54 



C. B. Sledge 



« 



unchanged from control figures. What about the cell membrane? Allison (1965), 

 working with cell cultures in 95'''o oxygen, found increased permeability of the 

 lysosomes to the Gomori substrate 12 hrs. before the cell membrane became permeable 

 to eosin. It was, therefore, suspected that the effect of hyperoxia was a direct one on 

 the lysosomal membrane. Mr. Dingle and I (Slldge and Dingle, 1965) prepared 

 lysosome-rich fractions from chick embryonic cartilage and incubated them with 

 oxygen or nitrogen. Similar fractions from liver, kidney, spleen and brain were run 



as controls. Only in the case of 

 lysosomes from cartilage was there 

 activation by oxygen. 



When Vitamin E, a lipid solu- 

 ble anti-oxidant, or vitamin C, a 

 water-soluble anti-oxidant, were 

 added to the culture medium, pro- 

 tection from hyperoxia was ob- 

 served (Fig. 3). Tappel et al. (1963) 

 have found increased lipid per- 

 oxidation in vitamin E deficiency. 

 It therefore seems likely that the 

 effect of hyperoxia is mediated 

 through increased release of hydro- 

 lytic enzymes by lipid peroxida- 

 dation of the lysosomal membrane. 

 The protective effect of vitamin C 

 IS perhaps due to a re-cycling phenomenon whereby a membrane constituent, sus- 

 ceptible to oxidation, is protected by lipidsoluble reducing substance (perhaps vit- 

 amin E) which, in turn, is maintained in a reduced state by vitamin C in the cytoplasm. 

 The role of the osteoclast in these experiments has been most interesting. They are 

 not normally seen in chick limb-bones in culture. However, in all of the rudiments 

 exposed to hyperoxia alone, osteoclasts appear — never in great number and always 

 after resorption is well underway. They are not due to hyperoxia, per se, as they are 

 not seen in rudiments exposed to hyperoxia but protected from matrix degradation 

 by Cortisol, EACA, or vitamins E or C. From this it would appear that some 

 product of matrix degradation is responsible for the induction of osteoclast formation. 

 A possible scheme might be: 



oxygen + lysosome '*!", . — acid protease 



^ ^ ^ peroxidation '^ 



acid protease + protein-polysaccharide 



Fig. 3. Top: control rudiment exposed to 85% oxygen tor 

 6 days. Toluidine blue. XIO. — Bottom: contralateral rudi- 

 ment exposed to 85°/o oxygen for 6 days. 40 yJm\. vitamin E 

 added. Toluidine blue. ^10 



arginine bond 

 hvdrolvsls"' 



lon-collagenous protein + polysaccharide 



non-coilagenous protein 



undifferentiated 



mesenchymal 



cell 



osteoclast 



Using 85''/o oxygen and natural medium (plasma clot-embryo extract), a marrow 

 cavity has been produced in chick limb bones in culture for the first time (Fig. 4). As 

 no vessels were present at the time of explantation (8 days), this is a direct effect of 

 oxygen on the tissues. 



