Toluene permeabilization differentially affects F- and P-type ATPase activities present in the plasma membrane of Streptococcus mutans

Streptococcus mutans membrane-bound P- and F-type ATPases are responsible for H+ extrusion from the cytoplasm thus keeping intracellular pH appropriate for cell metabolism. Toluene-permeabilized bacterial cells have long been used to study total membrane-bound ATPase activity, and to compare the properties of ATPase in situ with those in membrane-rich fractions. The aim of the present research was to determine if toluene permeabilization can significantly modify the activity of membrane-bound ATPase of both F-type and P-type. ATPase activity was assayed discontinuously by measuring phosphate release from ATP as substrate. Treatment of S. mutans membrane fractions with toluene reduced total ATPase activity by approximately 80 percent and did not allow differentiation between F- and P-type ATPase activities by use of the standard inhibitors vanadate (3 µM) and oligomycin (4 µg/mL). Transmission electron microscopy shows that, after S. mutans cells permeabilization with toluene, bacterial cell wall and plasma membrane are severely injured, causing cytoplasmic leakage. As a consequence, loss of cell viability and disruption of H+ extrusion were observed. These data suggest that treatment of S. mutans with toluene is an efficient method for cell disruption, but care should be taken in the interpretation of ATPase activity when toluene-permeabilized cells are used, because results may not reflect the real P- and F-type ATPase activities present in intact cell membranes. The mild conditions used for the preparation of membrane fractions may be more suitable to study specific ATPase activity in the presence of biological agents, since this method preserves ATPase selectivity for standard inhibitors.

Neurobehavioral development of rats exposed to toluene through maternal milk

Organic solvents have been detected in the milk of workers in the rubber industry exposed during gestation to a mixture of solvents at average concentrations lower than the currently accepted occupational limit of exposure (100ppm). The objective of the present study was to determine if exposure of rat offspring to toluene during laction, through maternal milk, would affect the developing brain. There month old, lactating Wistar rats were injedted with toluene (1.2 g/Kg, sc, N = 10) daily from laction day 2(day of delivery - day 1) to day 21. Controls (N=9) were injected with the vehicle (c0rn oil). Offspring (7 pups per litter) were evaluated form neurosomatic development and exploratory behavior before weaning and behavior in the open field. A second group of toluene treated ratas (N=6) and controls (N=6) was used to evaluate behavior of the offspring in the open-field on day 35 and performance in a shuttle box in adulthood. Toluene levels in blood and milk after a single 1.2 g/Kg sc injection were studied in a third group of rats on laction day 10. Toluene levels in milk 4 h after a single injection (10.3ñ6.2) were 5 times higher than in blood (2.1ñ0.8). No effects of treatment on offspring development or on any of the behavioral tests were observed. Sex differences were observed in open-field behavior and performance in the shuttle box. The present results suggest that exposure of pups to high concentrations of toluene through maternal milk does not result in blood levels high enough to affect growth or development