Occupational exposure to crystalline silica and the risk of lung cancer in Canadian men.

Crystalline silica is a recognized carcinogen, but the association with lung cancer at lower levels of exposure has not been well characterized. This study investigated the relationship between occupational silica exposure and lung cancer and the combined effects of cigarette smoking and silica exposure on lung cancer risk. A population-based case-control study was conducted in eight Canadian provinces between 1994 and 1997. Self-reported questionnaires were used to obtain a lifetime occupational history and information on other risk factors. Occupational hygienists assigned silica exposures to each job based on concentration, frequency and reliability. Data from 1681 incident lung cancer cases and 2053 controls were analyzed using logistic regression to estimate odds ratios (OR) and their 95% confidence intervals (CI). Models included adjustments for cigarette smoking, lifetime residential second-hand smoke and occupational exposure to diesel and gasoline engine emissions. Relative to the unexposed, increasing duration of silica exposure at any concentration was associated with a significant trend in lung cancer risk (OR ≥ 30 years: 1.67, 1.21-2.24; ptrend = 0.002). The highest tertile of cumulative silica exposure was associated with lung cancer (OR = 1.81, 1.34-2.42; ptrend = 0.004) relative to the lowest. Men exposed to silica for ≥30 years with ≥40 cigarette pack-years had the highest risk relative to those unexposed with <10 pack-years (OR = 42.53, 23.54-76.83). The joint relationship with smoking was consistent with a multiplicative model. Our findings suggest that occupational exposure to silica is a risk factor for lung cancer, independently from active and passive smoking, as well as from exposure to other lung carcinogens.

Assessment and quantification of crystal-induced lysosomal damage.

Lysosomes are organelles that degrade endocytosed, phagocytosed, or autophagocytosed materials. Lysosomal degradation of engulfed material is a highly controlled mechanism, which is vital in the control of infection, recycling of cellular organelles, and the breakdown of larger material. Following ingestion, lysosomes acidify and mature, leading to the activation of a range of proteolytic enzymes. Lysosomes are considered stable organelles that separate their highly hydrolytic enzymes from the cytosol to prevent digestion of self-molecules and host cell damage. Some substances, mostly of a particulate or aggregated nature, are able to damage lysosomes. Lysosomal damage and rupture, with subsequent release of lysosomal content into the cytosol can have dramatic consequences for the cell, such as the induction of cell death or activation of the NLRP3 inflammasome. In this chapter, we provide methods for the induction, assessment, and quantification of lysosomal damage by flow cytometry and confocal microscopy.

Melanoma cell growth inhibition by betagamma-CAT, which is a novel non-lens betagamma-crystallin and trefoil factor complex from frog Bombina maxima skin.

In vertebrates, non-lens betagamma-crystallins are widely expressed in various tissues but their functions are unknown. The molecular mechanisms of trefoil factors, initiators of mucosal healing and being greatly involved in tumorigenesis, have remained elusive. betagamma-CAT, which is responsible for the potent hemolytic activity and lethal toxicity on mice of frog Bombina maxima skin secretions, is the first example of a non-lens betagamma-crystallin and trefoil factor complex. Its alpha- and beta-subunits show significant sequence homology to human Absent In Melanoma 1 and trefoil factors, respectively. Here, we showed that betagamma-CAT triggered two types of cellular responses in human melanoma cells. On one hand, the protein (25-200 pM) was able to stimulate cell migration in melanoma A375 cells. On the other hand, it inhibited cell proliferation by delaying S-G2/M cell phase transition. Blockade of the cell cycle was associated with increased p21/WAF1 expression, and reduced amounts of Cdc2 and Cdc25C. betagamma-CAT also reduced Cdc2 function by increasing the level of inactivated phospho-Cdc2. In addition, the expression of JunB, a well-known cell proliferation inhibitor, was significantly up-regulated. These results provide the first evidence of an anti-proliferative role for a non-lens betagamma-crystallin member and action mechanism via association with a trefoil factor.

Betagamma-CAT, a non-lens betagamma-crystallin and trefoil factor complex from amphibian skin secretions, caused endothelium-dependent myocardial depression in isolated rabbit hearts.

Previous in vivo study demonstrated that betagamma-CAT, a newly identified non-lens betagamma-crystallin and trefoil factor complex from frog Bombina maxima skin secretions, possessed potent lethal toxicity on mammals resulted from hypotension and cardiorespiratory arrest. However, the mechanism of cardiac dysfunction induced by the protein is unknown. Here, we report that betagamma-CAT, with dosages of 0.8-3.0 nM, elicited an acute negative inotropic effect in isolated rabbit heart Langendorff preparations, which mimicked acute heart failure. In addition, the effect of betagamma-CAT on the hearts was mediated by endothelium-dependent coronary vasoconstriction (P<0.01, compared between endothelium-intact and removal hearts). After betagamma-CAT (3.0 nM) treatment, the positive signal of tumor necrosis factor-alpha (TNF-alpha) was detected mainly around the endothelial cell layer as detected by in situ indirect immunofluorescence, indicating that the release of TNF-alpha occurred. At the same time, a rapid TNF-alpha release was detected in primary cultured rabbit endocardial endothelial cells (REECs) treated with betagamma-CAT. After addition of betagamma-CAT (3.0 nM) for 10 min and 30 min, the TNF-alpha levels were increased to 57.33+/-3.22 pg/ml and 60.00+/-5.35 pg/ml (P<0.05, compared with the control values of 21.67+/-3.45 pg/ml and 33.70+/-6.24 pg/ml, respectively). At high concentrations, betagamma-CAT interfered with the cell viability of REECs (CC(50) about 25 nM). Taken together, betagamma-CAT was able to induce acute myocardial depression and the toxic effect might be partially explained by the release of TNF-alpha. The finding provides new information to understand the patho-physiological roles of non-lens betagamma-crystallins and trefoil factors.

Acute toxicity of betagamma-CAT, a naturally existing non-lens betagamma-crystallin and trefoil factor complex from frog Bombina maxima skin secretions.

In vertebrates, non-lens betagamma-crystallins are widely expressed in various tissues, but their functions are unknown. The molecular mechanisms of trefoil factors, initiators of mucosal healing and being greatly involved in tumorigenesis, have remained elusive. betagamma-CAT is the first example of a naturally existing multifunctional protein complex of a non-lens betagamma-crystallin and a trefoil factor from frog Bombina maxima skin secretions. Here we report the investigation of its in vivo toxic effects on mice and rabbits. The LD(50) values of betagamma-CAT on mice were determined to be 0.4 mg/kg and 20 microg/kg under intraperitoneal (i.p.) and intravenous (i.v.) injection, respectively. The mice subcutaneously injected with betagamma-CAT (6 microg/g body weight) showed strong hyperaemia of subcutaneous capillary vessel, but no hemorrhagic spots were observed. Intravenous injection of betagamma-CAT in rabbits (8-22 microg/kg body weight) caused a rapidly hypotensive effect and followed with cardiovascular collapse. Injection with betagamma-CAT (22 microg/kg, i.v.) significantly decreased hematocrit (P<0.05) and mean corpuscular volume (P<0.05) of the rabbits in 5 min. At the same time, the counts of platelets and white blood cells were significantly decreased (P<0.05), while the blood levels of glucose, lactate dehydrogenase and serum glutamic-oxaloacetic transaminase were significantly increased (P<0.05). Furthermore, serials of tissues edema and damages were also observed. These results indicate that betagamma-CAT rapidly caused several in vivo toxic effects on mammals and its lethal toxic potency was mainly contributed by hypotension and cardiovascular collapse, providing new clues for the understanding of the patho-physiological roles of non-lens betagamma-crystallins and trefoil factors.

Differences in the composition of inflammatory cell infiltrate in lens-induced uveitis under therapy with allopurinol or steroids.

PURPOSE: The aim of this study was to compare the qualitative changes in the composition of inflammatory cell infiltrate in lens-induced uveitis (LIU) under treatment with allopurinol (Allo), methylprednisolone (Pred) or the two drugs combined (Allo/Pred). METHODS: Twenty male Wistar rats were sensitized with lens proteins for eight weeks. Intravenous (IV) therapy was started after anterior capsule disruption in one eye of each animal. Five rats were randomly assigned to each of the four groups: controls, Allo (50 mg/kg bw), Pred (7.5 mg/kg bw) and Allo/Pred (50 mg/7.5 mg per kg bw). Eyes were enucleated 24 hours later and fixed in paraformaldehyde/glutaraldehyde. Sections at three levels were stained with Giemsa and examined using a 0 to 4+ score for each type of inflammatory cell. Granulocytes were seen as neutrophils and eosinophils. Neutrophils were divided into polymorphs and "others", and graded with lymphocytes. RESULTS: In all therapy groups there was a significant reduction of polymorphs (p<0.05) in comparison to the control group. There was also a significant reduction in lymphocytes (p<0.05) in the Pred and Allo/Pred groups as compared to the control group and the Allo group. CONCLUSIONS: Single-dose IV allopurinol significantly reduced the overall number of polymorphonuclear leucocytes in LIU. Unlike methylprednisolone, allopurinol did not have any significant impact on lymphocytes.