Page 68 - 66_04

ARMIN WOLF ANAL REAL ACAD. FARM.

Transmission electron microscopy

The cell cultures were fixed with 1% glutaraldehyde in 0.1 M
phosphate buffer, pH 7.4, for 1 hour or overnight at 4 °C. After
postfixation with 1 % OsO4 in 0.1 M cacodylate buffer, pH 7.4, for 1 hour
at 4 °C, the cell cultures were dehydrated in graded ethanol solutions and
embedded in Epon according to the method of Pease (Pease 1984).

Ultrathin sections of hepatocyte cultures from at least two selected
tissue blocks per well were counterstained with uranyl acetate and lead
citrate, and were examined with a Philips CM10 transmission electron
microscope.

Alterations in the treated cell cultures were expressed by different
scores depending on their degree of intensity. The following scores were
used: changes observed in one cell per mm2 = marginal; changes in two
cells per mm2 = slight; alterations in three to nine cells per mm2 =
moderate; alterations in ten or more cells per mm2 = marked.

Determination of cytotoxicity

Lactate dehydrogenase (LDH) activity in the culture media was
measured spectrophotometrically as an index of plasma membrane
damage and loss of membrane integrity (Wedler and Acosta 1994).
Enzyme activity was expressed as the percentage of extracellular LDH
activity of the total LDH activity on the plates.

Determination of chromatin condensation and degradation

Chromatin condensation and fragmentation were determined by
Feulgen staining and using light microscopy to count the percentage of
cells containing alterations in the nuclear structure (Lillie and Fullmer
1976).

Hepatocyte samples were treated as follows: after the cell culture
medium was removed, the cells were fixed overnight with 4 %
formaldehyde in PBS. After hydrolyzing in 5 N HCl for 90 min at room
temperature, the cells were rinsed in distilled water and stained for 30 min
in Schiff reagent4. After this, the hepatocytes were rinsed in 0.05 M
Na2S2O3 solution for 2 min, washed first in tap water, and then washed in

6