Toxicodynamics of rigid polystyrene microparticles on pulmonary gas exchange in mice: implications for microemboli-based drug delivery systems.

The toxicodynamic relationship between the number and size of pulmonary microemboli resulting from uniformly sized, rigid polystyrene microparticles (MPs) administered intravenously and their potential effects on pulmonary gas exchange were investigated. CD-1 male mice (6-8 weeks) were intravenously administered 10, 25 and 45 µm diameter MPs. Oxygen hemoglobin saturation in the blood (SpO(2)) was measured non-invasively using a pulse oximeter while varying inhaled oxygen concentration (F(I)O(2)). The resulting data were fit to a physiologically based non-linear mathematical model that estimates 2 parameters: ventilation-perfusion ratio (V(A)/Q) and shunt (percentage of deoxygenated blood returning to systemic circulation). The number of MPs administered prior to a statistically significant reduction in normalized V(A)/Q was dependent on particle size. MP doses that resulted in a significant reduction in normalized V(A)/Q one day post-treatment were 4000, 40,000 and 550,000 MPs/g for 45, 25 and 10 µm MPs, respectively. The model estimated V(A)/Q and shunt returned to baseline levels 7 days post-treatment. Measuring SpO(2) alone was not sufficient to observe changes in gas exchange; however, when combined with model-derived V(A)/Q and shunt early reversible toxicity from pulmonary microemboli was detected suggesting that the model and physical measurements are both required for assessing toxicity. Moreover, it appears that the MP load required to alter gas exchange in a mouse prior to lethality is significantly higher than the anticipated required MP dose for effective drug delivery. Overall, the current results indicate that the microemboli-based approach for targeted pulmonary drug delivery is potentially safe and should be further explored.

Characterization of Chinese hamster ovary cells with impaired spreading properties on fibronectin.

The development of receptor-defective or -deficient mutants can be applied to the investigation of cell-matrix interactions including cell adherence and spreading. In the present study we developed a series of ethyl methyl sulfonate (EMS)-induced Chinese hamster ovary (CHO) cell mutants, which adhere to fibronectin but have impaired spreading characteristics. Using morphometric analysis, a significant suppression in the degree of cell spreading between the wild-type and the mutant cells (P less than 0.001) was seen. This inability of the mutant cells to spread adequately on fibronectin also resulted in a decreased number and diameter of stress fibers as compared to wild-type cells. The decreased cell spreading of the mutant cells was not due to inherent differences in cell size or volume, as determined by fluorescence-activated cell sorter (FACS) analysis. Since integrins, specifically the fibronectin receptor (alpha FN/beta 1), are important in cell adhesion and cell spreading, we carried out a comparative immunochemical analysis, using a monoclonal antibody to the beta 1 subunit of integrin (7E2). Western blot analysis of cell extracts and cell membranes indicated that both wild-type and mutant cells expressed the alpha and beta 1 subunits of the fibronectin receptor; the mutant cells displayed reduced levels of the subunit. Immunohistochemical analysis indicated that, despite the presence of the receptor in both cell types, their patterns of localization and aggregation were different. The wild-type cells showed a needle-like distribution of the receptor, in contrast to the clumped appearance in the mutants.(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation of biological processes by mineral fiber adsorption of macromolecules in vitro.

Three classes of macromolecules (i.e., DNA, RNA, and protein) were shown to be adsorbed to asbestiform minerals. The cytotoxicity exerted by the fibers on a normal human fibroblast cell line, which may be an indicator of the carcinogenic potential of mineral fibers, correlated positively with the degree of macromolecular adsorption of the fiber, namely: chrysotile greater than amosite greater than glass fiber. Asbestiform fibers also induce an alteration in in vitro DNA hydrolysis by bovine pancreatic deoxyribonuclease. This phenomenon suggests that adsorption by asbestiform minerals may modulate biological processes by inducing a conformational change in biological macromolecules as a result of coulombic interaction between the surface charge of the fiber and the hydrophilic groups on the macromolecule.

Morphological transformation in vitro of normal human fibroblasts by chrysotile.

Pathologic response of tissue to asbestos in vivo gives rise to fibromatoma, granuloma and mesothelioma. We are attempting to develop a model system in vitro using human cells in order to investigate the possible mechanisms responsible for these pathologies. Within the first 12 hr of exposure to chrysotile, the fibroblasts showed distinctive morphological changes. Cells appeared elongated with occasional vacuolated nuclei and granular cytoplasm. Cells showed no other obvious morphological changes by light microscopy and were serially passaged at confluence. The cells with vacuolated nuclei were successfully serially passaged. Binucleated cells were first observed 48 hr after passaging. As time in culture increased (3 days to 2 weeks) many cells lost their distinctive bipolar properties and developed "stress striations" and multiple vacuoles in the cytoplasm. Multinucleated giant cells (2-11 nuclei/cell) with lobate nucleoli became more numerous. With increasing passages, the confluent cell density decreased and cell size increased. Cells usually had condensed nucleoli and had lost all control of directional growth. Preliminary indications suggest that these in vitro morphological transformations are due--at least in part--to a lack of control over cytokinesis.

Interaction of erythrocyte membranes with particulates.

Chrysotile fibers promoted the fusion of human erythrocyte membranes and the hemolysis and fusion of fowl erythrocytes. Amosite and glass fibers did not show the same effect. These findings provide a model for the internalization of the fibers by animal cells.

Interactions of chrysotile and benzopyrene in a human cell culture systems.

The risk of lung cancer related to asbestos exposure has been shown to increase disproportionately by cigarette smoking, suggesting a synergistic effect. Differing lengths of NIEHS chrysotile with benzopyrene [B(a)P, B(e)P] (organic by-products of combustion) were applied on normal human fibroblasts (cell line CI) to test for cytotoxicity (survival determined by colony-forming efficiency), binding of benzopyrene to DNA, and the production of benzopyrene metabolites. At concentrations of 100 micrograms/mL, NIEHS short chrysotile was more cytotoxic than NIEHS intermediate chrysotile (3% and 17% survival, respectively); B(a)P and B(e)P concentrations up to and including 10 microM were not cytotoxic. Simultaneous application of NIEHS short chrysotile with B(a)P or B(e)P did not decrease survival synergistically. On the contrary, application of B(a)P simultaneously with NIEHS intermediate chrysotile resulted in increased survival over that of intermediate chrysotile alone (25% and 17% survival, respectively). There were low levels of B(a)P bound to DNA in the presence of NIEHS short chrysotile or NIEHS intermediate chrysotile. Measurable levels of B(a)P-DNA adducts were formed both in the absence and in the presence of each size of NIEHS chrysotile. However, there was no strong indication of a perturbation of the level of DNA-B(a)P binding following simultaneous administration of increasing levels of asbestos in addition to 1 microM hydrocarbon. The asbestos had no demonstrable influence on the level of B(a)P metabolism during the 24-hr period following simultaneous exposure of asbestos and hyrdocarbons.(ABSTRACT TRUNCATED AT 250 WORDS)

Elemental modifications and polycyclic aromatic hydrocarbon metabolism in human fibroblasts.

Mineral fibers and particulates represent one of the best documented, economically important, and ubiquitously occurring categories of human carcinogens. Yet, while a wealth of information exists concerning the mechanism of action of physical, chemical, and viral carcinogens, virtually nothing is known relative to the mechanism of action of this economically important class of carcinogenic compounds known as mineral fibers and particulates. While the length and diameter of various forms of asbestos have been associated with both cellular toxicity in vitro and tumor occurrence in vivo, nothing is known about whether or not these same physical properties are responsible for the purported synergistic interaction between cigarette smoking and asbestos exposure relative to the induction of bronchogenic carcinoma. Thus, while the risk of bronchogenic carcinoma for nonsmokers exposed to asbestos appears to be only slightly greater than that for unexposed nonsmoking populations, the risk of occurrence of this same tumor in asbestos workers who also smoke is approximately 100-fold greater than in nonsmoking asbestos workers. The risk of bronchogenic carcinoma is increased approximately 8-fold in asbestos workers who smoke over non-exposed smokers. Since it is clear that cigarette smoke contains over 150 polycyclic aromatic hydrocarbons, some of which are known animal carcinogens, and since other laboratories have reported that metals associated with asbestos redirect the metabolism of these agents, it was of interest to us to investigate the effects of mineral fibers on the metabolism and biochemistry of polycyclic aromatic hydrocarbons.