Mitochondrial Lesions in Reversible

Erythropoietic Depression Due to Chloramphenicol

FRANKC. FumaN

From the University of Melbourne Department of Medicine, St. VincentsHospital, Fitzroy, Victoria 3065, Australia

A B S T R A C T The mechanism underlying the reversibledepression of erythropoiesis by chloramphenicol has beeninvestigated in rabbits in which hemolytic anemia hadbeen induced by phenyihydrazine so that the compensa-tory erythroid hyperplasia would provide a situationwhere abnormalities in the bone marrow cells reflectedpredominantly those of erythroid precursors.

Maintenance of chloramphenicol in the serum of theseanimals at concentrations in the order of 15 /Ag/ml re-sulted in erythropoietic depression after several days.The onset of this depression corresponded to the develop-ment of a cellular respiratory defect in the erythroidprecursors which was associated with an abnormality inthe composition of the mitochondrial respiratory path-way. The abnormality took the form of a selective de-pletion of cytochromes a + as and b which can be ex-plained by an inhibitory effect of the antibiotic on theirformation by the mitochondrial protein-synthesizing sys-tem. The relationship between the nmitochondrial lesionand the depression of proliferative activity was furtherindicated by the correlation between the restoration ofthe cytochrome deficit and the recovery of erythropoiesisafter chloramphenicol administration was ceased.

The features of the reversible depression of erythro-poiesis corresponded closely to those in man, so that aspecific action of chloramphenicol on mitochondrialformation provides a reasonable explanation for thisimportant manifestation of chloramphenicol toxicity.

INTRODUCTION

Chloramphenicol therapy has been frequently connectedwith toxicity in hemopoietic tissue, and the most com-monly encountered form of toxicity in animals and man

This work was presented in part at the 2nd Meeting ofthe Asian and Pacific Division of the International Societyof Haematology, Melbourne, Australia, May 1971.

Received for publication 15 November 1971 and in re-vised form 10 March 1972.

is a reversible depression of erythropoiesis (1-3). Thisdepression of erythropoiesis develops regularly in manwhen the concentration of free chloramphenicol in theserum is maintained at or above 15-20 /g/ml for severaldays (2). As recovery of erythropoiesis takes placerapidly after the suspension of chloramphenicol adminis-tration, the antibiotic has been considered to exert areversible and concentration-dependent toxic effect inerythroid precursors (2, 3). Several biochemical. proces-ses in bone marrow cells are known to be inhibited bychloramphenicol in vitro (4-8), but the process whoseinhibition by chloramphenicol is responsible for the re-versible depression of erythropoiesis in vivo has not yetbeen identified.

The purpose of the present study has been to determinewhether a specific effect of chloramphenicol on the forma-tion of mitochondria is related to the depression of eryth-ropoiesis. Such a mechanism was considered becausechloramphenicol has been shown to selectively inhibitthe synthesis of the mitochondrial cytochromes a + as andb in HeLa cells at concentrations similar to those pro-ducing erythropoietic depression in man (20 Mtg/ml).The physiological significance of this action of the drugwas that it eventually led to impaired function of the mito-chondrial respiratory pathway which in turn resulted indepression of proliferative activity after the cells hadcompleted two divisions in the presence of chlorampheni-col (9).

This study has been performed in the rabbit with theobject of determining the relationship between chlor-amphenicol-induced changes in the mitochondrial re-spiratory pathway of erythroid precursors and the stateof erythropoietic activity. It was considered that the taskof examining mitochondria from erythroid cells wouldbe greatly simplified by inducing erythroid hyperplasiain the experimental animals, as mitochondria isolatedfrom bone marrow tissue under these conditions wouldessentially represent those of erythroid precursors.Erythroid hyperplasia was induced as a compensatory

The Journal of Clinical Investigation Volume 51 August 1972 2085

response to hemolytic anemia produced by the regularadministration of phenyihydrazine. It was also decidedto maintain the concentration of free chloramphenicolin the serum of the experimental animals at approxi-mately 15 ig/ml in order to provide a comparable situa-tion with that in man where sustained concentrations ofthis order produce erythropoietic depression (3). Dueconsideration was given to the relatively greater ca-pacity of the rabbit to eliminate chloramphenicol, whichnecessitated the administration of larger doses of the drugon a body weight basis to produce similar serum chlor-amphenicol concentrations to those in man (10, 11).

METHODSDrug administration. The study was performed in young

adult New Zealand white rabbits. Hemolytic anemia wasinduced by single daily subcutaneous injections of phenyl-hydrazine HC1 in a dose of 4 mg/kg body weight (12).Chloramphenicol was administered in a depot form bysingle daily subcutaneous inj ections of microcrystallinechloramphenicol suspended in 0.5% carboxymethylcellulose,at a dose of 600 mg/kg body weight.

Measurement of hematological indices. The packed eryth-rocyte volume (PCV)' and reticulocyte count were esti-mated in venous blood using conventional techniques andthe films of bone marrow particles were stained withLeishman's stain.

Preparative procedures. Animals were sacrificed by ex-sanguination via cardiac puncture and the blood collectedusing sodium citrate at a final concentration of 0.38%o asthe anticoagulant. Erythrocytes were sedimented by cen-trifugation at 200 g for 10 min. The supernate and buffycoat were carefully discarded to reduce leukocyte contami-nation. The erythrocyte pellet was washed twice in isotonicsaline and the supernate and buffy coat discarded as before.Fewer than 200 leukocytes/mm8 were present in the finalerythrocyte pellet after completion of the washing steps.Red bone marrow was obtained from the femur, humerus,and upper third of the tibia. Bone marrow was suspended ina medium consisting of 0.25 M sucrose, 0.1 mMEDTA,and 10 mmtris buffer, pH 7.4, and gently homogenized ina Potter-Elvehj em homogenizer to disperse the cells. Thecell suspension was then centrifuged at 600 g for 10 minand the floating layer of fatty tissue discarded. Sedimentedbone marrow cells were resuspended in the same mediumfor cell fractionation, or in 137 mmNaCl, 3 mmKCI, and 10mm Na phosphate, pH 7.4 for estimation of respiratoryactivity.

Mitochondrial fractions were isolated from peripheralblood erythrocytes which comprised 30-35%o reticulocytes,and from bone marrow cells. The cells were disrupted byhigh speed homogenization which was continued untilexamination of the homogenate by phase contrast micros-copy indicated that greater than 95%o of the cells hadbeen ruptured. Mitochondrial fractions were prepared bysedimentation at 8000 g for 15 min from the supernateremaining after low speed centrifugation of the homoge-nate had removed intact cells, nuclei, and large membranefragments (13). The mitochondrial fractions were washedtwice under the same conditions for biochemical studies,

'Abbreviation used in this paper: PCV, packed erythro-cyte cell volume.

and three times before the determination of amino acidincorporation into protein.

Assay procedures. Respiratory activity of bone marrowcells was determined polarographically with an oxygenelectrode (Rank Brothers, Bottisham, U. K.). Respirationwas supported by succinate at a concentration of 5 mM.Amino acid incorporation into protein by isolated mito-chondria was assayed at 30'C during a 30 min period bypreviously described methods (14) using uniformly labeledL-leucine-J'C (151 jCi/Amole) obtained from C.E.A., Gif-sur-Yvette, France. Aseptic techniques, autoclaved equip-ment, and millipore-filtered solutions were used throughoutthis part of the study to minimize contamination by bac-teria. Total bacterial counts were determined routinely inthe final incubation mixtures. Activities of the followingenzymes were determined at 30°C: malate dehydrogenase(15), fumarase (16), succinate dehydrogenase (17) andcytochrome oxidase (18). Mitochondrial cytochromes wereestimated by difference spectroscopy using the extinctioncoefficients of Estabrook and Holowinsky (19). Proteinwas measured by the method of Lowry, Rosebrough, Farr,and Randall (20). The concentration of chloramphenicol inthe serum was determined as the free form by the methodof Levin and Fishbach (21).

RESULTS

Effect of drug administration on hematologicalstatus

Stimulation of erythropoiesis by phenylhydrazine ad-ministration. Daily phenylhydrazine administration tocontrol animals produced hemolytic anemia with a pro-gressive fall in the PCV until compensation of the he-molytic state developed on the 5th treatment day andsubsequently maintained the PCV at approximately 24%(Fig. 1). The reticulocyte counts rose to between 30and 40% on the 4th treatment day and remained at thislevel while phenylhydrazine was being administered.Section of the long bones on the 5th and 7th days demon-strated that red marrow had extended below the normallower limit in the upper half of the femur and humerusto the midshafts of the distal long bones. Smears of redmarrow from various sites in the long bones at thesetimes revealed hypercellular particles and erythroid hy-perplasia in which erythroid precursors comprised ap-proximately 80% of the nucleated cells (Table I), anincrease from the value of approximately 50% in normalanimals.

Serum chloramphenicol concentrations during chlor-amphenicol administration. The serum chloramphenicolconcentration was found to be 17+4 ,ig/ml (;mean +SE)6 hr after the daily injections of 600 mg chloramphenicol/kg body weight in rabbits receiving phenylhydrazine.24 hr after the injections, the value was 12±2 Ag/ml, andwhen chloramphenicol administration was ceased, theconcentration fell to 9±4 /Lg/ml after 48 hr and 0 gg/mlafter 72 hr. indicating a relatively slow rate of elimina-tion under these circumstances.

Effect of chloramphenicol on erythropoiesis in phenyl-

2086 F. C. Firkin

4O.

IFKEDCELL

(%)10 PHBITUIYNZIIIE_U

0 1 2 4567 8

DURAI1ON OF 0OM0 ADMMISTRATION(DAYS)

FIGURE 1 Effect of chloramphenicol administration on theerythrocyte packed cell volume in the phenylhydrazine-treated rabbit. Values are means ±+SE from six experimentalanimals in each category.

hydrazine-treated rabbits. The PCV fell to a mean of23% and the reticulocyte counts rose to 30-40% duringthe first 4 days of chloramphenicol administration tophenylhydrazine-treated rabbits. This was identical withthe sequence of events in the control animals (Fig. 1),and indicated that chloramphenicol did not prevent theinitial reticulocyte response to phenylhydrazine-inducedhemolytic anemia.

However, it is evident that continued administrationof chloramphenicol prevented the development of a com-pensated hemolytic state after this period. The PCV fellsteadily after the 4th day in the chloramphencol-treatedanimals and they became progressively weaker and diedwhen the PCV had fallen to values in the order of 8%.During this phase the reticulocyte count remained be-tween 30 and 40%, so that with the fall in the PCVtherewas a proportional decline in the number of reticulo-cytes in the circulation.

Section of the long bones on the 5th and 7th daysdemonstrated the same degree of extension of the redmarrow as in the control animals. Cellularity of aspi-rated particles from the red marrow was also similarto that in control bone marrow, and erythroid precursorscomprised approximately 80% of the nucleated cell popu-lation. In contrast to the distribution of erythroid pre-cursors in control bone marrow, there was a decrease inthe proportion of orthochromatic and polychromaticnormoblasts and an increase in the proportion of lessmature erythroid precursors (Table I). This was simi-lar to the picture of arrested maturation in the erythroidseries of patients immediately after the onset of erythro-poietic depression due to chloramphenicol (2). Cyto-plasmic vacuolation was present in some basophil nor-moblasts, pronormoblasts, and myeloid precursors asreported in chloramphenicol-affected human bone marrow(2).

TABLE IEffect of Chloramphenicol on the Bone Marrow Cell Population

in Phenylhydrazine-Treated Rabbits

Proportion of erythroid and myeloid precursors*

Ortho-chromatic

andpoly- Myeloid

chromatic Basophil series andnormo- normo- Pronormo- other cell

blasts blasts blasts lines

Control bone marrow 44±4 2843 742 2142Chloramphenicol-

affected bone marrow 2845 40±4 10±3 22±2

Values are means ±sE from four experimental animals in each category.Differences between the control and chloramphenicol-affected bone marrowwere statistically significant in the proportions of orthochromatic andpolychromatic normoblasts (0.02 < P < 0.05) and basophil normoblasts(0.02 < P < 0.05), but were not statistically significant in the case ofpronormoblasts and other cell series (P > 0.05).* Per cent of total cell polulation.

Recovery of erythropoiesis after chloramphenicol with-drawal. When chloramphenicol and phenylhydrazineadministration was suspended, the PCV continued tofall for several days at a similar rate to that in animalswhose treatment had been continued. The PCVeventuallybegan to rise but the delay before this occurred wasconsiderably greater than after the suspension of phenyl-hydrazine administration to the control animals (Fig. 2).During the fall in the PCV after a 5 day course ofchloramphenicol and phenylhydrazine as illustrated inFig. 2, the reticulocyte count remained between 30 and40% for 3 days until the nadir was reached and per-sisted at this level for 4-5 days during the subse-quent rise in the PCV. There was thus a progressive fallin the number of reticulocytes in the circulation before

OmU ADOMISTRATIUCOMMED CEASEDAJe &

TIME(DYS)

FIGURE 2 Response of the erythrocyte packed cell volumeto a limited course of drug administration in the rabbit.Values are means ±SE from four experimental animals.

Mitochondrial Lesions in Chloramphenicol Toxicity 2087

the number began to increase 4 days after the suspen-sion of chloramphenicol administration.

On the 1st day that the PCVand number of circulatingreticulocytes began to rise, bone marrow aspiration re-vealed that the pattern of maturation in erythroid pre-cursors had been restored to that in the correspondingcontrols. The degree of erythroid hyperplasia and theproportions of the various erythroid precursors wereidentical with those in control animals on the 5th dayof phenylhydrazine treatment as illustrated in Table I.Cytoplasmic vacuolation was no longer present.

Suspension of treatment after a longer period ofchloramphenicol and phenyihydrazine administration re-sulted in the PCV falling to levels which were incom-patible with survival. Hence studies on the recovery oferythropoiesis were performed on animals whose treat-ment was limited to a 5 day course.

Mitochondrial and respiratory abnormalities inchloramphenicol-affected erythroid tissue

Two stages in the response of erythropoiesis to chlor-amphenicol were selected for evaluating changes in mito-chondrial composition and respiratory activity in ery-throid tissue. These were when erythropoietic depressionhad become established, and when recovery of erythro-poiesis had clearly developed after the suspension ofchloramphenicol administration. The hematological pic-ture on the 5th treatment day was considered representa-tive of the former situation as compensation of anemiacould not be sustained, the circulating number of reticu-locytes was declining in spite of worsening anemia, anda picture of arrested erythroid maturation was present-in the bone marrow. The hematological picture on the4th day after the suspension of a 5 day course of chlor-amphenicol administration was considered consistentwith resumption of active erythropoiesis as the PCVandnumbers of circulating reticulocytes had started to riseand the pattern of erythroid maturation in the bone mar-row had reverted to that in control animals.

Respiratory activity of chloramphenicol-affected eryth-roid tissue. The rate of oxygen consumption in chlor-amphenicol-affected bone marrow cells was 0.88±0.07 -L02/10' nucleated cells/hr (mean +SE, four experiments),and represented a statistically significant depression(P < 0.01) of 27% in the value of 1.21±0.05 Al 02/106nucleated cells/hr for bone marrow from control ani-mals on the 5th treatment day. 4 days after the suspensionof treatment the values were 1.28±05 Al 02/106 nucleatedcells/hr in bone marrow from the animals which hadreceived chloramphenicol, and 1.27±0.06 Al 02/106 nu-cleated cells/hr in the controls.

Mitochondrial composition in chloramphenicol-affectederythroid tissue. The level of a number of respiratorychain cytochromes was significantly reduced in mito-

chondria from the bone marrow of chloramphenicol-treated rabbits on the 5th treatment day as illustratedin Table II. This change involved cytochromes a + asand b, and the decrease in the content of cytochromesa + as demonstrated by difference spectroscopy was cor-roborated by a similar decrease in the specific activityof cytochrome oxidase in keeping with its role as theenzymatic function of cytochromes a + as.

On the other hand, there was no significant change inthe content of the c group cytochromes or in the specificactivities of other representative mitochondrial enzymesassayed, such as fumarase and malate and succinate de-hydrogenases. There was also no detectable decrease inthe total protein content of the mitochondrial fractionfrom chloramphenicol-affected bone marrow cells (8-10mg/109 nucleated cells), so that the decreased contentof cytochromes a + as and b represents an alteration inmitochondrial composition rather than an over-all de-crease in the mitochondrial content of chloramphenicol-affected bone marrow cells. It is of interest that thecytochrome deficit appeared to be specific for cyto-chromes a + as and b, as the c group cytochromes werepresent in normal amounts.

4 days after suspension of drug administration, thecytochrome levels and enzyme-specific activities in mito-chondria isolated from the bone marrow of animals whichhad received chloramphenicol were identical with thosefrom control animals. There was no statistically sig-nificant difference between these values and the valuesin the mitochondria of control animals on the 5th treat-ment day as illustrated in Table II.

Mitochondrial composition in peripheral blood reticu-locytes of chloramphenicol-treated animals. Cytochromeoxidase activity was significantly decreased in mitochon-dria isolated from peripheral blood erythrocytes of chlor-amphenicol-treated animals on the 5th treatment day(Table II). The mitochondria originated from thereticulocytes which comprised from 30 to 40% of theerythrocytes in the cells which had been subjected tofractionation after leukocytes had been extensively re-moved by repeated washing. This decrease in cytochromeoxidase activity was similar to that in the bone marrowmitochondria, and the selective nature of the defect wasagain illustrated by the lack of change in the specific ac-tivity of succinate dehydrogenase. As in the bone marrowmitochondria, the specific activity of cytochrome oxidasein reticulocyte mitochondria had reverted to controlvalues on the 4th day after the suspension of chlor-amphenicol administration.

Action of chloramphenicol on amino acid incorporationinto protein by isolated bone marrow mitochondria.Chloramphenicol inhibited leucine-14C incorporation intoprotein by mitochondria isolated from control bonemarrow on the 5th treatment day. This effect was evi-

2088 F. C. Firkin

TABLE I IMitochondrial Composition in Chloramphenicol-Affected Erythroid Tissue

Cytochrome levels and enzyme-specificactivities in mitochondria

Chlor-Source of Nature of amphenicol

mitochondria mitochondrial component affected Control

P valueBone marrow cells Cytochromes a + a3 0.023 0.074 <0.01

Cytochrome b 0.117 0.146 0.01 < P < 0.02Cytochromes c + cl 0.149 0.166 >0.05Cytochrome oxidase 0.32 1.15 <0.01Succinate dehydrogenase 0.29 0.30 >0.05Malate dehydrogenase 0.81 0.71 >0.05Fumarase 0.41 0.40 >0.05

Peripheral blood Cytochrome oxidase 0.28 1.21 <0.01Erythrocytes Succinate dehydrogenase 0.35 0.36 >0.05

These values are the means from four experimental animals in each category. The levels of the cyto-chromes in the mitochondria are expressed as micromoles per milligram protein in the mitochondrialfraction. Enzyme activity is expressed as micromoles substrate utilized/minute for malate dehydrogenaseand fumarase, as the A ODat 600 mM&/min for the decolorization of phenazine methosulphate (0.17 mg/ml)for succinate dehydrogenase, and as the first order rate constant/minute for cytochrome oxidase. Specificactivities are per milligram protein in the mitochondrial fraction.

dent at a concentration of 20 Ag/ml and the degree ofinhibition increased with the concentration of the anti-biotic (Table III). Cycloheximide, an inhibitor of aminoacid incorporation into protein by mammalian cytoplas-mic ribosoxnes (22), did not affect the process which in-dicated that contaminating cytoplasmic ribosomes didnot contribute to the amino acid incorporation. The verylow contamination by bacteria in the final incubationsystems (routinely less than 40 organisms/ml) indi-cated that amino acid incorporation was also not due tocontaminating bacteria, as such small numbers of or-ganisms do not significantly contribute to amino acid in-

TABLE IIIAntibiotic Sensitivity of Amino Acid Incorporation into

Protein by Isolated Bone Marrow Mitochondria

InhibitionSpecific activity of amino

Concen- of mitochondrial acid incor-Antibiotic tration protein poration

pg/ml cpm/mg %Nil - 198 10 -

Chloramphenicol 20 140a=5 27Chloramphenicol 50 121 : 7 38Chloramphenicol 100 71 1 12 64Lincomycin 100 197 8 0Cycloheximide 100 200:111 0

Values are means A1SE from three experiments.

corporation into protein under the experimental condi-tions employed (23). The lack of effect of lincomycinon amino acid incorporation indicates that the sensitivityto chloralmphenicol does not represent sensitivity to anti-biotics in general, in keeping with the limited antibioticsensitivityof mitochondrial protein-synthesizing systemsin the tissues of other mammalian species (14).

Action of chloramphenicol on bone marrow cell res-piration. The capacity of chloramphenicol to directlyaffect cellular respiration was also examined using bone

100

75-

RESPIRATORYACTIVITY 5O

(%)

25-

100 200 300 400CHLORAMPENICO GOUCENUTRAIMO

FIGURE 3 Action of chloramphenicol on the respiratoryactivity of bone marrow cells isolated from the phenyl-hydrazine-treated rabbit. Values are means +SE from fourexperimental animals.

Mitochondrial Lesions in Chloramphenicol Toxicity 2089

marrow cells from control animals on the 5th treatmentday. Chloramphenicol did inhibit respiratory activity,but this effect was manifested only at concentrationsgreater than those attained in the serum of the experi-mental animals (Fig. 3). The degree of inhibition wasonly 5% at a concentration of 80 /Lg/ml but increasedprogressively to 30% at a concentration of 320 /g/ml.

DISCUSSION

Chloramphenicol administration has been shown to pro-duce a selective depression in the content of certainrespiratory enzymes in the mitochondria of erythroid pre-cursors in the rabbit. The significance of this mitochon-drial lesion is indicated by its close relationship to thereversible depression of erythropoiesis by chlorampheni-col.

The experimental approach involved the treatment ofthe rabbits with phenylhydrazine in order to producehemolytic anemia and compensatory erythroid hyper-plasia in the bone marrow. Under these conditions theproportion of erythroid precursors increased to approxi-mately 80%, so that the mitochondrial and respiratorycharacteristics of the bone marrow tissue predominantlyreflected those of erythroid precursors. Administrationof chloramphenicol was commenced with the phenylhy-drazine so that erythroid precursors would be exposed tochloramphenicol from the start of their active prolifera-tive response to the hemolytic anemia. It was reasonedthat development of erythropoietic depression underthese conditions would be manifested by abrupt changesin the peripheral blood picture which would clearly indi-cate a suitable time to evaluate underlying changes inthe erythroid precursors.

The number of circulating reticulocytes relative to thatin the control animals did in fact begin to declineabruptly on the 5th day after the institution of chlor-amphenicol treatment, and the underlying arrest of matu-ration of erythroid precursors indicated that this re-sponse was due to depression of reticulocyte production.Studies on the bone marrow at this stage demonstratedthat cellular respiration was depressed, and it wasshown that this did not simply represent a direct effectof free chloramphenicol on the cells. A specific causefor depressed respiratory activity was present in theform of an abnormality in the mitochondrial respiratorychain, as there was a considerable decrease in the mito-chondrial content of cytochromes a + a3 and b, and inthe specific activity of cytochrome oxidase. Cytochromeoxidase activity in the reticulocyte mitochondria wasalso considerably reduced, and as the reduction wasequivalent to that in bone marrow mitochondria it wasevident that mitochondrial composition had been affectedthroughout the erythroid series in the chloramphenicol-treated animals.

The mechanism of oxidative energy production bymitochondria is involved by such a lesion in the respira-tory pathway, which indicates how the abnormality caninterfere with proliferation of erythroid precursors. As asimilar degree of depletion of these cytochromes fromthe mitochondria of HeLa cells is responsible for thedepression of proliferative activity of these cells by chlor-amphenicol (9), it has to be considered that erythro-poietic depression was also produced by this means.When turnover rates of erythroid precursors are takeninto account, it would be expected by analogy with theresponse of HeLa cells that proliferation of pronormo-blasts and basophil normoblasts would become depressedafter two divisions, which would occur after approxi-mately 2 days (24). However, the maturation of poly-chromatic and orthochromatic normoblasts which occurswithout further cell proliferation would still continue toyield reticulocytes for a further 2-3 days before reticulo-cyte production became depressed. Such a course ofevents is consistent with that observed in the experi-mental animals. After the suspension of chloramphenicoladministration, the period before the number of circu-lating reticulocytes began to rise is also consistent withthat required for maturation of cells derived from theresumption of basophil normoblast proliferation afterchloramphenicol had been cleared from the serum. Onthe grounds that the mitochondrial lesion was causallyrelated to erythropoietic depression, its recovery through-out the erythroid series should have occurred at thestage when the cohort of newly formed reticulocytes hadreached the circulation, and this is in agreement withthe experimental findings.

The means by which chloramphenicol selectively in-terferes with the synthesis of cytochromes a + a3 and bin erythroid precursors is suggested by findings in stud-ies with other mammalian tissues. It had been shownin these studies that the drug inhibited the synthesis ofa small proportion of mitochondrial proteins in the ab-sence of an inhibitory effect on the synthesis of the ma-jority of mitochondrial and other cellular proteins (9,13, 22). Cytochromes a + aa and b were among the,mitochondrial proteins whose synthesis was affected bychloramphenicol. The selective nature of the inhibitoryeffect was attributed to the capacity of chloramphenicolto inhibit protein synthesis by mitochondria but not bycytoplasmic ribosomes. Such a mechanism is also ap-plicable to the erythroid precursors as protein syntheticactivity in the isolated mitochondria was inhibited bychloramphenicol in vitro, and isolated cytoplasmic ribo-somes from this tissue have been shown elsewhere to beinsensitive to the drug (25). The ability of chlorampheni-col to affect protein-synthetic activity in isolated mito-chondria has been known for some time, and providedthe basis for the speculation that such an effect was re-

2090 F. C. Firkin

sponsible for toxicity (7, 13, 23, 26, 27). The importanceof the present findings is in providing evidence that aneffect of chloramphenicol on this system is operativein erythroid precursors in vivo.

Development of such highly specific changes in themitochondria is not consistent with the consequencesof certain alternative mechanisms proposed to accountfor chloramphenicol toxicity, because the suggestedmechanisms invoked an inhibition of major cellular syn-thetic pathways which would have led to a generalizedrather than specific inhibition of the synthesis of mito-chrondial components. These include the inhibition ofprotein synthesis by cytoplasmic ribosomes proposed byWeisberger and Wolfe (4), and the inhibition of ribo-nucleic acid synthesis proposed by Ward (5). Depressionof ferrochelatase activity (8) also cannot account for thechanges produced in the mitochondria, as this wouldhave interfered with heme synthesis and thereby gen-erally inhibited the formation of cytochromes. -Such asituation was shown not to be the case as the c groupcytochromes were formed in normal amounts as in otherstudies on the action of chloramphenicol in mammaliantissues (9, 13).

Erythropoietic depression due to chloramphenicol inthis experimental situation closely resembles that inman under clinical conditions in the pattern of develop-ment and recovery, as well as in the morphologicalchanges in erythroid precursors (2, 3). The effectivechloramphenicol concentration in the rabbit is also com-parable with the level of 15-20 Ag/ml at or above whichdepression regularly develops in man (3). The serumconcentration rather than the administered dose ofchloramphenicol is believed to be the determining factorin the development of erythropoietic depression (3, 11),and it was only to compensate for the greater capacityof the rabbit to eliminate chloramphenicol that a rela-tively greater dosage on a body weight basis was ad-ministered to them (10). Thus, in all essential features,the response in the rabbit resembles that in man, so thata similar mechanism can be proposed to account for thereversible erythropoietic depression by chloramphenicolin man.

Chloramphenicol also produced a direct effect on bonemarrow cell respiration at concentrations which wereconsiderably greater than those attained in the experi-mental animals, and this second type of effect may berelated to a different form of human toxicity manifestedonly at very high serum concentrations of the drug. In-hibition of cell respiration has also been observed athigh concentrations in other mammalian tissues (14, 22,28), and was shown to be due to inhibition of NADHdehydrogenase which in turn led to depression of energy-dependent synthetic processes (28). This mechanism pro-vides an explanation for the potentially lethal generalized

toxicity known as the Grey syndrome, which developswhen chloramphenicol accumulates to very high con-centrations (75-180 ug/ml) in the serum of neonatesas a consequence of their impaired capacity to detoxifythe drug (29).

ACKNOWLEDGMENTSThe technical assistance of Miss V. Squires is gratefullyacknowledged.

This work was supported by the National Health andMedical Research Council of Australia.

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Mitochondrial Lesions in Chloramphenicol Toxicity 2091

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