Human monocytes (MN) produce O2- and H2O2 when stimulated by agonists. Dichlorofluorescin diacetate (DCFH-DA) has been used as a substrate for measuring intracellular oxidant production in neutrophils. DCFH-DA is hydrolyzed by esterases to dichlorofluorescin (DCFH), which is trapped within the cell. This nonfluorescent molecule is then oxidized to fluorescent dichlorofluorescin (DCF) by action of cellular oxidants. DCFH-DA can not be appreciably oxidized to a fluorescent state without prior hydrolysis. We have examined the utility of DCFH-DA for the assessment of monocyte oxidative responses. The levels of intracellular fluorescence measured by flow cytometry were considerably less than expected from reported levels of O2--production or chemiluminescence assays. Compared with neutrophils, monocytes produced minimal increases in DCF fluorescence after stimulation with phorbol myristate acetate as measured by flow cytometry, but both cell types showed increases in fluorescence when bulk cell suspensions were measured by spectrofluorometry. To determine the intracellular location of the DCFH, bulk fluorescence measurements were made on both whole and sonicated cell preparations. When intact mononuclear cells were preloaded with DCFH-DA, then sonicated and oxidized with added excess H2O2, the increase in fluorescence was only 30% of the fluorescence of mononuclear cell sonicates to which DCFH-DA was added and oxidized in a similar manner. These results suggest that a portion of the DCFH-DA incorporated by intact cells, is not susceptible to oxidation by the added H2O2. Addition of NaOH to induce hydrolysis of any residual DCFH-DA in the sonicates of DCFH-DA-loaded intact mononuclear cells resulted in a further increase in fluorescence upon addition H2O2, suggesting that a significant portion of the DCFH-DA was not hydrolyzed despite ample uptake of this dye by these cells. In contrast, no further increase in fluorescence was observed in sonicates of DCFH-DA-loaded intact neutrophils, suggesting complete hydrolysis of all incorporated DCFH-DA to DCFH. When monocytes were allowed to phagocytose DCFH-DA-loaded Staphylococcus aureus, intracellular fluorescence was measurable by flow cytometry, indicating intracellular oxidation of the fluorochromes. We therefore propose that in monocytes the mechanism of intracellular processing of these fluorochromes differs from that in neutrophils owing to differences in intracellular localization of fluorochromes, site of oxidant production, and/or accessibility of the DCFH-DA to esterolysis.
Eye irritation potency of a compound or mixture has traditionally been evaluated using the Draize rabbit-eye test (Draize et al., 1944). In order to aid predictions of eye irritation and to explore possible corresponding mechanisms of eye irritation, a methodology termed "membrane-interaction QSAR analysis" (MI-QSAR) has been developed (Kulkarni and Hopfinger 1999). A set of Draize eye-irritation data established by the European Center for Ecotoxicology and Toxicology of Chemicals (ECETOC) (Bagley et al., 1992) was used as a structurally diverse training set in an MI-QSAR analysis. Significant QSAR models were constructed based primarily upon aqueous solvation-free energy of the solute and the strength of solute binding to a model phospholipid (DMPC) monolayer. The results demonstrate that inclusion of parameters to model membrane interactions of potentially irritating chemicals provides significantly better predictions of eye irritation for structurally diverse compounds than does modeling based solely on physiochemical properties of chemicals. The specific MI-QSAR models reported here are, in fact, close to the upper limit in both significance and robustness that can be expected for the variability inherent to the eye-irritation scores of the ECETOC training set. The MI-QSAR models can be used with high reliability to classify compounds of low- and high-predicted eye irritation scores. Thus, the models offer the opportunity to reduce animal testing for compounds predicted to fall into these two extreme eye-irritation score sets. The MI-QSAR paradigm may also be applicable to other toxicological endpoints, such as skin irritation, where interactions with cellular membranes are likely.
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