Effect of trichloromethyl and trichloromethyl peroxyl free radicals on protein sulfhydryl content studies in model and in enzymatic carbon tetrachloride activation systems.

The effect of trichloromethyl and trichloromethyl peroxyl free radicals on protein sulfhydryl content was studied using both, model and enzymatic activation systems. In the model system activation of CCl4 to both free radicals was by UVC light and the target protein was either delipidated or undelipidated albumin. Under air, the CCl3O2. radicals were able to significantly decrease the protein SH in both albumin preparations. A small but signficant effect of UVC alone was observed with defatted albumin. No significant decreases in protein sulfhydryl were observed by .CCl3 attack on the defatted albumin. Reaction of CCl3O2. on cysteine SH led to chloroform formation indicating that a H abstraction reaction is involved in the process. UVC light has an own effect on SH group content. Similar results were obtained when the interaction was with undelipidated albumin rather than with cysteine. Their formation was significantly prevented by Trolox 1 mM in incubation mixture. When the CCl3O2. were generated by liver microsomal activation of CCl4 under air, a significant decrease in microsomal protein SH content was observed. NADPH also exerted an effect of its own. These decreasing effects were fully prevented by either Trolox or EDTA addition to incubation mixtures but not by alpha-tocopherol free or as a succinate ester. Incubation mixtures containing nuclear suspensions and NADPH led to a decrease in protein SH content. This decrease was not enhanced further by the presence of CCl4. No effect on the protein SH content was observed when either mitochondrial or cytosolic fractions were employed to attempt activation of CCl4 to .CCl3/CCl3O2. free radicals. The ability of CCl4 derived free radicals to decrease protein SH in liver microsomes could be involved in loss of activity of key SH enzymes of relevance such as microsomal calcium pump. This pump is known to be damaged during CCl4 poisoning. This effect was blamed to initiate alterations in calcium homeostasis later leading to CCl4 induced liver cell death.

Carbon tetrachloride-induced free radical mediated protein oxidation in vitro and in vivo.

Trichloromethyl and trichloromethylperoxyl free radicals generated by UVC light activation of carbon tetrachloride promote the formation of protein carbonyls in chicken egg albumin in vitro. Further, UVC light itself exerts a highly significant equivalent effect of its own. The reaction was significantly blocked by Trolox but not by α-tocopherol at a concentration of 1 mM. EDTA (3 mM) partially prevented the oxidative effects of (•)CCl3 or CCl3O2(•) in proteins but not those of UV light. Rat liver microsomes in the presence of NADPH also led to the formation of carbonyl groups in microsomal proteins. That effect was significantly enhanced by the presence of CCl4 in the incubation mixture. Liver microsomes from CCl4 poisoned animals exhibited significantly increased levels of protein carbonyls at 1 and 2 h after administration but not at 3 h. These results suggest that proteins might be targets of CCl4 oxidative effects. Protein carbonyls might arise from either direct attack by (•)CCl3 or CCl3O2(•), or from 4-hydroxynonenal reaction via Michael addition with amino acids at the aldehyde double bond and retaining the carbonyl group attached to the target protein.