Restoration of hepatic mitochondria during recovery from carbon tetrachloride intoxication.

-During CC14 intoxication in rats, a disruption of hepatic mitochondrial structure and function occurs, which is characterized by a loss of respiratory activity, loss of phosphorylation coupled to respiration, and mitochondrial swelling, attended by loss of cristae structure. Within 15-25 hr, after full development of the mitochondrial lesion, the function and structure of the mitochondria are largely restored. Studies of the turnover of mitochondrial DNA and the rates of synthesis of mitochondrial DNA and protein indicated that the CC14-insulted hepatocyte is repairing the mitochondrial damage by the insertion of specific elements into the damaged organelle, rather than by proliferation of undamaged mitochondria for replacement. The failure of ethidium bromi~e, oxytetracycline and chloramphenicol, specific inhibitors of mitochondrial protein, and/or nucleic acid synthesis, to block this restoration substantiates the postulated repair process, and also indicates the non-critical nature of the respective mitochondrial functions during the repair process. Cytochrome measurements made during the period of acute damage revealed normal levels of cytochrome c, c~ and aa a. The observed elevation of cytoehrome b is attributed to contamination of the preparation by hemoglobin. THE HEPATIC m i t o c h o n d r i o n is one of the charac ter i s t ic sites of CC14-induced cell damage . 1 Ul t ras t ruc tu ra l ly , the m i t o c h o n d r i a of an in tox ica ted an ima l a p p e a r to be swollen in situ, and the mat r ix is a b n o r m a l l y lucid. 2 The m i t o c h o n d r i a are ca lc ium loaded, and amorph ic , dense bodies , p r o b a b l y aggrega t ions of ca lc ium phospha te , are no ted in the mat r ix of the swollen organelles , a The a l te red in situ a ppe a ra nc e is a c c o m p a n i e d by a loss of r e sp i r a to ry activity, especial ly on N A D ÷ l inked substrates , and by an impa i rmen t of energy-coupled functions, which is de tec tab le when i so la ted m i t o c h o n d r i a are assayed. 4 This lesion is not necessar i ly assoc ia ted wi th lethali ty, since the lesion can be deve loped by a dose of CC14 from which the an imal recovers. U n d e r such condi t ions , m i tochond r i a l degenera t ion ceases and a re turn of no rma l s t ructure and funct ion is observed. O f interest to this l a b o r a t o r y is the route by which the CC14-damaged hepa tocy te affects the r e s to ra t ion of its d a m a g e d mi tochondr i a . In cons ide ra t ion of recent p ro posa ls re la ted to m i tochond r i a l biogenesis , it is necessary to cons ider a m i n i m u m of three poss ib le rou tes of r e s to ra t ion : (1) Rep lacemen t of d a m a g e d organelles . Since it is poss ib le tha t all m i t o c h o n d r i a wi th in a surviving hepa tocy te have no t suffered mass ive damage , a repl icat ive burs t o f m i t o c h o n d r i a l b iogenesis by the sl ightly damaged , or u n d a m a g e d m i t o c h o n d r i a cou ld serve to replenish the m i t o c h o n d r i a l B,P. 23/23--A 3 2 2 7 3228 M.J. BRABEC, R. H. GRAY and I. A. BERNSTEIN population depleted by CCl4-induced destruction. (2) Repair of the damaged organelles. The CC14-induced damage, while causing obvious mitochondrial derangements, might be localized in a relatively simple unit of the mitochondrion, amenable to repair by elements generated by the recovering hepatocyte. (3) Repair and replacement. A combination of both operations could repair the less damaged organelles, and replace those that have been irreversibly damaged. This study of the synthesis and turnover of mitochondrial elements during CC14 intoxication, complemented with the use of selective inhibitors of mitochondrial protein and nucleic acid synthesis, was designed to indicate which of the above three mechanisms was responsible for restoration. The data indicated that the restoration of the damaged organelles was primarily a repair process, not involving mitochondrial DNA, RNA or protein synthesis. However, a late acceleration in mitochondrial DNA turnover rates does indicate that mitochondrial replication can be occurring during the late phase of recovery. Normal levels of mitochondrial cytochromes were found, thereby tentatively eliminating these elements as being sites of CC14-induced damage. MATERIALS AND METHODS Materials. Heavy metal-free sucrose, ADP, Tris and oligomycin were obtained from Sigma Chemical Co. Ethidium bromide, B grade, was purchased from Calbiochem. Chloramphenicol (Chloromycetin) was a generous gift from Mr. Stanley Kurtz of Parke, Davis & Co. Oxytetracycline (Liquamycin) was purchased from Chas. Pfizer Co. Radioisotopes were obtained from New England Nuclear or SchwarzMann. PCS solubilizer (Schwarz-Mann) was used as a scintillation mixture for radioactivity determinations. Carbon tetrachloride was reagent grade, as were all other chemicals used. Solutions used for mitochondrial assay and isolation were prepared with double-distilled water. Mitochondrial preparation and assays. Rats were 180-220 g males, CFN strain, reared in this laboratory's colony. CC14 was injected i.p., without dilution, at 1-4 ml/ kg. Eater in the study, the level of CC14 was reduced to 1.2 ml/kg, which reduced the rate of barly mortality without affecting the mitochondrial response. At desired intervals, the' intoxicated rats were killed by stunning and decapitation, the liver, or a selected lobe, was removed, minced, rinsed and then homogenized in 0.25 M sucrose, 1 mM EDTA, l0 mM KC1 and l0 mM Tris (pH 7.0 at 0°). The homogenate was centrifuged at 450 g for 10 min to remove nuclei, unbroken cells and debri. The mitochondrial fraction was prepared from the post-nuclear supernatant by centrifugation at 8800 g for 10 min. The mitochondrial pellet was resuspended in the original homogenate volume of 0.25 M sucrose and centrifuged for 10min at 480g. The supernatant was centrifuged at 7200g for 10 min. The resulting pellet was resuspended in 0-25 M sucrose, and the 7200 g wash routinely repeated once except for those experiments that involved radioisotopes, in which case four additional 7200 g washes were performed. Aseptic techniques using sterile solutions were practiced in the L-leucine incorporation experiments in vitro to diminish the problem of bacterial contamination. The mitochondria were assayed with an oxygen electrode apparatus in a 2-ml water-jacketed chamber stirred with a magnetic bar. The respiratory rate and respiratory control ratio (RCR) were determined essentially as described by Estabrook 5 Restoration of CC14-damaged hepatic mitochondria 3229 in a medium containing 0-15 M sucrose, 20 mM KH2PO4, 20 mM Tris (pH 7.4 at 25°), 5 mM MgC12 and 100 mM succinate. Oligomycin was added to a final concentration of 1 #M. Protein was determined by the Biuret method as modified by Jacobs et al. 6 or by the procedure of Lowry et al. 7 Inhibitor studies. Chloramphenicol, as the sodium succinate salt dissolved in water, was injected i.p., at a level of 200 mg/kg body weight every 6 hr, commencing 30 hr after the injection of CC14. Oxytetracycline was administered i.p., 100 mg/kg, every 8 hr beginning 30 hr after CC14 administration. Ethidium bromide, dissolved by warming in chloroform-saturated water, was injected i.p., 25 mg/kg, twice daily, in a manner similar to chloramphenicol and oxytetracycline. Mitochondrial D N A synthesis and turnover. Control and CC14-intoxicated rats were injected with 250 ~tCi/kg body weight of [3H]thymidine (sp. act. 30-50 Ci/mmole) at the selected intervals and the mitochondria isolated 30 min later, with the additional washes described above. The final pellet was dissolved in 4 ml of 1~o sodium deoxycholate (DOC), and the nucleic acids and protein were precipitated with 4 ml of 0.5 N perchloric acid (PCA). A pellet was obtained by centrifuging for 5 min at 9000 g. The pellet was washed twice by resuspension in 10 ml of 0.5 N PCA. The supernatant of the second wash was essentially non-radioactive. The pellet was dissolved in 3 ml of 88~o formic acid and aliquots were removed for estimation of protein and for determination of radioactivity in a Packard Tri-Carb 3300 liquid scintillation counter. Counts/min were corrected to dis./min by use of an efficiencyquench curve, using the instrument's Automatic External Standard, based on an unquenched efficiency of 38"3 per cent for the scintillation mixture. The mitochondrial DNA was labeled for the turnover studies by three i.p. injections of a total of 170 #Ci [3H]thymidine administered at 2-hr intervals, a CC14 was administered to the rats 2 hr after the last [3H]thymidine injection. At the indicated time intervals, pairs of animals were sacrificed, and the mitochondria isolated as above, precipitated and washed by the above protocol, dissolved in 0.4 N K O H I ~ sodium dodecylsulfate (SDS), and the radioactivity and protein determined on duplicate aliquots, as above. L-[14C]leucine incorporation, in vitro. Mitochondria were isolated aseptically from control and intoxicated rats with the additional washes as described above. The assay for L-[14C]leucine incorporation was carried out as described by Simpson et al., 9 except that the incubation period was shortened to 20 rain. Mitochondria (10 mg protein/ml) were incubated in 0.15 M sucrose, 3 mM KC1, 1 mM EDTA, 8 mM succinate, 12 mM KH2PO4, 3 mM AMP, 3"5 mM NAD, 6 mM MgSO4 (pH 7.0), complete amino acid mixture (minus L-leucine) and L-[14C]leucine (sp. act. 300-326 Ci/mole) at 30 °. Bacterial contamination was monitored by plating 0-05 ml of incubation mixture on nutrient agar plates immediately after the assay and incubating for 24 hr at 37 °. Experiments that yielded over 1000 colonies/ml were discarded. L-[14C]leucine incorporation, in vivo. Control and CC14-intoxicated groups of rats were given 250/2Ci/kg body weight t-[3H]leucine (sp. act. 53 Ci/m-mole) i.p. at the time period of interest. Sixty min later, the animals were sacrificed, paired livers pooled, and the mitochondria were isolated as described above. The final pellet was suspended in 0-I M Tris (pH 8.0)-0.1 M NaC1, 1~ SDS, then precipitated with 10~ trichloroacetic acid (TCA)-0.05 M L-leucine, collected at 2000 9, extracted once with 5~ TCA, dissolved in 1 N NaOH, reprecipitated with 10~ TCA-0.05 M L-leucine, 3230 M.J . BRABEC, R. H. GRAY and I. A. BERNSTEIN sedimented, and finally dissolved in 2 N NaOH. Aliquots were removed for protein determination and scintillation counting. An aliquot of the final TCA supernatant was also monitored for radioactivity. Electron microscopy. Tissue was finely minced, fixed in 3~ glutaraldehyde-0"2 M NaHPO4, pH 7"3, stained with 2% OsO4-0"l M NaHPO4, pH 7-3, dehydrated in ethanol and propylene oxide and embedded in Epon. Thin sections were obtained with a Reichart OMU-2 ultramicrotome equipped with a diamond knife, placed on carbon-coated Formvar grids, post-stained with Reynolds lead citrate 1° and 7~o uranyl magnesium acetate, and examined in an AEI Corinth electron microscope. Cytochrome assays. Mitochondrial cytochrome content was measured by difference spectroscopy on DOC-solubilized mitochondria with an Aminco DW-2 spectrophotometer operated in the split-beam mode as described by Williams. 11