Samir Abou-Khalil

Interaction of rhodamine 123 with mitochondria isolated from drug-sensitive and -resistant friend leukemia cells

Mitochondria isolated from Friend leukemia cell lines sensitive (FS) and resistant (FR) to rhodamine 123 (Rho123), showed respiratory control and ADP/O ratios indicative of well-coupled oxidative phosphorylation activity. When Rho123 was added to mitochondria from both cell lines, respiratory State 4 increased. The increase was higher in mitochondria isolated from resistant than from sensitive cells. Respiratory State 3 was slightly more inhibited by Rho123 in resistant than in sensitive cell mitochondria (98 and 82% inhibition, respectively). While it is not clear how the uncoupling-like effects of Rho123 on State 4 contribute to cellular toxicity, our results indicate that differential cellular sensitivity to the drug does not correlate with inhibition of oxidative phosphorylation in mitochondria isolated from drug-sensitive and -resistant cells.

Differential effects of chloramphenicol and its nitrosoanalogue on protein synthesis and oxidative phosphorylation in rat liver mitochondria.

Abstract Protein synthesis and oxidative phosphorylation were chosen as measures to study the differences between the effects of chloramphenicol (CAP) and its derivative nitroso-chloramphenicol (NO-CAP) on rat liver mitochondria. [ 14 C]Leucine incorporation into mitochondrial protein was inhibited 83 per cent by 30 μM CAP and was equally inhibited by a similar concentration of thiamphenicol; 30μM NO-CAP, however, inhibited [ 14 C]leucine incorporation only 34 per cent and 30μM nitrosobenzene had no effect. A millimolar concentration of CAP was required to inhibit oxidative phosphorylation, whereas 75 μM NO-CAP was inhibitory. Unlike CAP, NO-CAP at 100 μM slightly inhibited state 4 respiration with glutamate as substrate, but slightly activated it with succinate. Respiratory state 3 with glutamate was completely inhibited by 75 μM NO-CAP, whereas the same concentration of CAP was only 10 per cent inhibitory. With succinate, 250 μM NO-CAP was required to inhibit state 3, whereas 600 μM CAP had no effect. The uncoupled state triggered by 2,4-dinitrophenol in the presence of either glutamate or succinate was inhibited totally by NO-CAP, but not by CAP. The inhibition by NO-CAP was mitochondrial protein dependent, for more NO-CAP was required for inhibition with a larger amount of protein. NO-CAP effects could be prevented or released by cysteine, but not by washing. Oxidative phosphorylation was also inhibited by another nitroso compound, nitrosobenzene, which, however, did not affect mitochondrial protein synthesis. The results indicate that, unlike CAP, NO-CAP is a potent inhibitor of the energy conserving mechanism.

Aerobic nitroreduction of dehydrochloramphenicol by bone marrow

It has been previously demonstrated that dehydrochloramphenicol (DH-CAP), a bacterial metabolite of chloramphenicol, induces DNA single strand breaks in intact cells and is profoundly more cytotoxic than chloramphenicol (CAP). In view of previous observations relating genotoxicity of nitrocompounds to their nitroreduction by the target tissue, we studied the nitroreduction of DH-CAP by human and rabbit bone marrow. Nitroreduction by tissue homogenates was determined by the Bratton Marshall colorimetric assay and by high-performance liquid chromatography (HPLC). Nitroreduction of DH-CAP by bone marrow cell homogenates was observed under aerobic conditions and the reduction was both cell concentration- and time-dependent. The formation of the amino product aminodehydrochloramphenicol was confirmed by HPLC. Reduction by other tissues including human liver, Raji cells, and HL-60 tumors was also observed. These results suggest that genotoxicity of DH-CAP may be related to its nitroreduction by the target tissue with in situ production of toxic intermediates. Together with previous studies, these observations lend support to the thesis that the p-NO2 group may be the structural feature underlying aplastic anemia from CAP.

Inhibition by rhodamine 123 of protein synthesis in mitochondria of normal and cancer tissues

The effect of the mitochondrial dye rhodamine 123 (Rho 123) on protein synthesis (PS) activity was investigated in mitochondria isolated from liver and from both chloroma and erythroleukemia tumors. Incorporation of labelled leucine into mitochondrial protein was used to measure the rate of PS. While PS specific activity was much higher in hematopoietic tumors mitochondria as compared to that of liver, the addition of increased concentration of Rho 123 in all tested organelles resulted in increased inhibition of PS to reach 75-82% with 10 micrograms/ml of the dye. Similar results were obtained with 10 micrograms/ml of chloramphenicol, the specific inhibitor of mitochondrial PS. Moreover, under the conditions of the study, the addition of Rho 123 to mitochondria did not trigger any ATPase activity, thus eliminating any competition for the energy source ATP between PS and ATPase. These results demonstrate that, in addition to its known inhibitory action on oxidative phosphorylation, the mitochondrial dye Rho 123 has a potent inhibitory effect on PS in both liver and hematopoietic tumors mitochondria.

Mechanism of interaction of ticlopidine and its analogues with the energy-conserving mechanism in mitochondria

It was shown recently that the antiaggregating agent ticlopidine and some of its analogues inhibit the energy-conserving mechanism in mitochondria [Abou-Khalil et al., Biochem. Pharmac. 33, 3893 (1984)]. In the present investigation, the mechanism of inhibition by these drugs was investigated by studying their effects on key reactions of oxidative phosphorylation. Liver mitochondria were isolated from Sprague-Dawley male rats, and the interactions of ticlopidine and six of its analogues with those key reactions were tested. We found: The transport of phosphate, glutamate and succinate into mitochondria was not affected significantly by ticlopidine or any of its analogues; however, it was inhibited by both mersalyl and N-ethylmaleimide as expected. There was no inhibitory effect of the tested drugs on the mitochondrial [3H]ADP translocation activity; rather, ticlopidine produced a concentration-dependent increase of that activity, reaching 54% with 20 micrograms/ml. Ticlopidine and its analogue, PCR 5325, increased the latent ATPase activity by about 400% and the DNP-dependent ATPase by about 50%. Also, PCR 4099 caused a 115% increase in the latent activity, whereas the effects of the remaining analogues varied from slight activation to slight inhibition. Under nonphosphorylation conditions, the mitochondrial H+ extrusion resulting from succinate oxidation was inhibited by ticlopidine in a concentration-dependent manner reaching a quasi total inhibition with 40 micrograms/ml. While PCR 5325 gave results similar to ticlopidine, PCR 4099 was less inhibitory and the other analogues were ineffective. These data indicate that the inhibitory action caused by ticlopidine and some of its analogues on oxidative phosphorylation does not reside at one particular site in the mitochondrial membrane; rather, the inhibition seems to be the outcome of profound alterations in mitochondrial ADP translocase, latent ATPase, and proton translocation in the respiratory chain.

On the mechanism of erythroid cell sensitivity to chloramphenicol: Studies on mitochondria isolated from erythroid and myeloid tumors

Abstract Erythroleukemia mitochondria (E. Mito) and chloroma mitochondria (C. Mito) were isolated from tumors grown in their hosts, DBA/2J mice and Long-Evans rats, respectively. Oxypolarographic tests showed respiratory control and ADP/O ratios typical for well-coupled mitochondria. Therapeutic concentration of chloramphenicol (CAP) had no effect on the energy transfer of those mitochondria. l -[14C]leucine incorporation into protein was comparable in both types of mitochondria. Although the incorporation at 15 min appeared higher in C. Mito, at 60 min it became similar to that in E. Mito. When CAP was used at the therapeutic concentration of 20 μg/ml about 80% inhibition was observed in both mitochondria. The exogenous amino acid mixture added to the medium was an important determinant in both the rate of leucine incorporation as well as the sensitivity to CAP. Thus, if no amino acids were added the incorporation was reduced to 18–25%. Under these conditions, however E. Mito were significantly more sensitive to the same concentration of CAP than C. Mito. The results suggest that mitochondrial amino acid pool may be involved in the greater sensitivity of erythroid precursors to CAP.

Stability of chloramphenicol metabolites in human blood and liver as determined by high-performance liquid chromatography

The pathogenesis of aplastic anemia from chloramphenicol (CAP) remains uncertain. Recent observations suggest that metabolites of CAP generated by intestinal bacteria may play an important role in mediating hematotoxicity from the drug. Thus, it is possible that once in circulation and after passage through the liver, some CAP metabolites may gain access to the marrow causing hematotoxicity. Based on this possibility, we have studied the stability of CAP and the three cytotoxic analogues dehydrochloramphenicol (DH-CAP), nitrophenylaminopropanedione (NPAP) and nitroso-chloramphenicol (NO-CAP) in human blood and liver. Determination of these compounds was accomplished by using a high-performance liquid chromatography system uniquely suited for their separation and detection. Several methods for deproteinization were utilized in order to ensure a full quantitative extraction of the drugs. At zero time, a 100% recovery of CAP, DH-CAP and NPAP was reached with acetonitrile (1 vol/3 vol); whereas NO-CAP was slightly or not detectable with all methods. Incubation of CAP and analogues with blood or liver at 37 degrees C for up to 30 min showed the following: CAP was stable in both tissues with full recovery; DH-CAP was stable for 5 min, then gradually decreased reaching 50 or 70% of the initial amount after 30 min of incubation with blood and liver, respectively; NPAP decreased at a faster rate than DH-CAP, and NO-CAP completely disappeared. The data suggest that if and when formed in the body, DH-CAP and NPAP may stay in the circulation long enough to reach the marrow and interact with its cellular components.

Discriminatory effects of gold compounds and carriers on mitochondria isolated from different tissues

Abstract Four different gold compounds and three gold carriers were used to study the toxicity of gold salts on mitochondria isolated from different tissues. Each of the following compounds—aurothiomalate (ATM), thiomalate (TM), aurothiosulfate (ATS), thiosulfate (TS), aurothioglucose (ATG), thioglucose (TG), and gold chloride (GC)—was tested with mitochondria extracted from rabbit (RbLM) or rat liver (RLM), and from rabbit bone marrow (RbMM) or chloroma tumor (CM). The toxicity of these compounds and carriers on mitochondria was evaluated by determining the inhibition of oxidative phosphorylation as measured by oxypolarography. With glutamate as substrate, mitochondria from hematopoietic tissues (e.g. RbMM and CM) showed a very high sensitivity to low concentrations of ATM, while liver mitochondria (RbLM and RLM) were slightly or not at all affected by those concentrations. Such difference of sensitivity was not observed when ATS, ATG or GC was used. Thus, identical concentrations of ATS or GC were needed to inhibit the oxidative phosphorylation in all mitochondria, and concentrations up to 2500 μM ATG were without effect on any of the mitochondria studied. While with glutamate ATM and TM primarily inhibited transition state 4/state 3, suggesting the involvement of site 1 in the respiratory chain, ATS, ATG and GC appeared to have the same effect on mitochondria oxidizing either glutamate or succinate. Furthermore, whereas the inhibitory effect of ATS and GC could be prevented or released by cysteine, inhibition by ATM could not. The interaction of the gold compounds and carriers with the highly reactive thiol groups involved in the energy conservation mechanism is discussed.

Inhibition by nitroso-chloramphenicol of the proton translocation in mitochondria

Abstract We have found recently that, unlike chloramphenicol (CAP), its nitroreduction derivative nitroso-chloramphenicol (NO-CAP) behaved as a potent inhibitor of the energy-conserving mechanism in mitochondria [Abou-Khalil et al. Biochem. Pharmac. 29, 2605 (1980)]. Concentrations of 75 and 250 μM NO-CAP were required to inhibit ATP formation with glutamate and succinate, respectively, whereas similar CAP concentrations were without effect. Testing several key reactions associated with the biosynthesis of ATP, inhibitory concentrations of NO-CAP were found to interfere as follows: (a) the transport of an NAD-linked substrate (e.g. glutamate) into mitochondria was only partially inhibited, whereas that of an FAD-linked substrate (e.g. succinate) was not inhibited but was rather slightly activated; (b) the transport of P i was only inhibited at about 50%; (c) mitochondrial ADP transport was not affected at all; (d) the ATPase activity, measured either by Pi release in the presence of an uncoupler or by H + ejection, was only slightly affected; and (e) under either phosphorylation or no phosphorylation conditions and in the absence of P i , NO-CAP was found to completely block mitochondrial H + extrusion resulting from the oxidation of either succinate or glutamate; however, under similar conditions the oxidation of the two substrates was not totally inhibited. The possibility of interference by NO-CAP with reactive mitochondrial thiols groups is discussed in the light of previous data and current experiments showing protection by P i against NO-CAP effects on respiration. Moreover, NO-CAP as compared to conventional inhibitors of oxidative phosphorylation (e.g. rotenone, antimycin A, oligomycin, mersalyl and others) appeared to have a distinct mode of action on that process. The results demonstrate that the inhibitory effect of NO-CAP is primarily located at the respiratory chain level where the proton translocation activity is fully blocked.

Swelling of mitochondria by the platelet antiaggregating agent ticlopidine

Our studies on the effects of ticlopidine on mitochondrial functions led us to an intriguing observation related to its interaction with mitochondrial membranes. Liver mitochondria were isolated from Sprague-Dawley rats and assayed for swelling by spectrophotometry. When ticlopidine was added to mitochondria preincubated in an isotonic test medium, an induced-swelling activity was observed. This activity was time and concentration dependent and occurred in different isosmotic solutions. Several analogues of ticlopidine, assayed under identical conditions, produced only a minor effect. Respiratory chain inhibitors, uncouplers, ATP, and phosphate protected the mitochondria against the ticlopidine-induced swelling, whereas oligomycin did not. Comparative studies with the drugs chloramphenicol, nitroso-chloramphenicol, and salicylate (known for their association with mitochondrial injury) showed the first two to have little effect while the third one caused swelling as expected. On the other hand, oxypolarographic tests of respiring mitochondria in the presence of ticlopidine showed that the drug is not an uncoupling agent. These results indicate that the antiaggregating agent ticlopidine interacts with mitochondrial membranes causing swelling which, in turn, may alter mitochondrial permeability; however, unlike some other swelling agents, it does not act as a classical uncoupler.