombomodulin, 9 of 63 (14%) were positive for CK5/6, and 27 of 60 (45%) were positive for CD44H. With calretinin, only 1 case of 59 (2%) showed positive nuclear staining. All four antibodies stained reactive mesothelium; thrombomodulin also stained endothelium; and CD44H variably stained lymphocytes, macrophages, and fibroblasts. We conclude that all four antibodies show high sensitivity for epithelioid mesothelioma, but only calretinin (98%), cytokeratin 5/6 (86%), and thrombomodulin (81%) show sufficient specificity for practical use in this situation.

The use of histological and immunohistochemical markers to distinguish pleural malignant mesothelioma and in situ mesothelioma from reactive mesothelial hyperplasia and reactive pleural fibrosis.

Distinguishing malignant mesothelioma from reactive mesothelial hyperplasia and reactive fibrosis can be a diagnostic problem in small pleural biopsies, made more difficult by the recent recognition of mesothelioma-in-situ. Antibodies to epithelial membrane antigen (EMA), p53, and bcl-2 have all been advocated for differentiating reactive from neoplastic conditions, but reports are inconsistent. These antibodies have therefore been applied to 31 cases of malignant mesothelioma, 34 cases of reactive pleural disease (20 reactive mesothelial hyperplasia and 14 reactive pleural fibrosis) and four small biopsies that were initially coded as suspicious, from patients who later developed frank mesothelioma. Thirty out of 31 (97 per cent) cases of mesothelioma showed positive nuclear staining for p53, with a higher incidence of positivity in epithelioid than in sarcomatoid elements and 30/31 (97 per cent) showed diffuse linear membrane staining for EMA, again more intense in the epithelioid elements. No mesothelioma was positive for bcl-2. In seven cases that contained both in situ and invasive mesothelioma, the in situ elements showed similar staining patterns to the invasive epithelioid elements. Thirteen out of 20 (65 per cent) cases of reactive mesothelial hyperplasia showed occasional nuclear positivity for p53 and 5/20 (25 per cent) cases showed focal weak membrane staining for EMA. Three out of 14 (21 per cent) cases of reactive pleural fibrosis showed positive nuclear staining for p53 and 6/14 (43 per cent) cases showed focal membrane staining with EMA. No reactive cases stained for bcl-2. All four suspicious cases showed diffuse linear staining with EMA and three showed focal staining for p53. It is concluded that strong diffuse linear staining for EMA is a good marker of malignancy when differentiating epithelioid malignant mesothelioma and mesothelioma-in-situ from reactive mesothelial hyperplasia, although weak focal staining may occur in reactive conditions. Nuclear staining for p53 is also suggestive of epithelioid mesothelioma, but should be regarded as no more than suspicious. The antibodies used in this investigation are less helpful in differentiating sarcomatoid mesothelioma from reactive pleural fibrosis.

Reproducibility of the WHO/IASLC grading system for pre-invasive squamous lesions of the bronchus: a study of inter-observer and intra-observer variation.

AIMS: Although many workers have graded pre-invasive squamous lesions arising in the bronchus, there has been no consensus classification system until the latest edition of the WHO/IASLC histological classification of pulmonary and pleural tumours. Because the value of any such system is dependent on its reproducibility, we have circulated a series of such lesions to a panel of histopathologists to assess interobserver and intra-observer variation when the WHO/IASLC classification was applied. METHODS AND RESULTS: Colour transparencies of 28 pre-invasive squamous lesions were assessed by six histopathologists (two with a special interest in pulmonary pathology, two generalists and two trainees) on three separate occasions over a period of 3 months, using the criteria of the WHO/IASLC (mild, moderate and severe dysplasia, and in-situ carcinoma). An additional category of metaplasia was added for those cases that showed no dysplasia. Weighted kappa coefficents of agreement (K(w)) were used to evaluate paired observations with a standard quadratic weighting being employed, such that kappa coefficients corresponded to intra-class correlation coefficients. Wilcoxon's sign-ranked test was used to measure the statistical significance of group trends, when comparing kappa values for the three grading systems. Various 3-point systems were also assessed, through combination of the above groups. Intra-observer agreement was substantially better than interobserver variation (mean: 0.71 vs. 0.55). Between the various pathologist groups, inter-observer variation was relatively minor, although intra-observer variation was higher within the trainee pathologist group. Using weighted kappa values, there was no significant difference in either inter-observer or intra-observer agreement between the five point grading system and a 3-point system of metaplasia/mild, moderate and severe/in-situ grades. However, there was a significant increase in variation when a 3-point system of metaplasia/mild, moderate/severe and in-situ carcinoma was used. CONCLUSION: This study shows levels of interobserver and intra-observer variation similar to those found in other grading systems in histopathology, with no significant decrease in variability found by abridging the system. The WHO/IASLC system is therefore recommended for future use in both clinical and research fields.

Urban air pollution enhances the formation of urethane-induced lung tumors in mice.

This paper investigates the association between air pollution and lung neoplasia in an animal model. The experimental exposures were done in two locations with different air pollution profiles: a polluted area (downtown São Paulo) and a "clean" environment (Atibaia). Swiss mice were employed and urethane (3 g/kg) was used as carcinogenic substance. Two experiments were performed: Experiment I was designed to verify whether air pollution acts as initiator and/or promoter of lung cancer, using 300 mice; Experiment II employed 250 animals and aimed to verify if the effects of air pollution on the development of lung tumors was dose dependent. A significant effect of air pollution in augmenting lung carcinogenecity induced by urethane was observed. This effect was shown to be dose-dependent and reproducible on two different occasions. In addition, morphometric studies revealed that pollution may influence tumor phenotype. These results support the hypothesis that air pollution plays a significant role in the development of lung tumors.