The major objective of this investigation was to determine the thiol s
tatus of chondrocytes and to relate changes in the level of glutathion
e and cysteine to maturation of the cells as they undergo terminal dif
ferentiation. Chondrocytes were isolated from the cephalic portion of
chick embryo sterna and treated with all-trans retinoic acid for one w
eek. We found that the addition of 100 nM retinoic acid to the culture
s decreased the intracellular levels of glutathione and cysteine from
6.1 to 1.6 and 0.07 to 0.01 nmol/mu g DNA respectively; retinoic acid
also caused a decrease in the extracellular concentration of cysteine.
The decrease in chondrocyte thiols was dose and time dependent. To ch
aracterize other antioxidant systems of the sternal cell culture, the
activities of catalase, glutathione reductase and superoxide dismutase
were determined. Activities of all of those enzymes were high in the
retinoic acid-treated cells; the conditioned medium also contained the
se enzymes and the cytosolic isoenzyme of superoxide dismutase. We pro
bed the specificity of the thiol response by using immature caudal cho
ndrocytes. Unlike the cephalic cells, retinoic acid did not change int
racellular glutathione and extracellular cysteine levels, although the
retinoid caused a reduction in the intracellular cysteine concentrati
on. Finally, we explored the effect of medium components on chondrocyt
e thiol status. We noted that while ascorbate alone did not change cel
l thiol levels, it did cause a 4-fold decrease in the extracellular cy
steine concentration. When retinoic acid and ascorbic acid were both p
resent in the medium, there was a marked decrease in the level of glut
athione. In contrast, the phosphate concentration of the culture mediu
m served as a powerful modulator of both glutathione and cysteine. Res
ults of the study clearly showed that there is a profound decrease in
intracellular levels of both cysteine and glutathione and that thiol l
evels are responsive to ascorbic acid and the medium phosphate concent
ration. These findings point to a critical role for thiols in modulati
ng events linked to chondrocyte maturation and cartilage matrix synthe
sis and mineralization.