Development of an in vitro rat nasal epithelial model for predicting upperrespiratory tract toxicity

Nasal toxicology is an important, expanding area of research, which has, in the past, centered on in vivo studies. There is now a need for in vitro me thodology to study this target organ, and the aim of this work was to devel op such a model using isolated rat nasal turbinates. Initially, optimal cul ture conditions for olfactory epithelium (OE) and respiratory epithelium (R E) were determined using lactate dehydrogenase (LDH) release as an indicato r of viability. The optimal culture conditions were found to be incubation at 31 degrees C under an atmosphere of 95%O-2:5%CO2, in six-well plates con taining 2 mt of William's E medium supplemented with glutamine (2 mM) and g entamycin (100 mu g/mL), with shaking at 60 strokes/min (amplitude 3.5 cm). Under these conditions LDH release from both tissues was 30-40% by 24 h. T he model was further characterized in terms of a range of viability paramet ers reflecting different aspects of cellular function. Initial values for i ntracellular potassium, ATI: and nonprotein sulfhydryl levels were 1 mu mol /mg protein, 4.43 nmol/mg protein, and 33 nmol/mg protein, respectively, fo r OE, and 0.84 mu mol/mg protein, 5.53 nmol/mg protein, and 31 nmol/mg prot ein, respectively, for RE. During a 24-h incubation period these parameters did not fall significantly below these initial values in either tissue. Pr otein synthesis was maintained throughout the 24-h incubation, and the rate of synthesis in RE was double that of OE. Histological examination of the tissues demonstrated that RE maintained normal morphology for 24 h, but OE showed signs of focal degeneration as early as 4 h after the start of the i ncubation period. Thus, RE was maintained in a viable state for 24 h, but O E for only 8 h.