Document B5JrOpQ9nZrzk51R2z3EB84Go

Biochemical Mechanisms of Liver Injury J. D. JUDAH, M.D. A. E. : M. McLEAN, M.D. E. K. McLEAN, M.D. London. England From the Department of Experimental Pathology, University College Hospital Medical School, London, WCIE 6JJ. En gland. Requests for reprints should be ad dressed to Dr. J. D. Judah. Department of Experimental Pathology, University College Hosoital Medical School, University Street, London W.C. 1, England. Mechanisms of liver injury are considered from the points of view of fat accumulation, necrosis and the role of toxic metabo lites. The actions of microsomal enzymes in producing such toxic metabolites, or else detoxicating other chemicals, are related to the nutritional control of microsomal enzyme activity and*the effects of enzyme inducers. The question whether the biochemical changes observed have any relevance to the development of pathologic states is dis cussed. It is concluded that useful conclusions can be derived only by comparative studies using either different poisons or animals in differing physiologic states. A great deal of work has been done on the mechanisms of liver injury using experimental models based on a variety of toxic agents of greater or lesser specificity. In this review we con sider three major topics which illustrate the advances and the difficulties of such an approach to disease. The accumulation of triglyceride fat in liver cells may be considered the result of imbalances imposed upon the system responsible for the trans port of these molecules. Since the normal mechanism for this is reasonably well known, the study has been rewarded with a fair degree of success. The question of necrosis is hedged about with formidable difficulties, and correspondingly little success has been experienced in the attempts to solve it Finally, we discuss the metabolism of some of the toxic agents used in these studies and show how the liver cells may be induced to act as mediators of their own fate. Several reviews have recently appeared on this and related topics [1-4]. ACCUMULATION OF TRIGLYCERIDES IN LIVER CELLS The mechanism of the production of fatty livers was first in vestigated by using toxic substances which induce the accumula tion of triglyceride fat within liver cells (e.g., carbon tetrachlo ride (COL), used by Cameron and Karunaratne [5]. It was not at first appreciated that the accumulation of fat could be function ally separated from the cell necrosis, which is also seen in many instances of experimentally produced liver injury, and it was not until these processes were separated in the 1960's that progress Volume 49, November. 1970 UMI Article permission prohibited. 609 fiFG36i24 BIOCHEMICAL MECHANISMS OF LIVER INJURY--JUDAH ET At_ was made. Christie and Judah [6] suggested a biochemical mechanism for CCI4 toxicity based on mitochondrial damage, which was thought to lead both to accumulation of fat and to death of the liver cell. This theory was rapidly shown to be untenable when electron microscopy [7] revealed that the endoplasmic reticulum (ER) was damaged within thirty minutes of the administration of CCI4 whereas the mitochondria survived apparently un altered for several hours. Biochemical evidence that endoplasmic reticulum was damaged was not hard to find. Smuckler, Iseri and Benditt [8] showed that protein synthesis was reduced within two hours of CCI4 poisoning and thought that this event could be responsible for the whole patho logic sequence caused by CCI4. Decisive evidence that cell necrosis and the accumulation of neutral fat were separate came from several observations: (1) drugs could reduce cell necrosis without affect ing the fat accumulation; (2) fat accumulation be gan within sixty minutes of the onset of intoxication by CCI4. whereas necrosis was not seen until ten to twelve hours had elapsed: (3) poisons which brought about fat accumulation without causing necrosis were used to separate many of the changes caused by CCI4 and to make a logical hypothesis v.hich accounted for these changes. To understand this we must return to the mechanisms proposed for the secretion of triglyceride by the hepatocytes [9-12]. Fatty acids move from the peripheral fat depots to the liver. There they are either oxidized or formed into triglyceride.: The triglycerides are then conjugated with a globulin to yield lipoproteins, which are then secreted. This mechanism is thought to give rise to the plasma lipoproteins. Figure 1 illustrates the cycle of events. If the synthesis of the carrier globulin is; blocked, lipoprotein will not be formed, and fat will accumulate in the liver cell. This is the sig nificance of the inhibition of protein synthesis early in CC1, poisoning. Triglyceride secretion is much reduced in isolated perfused livers examined as early as three hours after CCI4 poisoning in rats [13]. Investigations of ethionine, the ethyl analogue of methionine [14], seemed to support the view that inhibition of protein synthesis caused a large accumulation of triglyceride fat. Ethionine does not cause any detectable cellular necrosis, and the work done upon it has yielded the final evidence that cell necrosis and the accumulation of fat in the liver are separate phenomena. These studies also suggest that diminution of protein synthesis has no major role in the pathogenesis of cell death. Figure 1 indicates the general mech anism of the production of fatty liver, but the study of ethionine, whilst it increased our know ledge, also brought to light new problems. Role of Adenosine Triphosphate (ATP). Farber et al. [14] found that ethionine causes a profound fall in the level of adenosine triphosphate (ATP) in the livers of poisoned female rats. Male rats were not so susceptible to the poison and did not de velop any pathologic changes. The decrease in ATP is due to the reaction ATP + ethionine -> S-adenosylethionine + inorganic orthophosphate. This is analogous to the reaction undergone by methionine but, in contrast, S-adenosylethionine is inert and acts as an adenosyl trap, thus reduc ing the level of ATP. The administration of adenine to poisoned rats allowed a new synthesis of ATP and reversed the toxic effects of ethionine. It was thought that the chain of events following upon ethionine poisoning was low ATP -- reduced pro tein synthesis - reduced lipoprotein synthesis - fat accumulation. Role of the Ribosomes. The investigations of the effects of CCI4 and of ethionine here drew close together. The electron microscope has shown ex tensive damage to the endoplasmic reticulum, upon the membranes of which are attached the spirals composed of ribosomes (polysomes) that are the sites of protein synthesis. Shortly after administration either of CCI4 or of ethionine the ribosomes are seen to be detached from the mem branes. When cell-free preparations of poisoned livers are examined by ultracentrifugal analysis it is found that the polysomes are disaggregated into smaller assemblies and even into single par ticles [15]. All these findings were in accord with the diminished incorporation of p4C] amino acids Figure 1. The fatty eeid cycle. The scheme shows move ments of fatty acids from depots to liver and the return of surplus as triglyceride. 610 The American Journal of Medicine SDOZOGtZ BpG3612s ]*}?!?***>' BIOCHEMICAL MECHANISMS OF UVER INJURY--JUDAH ET AL. into liver proteins in vivo. When the cell-free preparations were tested, it was found that their ability to incorporate amino acids into proteins was also reduced. Plainly, a ribosomal lesion had been found. Since messenger RNA (ip-RNA) is thought to hold the polysomes in the spiral form [16], the nature of the lesion seemed to be clear. In ethionine poisoning the low level of ATP was thought to cause a block in m-RNA synthesis, which in turn would allow the polyribosomes to break up. Protein synthesis would thus be reduced. The nature of the initiating damage in CCI4 poison ing was not clear, but there was the possibility of direct attack on the endoplasmic reticulum mem branes: this possibility was supported by the work of Recknagel and his group [3]. Other Inhibitors. If this hypothesis is correct, it may be assumed that other inhibitors of protein synthesis would also give rise to fat accumulation in liver cells. Puromycin [17] when given to rats does indeed cause a fatty liver, but effective doses of cydoheximide [18] do not. The administration of actinomydn D, which blocks m-RNA synthesis, causes no fat accumulation. To confuse matters even more, Stewart and Farber [19] have shown that an injection of adenine given after a dose of actinomydn 0 still reverses the ill effects of ethionine and allows realignment of the ribosomes. This can only mean that the low level of ATP has its effect in some other way than through block of synthesis by actinomydn D. The Transport Problem. In a review [1] we drew attention to two aspects of the problem of fat ac cumulation in the liver. The first concerned syn thesis of the carrier protein (apoprotein) to which triglyceride is attached before it can be moved out of the ceil. If it had a relatively long "life" and could be re-used for formation of lipoprotein, then hypotheses that tried to account for the rapid development of fatty liver (within a few hours of poisoning Cd4 or by ethionine) could not be based on failure ofsynthesis of protein alone. Secondly, we pointed out that failure of cation transport also took piace in ethionine poisoning [20,21] and potassium ion is lost from the liver cells, an effect shown to be due to low levels of ATP. Could it be that the accumulation of tri glyceride fat in the liver is part of a more general failure.to transport .more than one material? Evidence suggesting that a re-usable apoprotein exists for triglyceride transport is provided by the work of Roheim et al. [22]. Administration of orotic acid causes a failure in lipoprotein secre tion, which is not associated with failure to syn- thesize protein but apparently with failure to form lipoprotein from the component triglyceride and apoprotein. New evidence has also been obtained to support the idea of a failure of "transport" of protein and lipoprotein in ethionine poisoning. Low potassium ion levels are sufficient by them selves to bring about considerable (50 to 70 per cent) reductions in the rate of transfer of serum albumin and of lipoproteins across the membrane of the liver cell [23]. When potassium ion levels are restored, the rate of appearance of both groups of protein in the extracellular fluid is restored after a delay of about thirty to forty minutes. This effect of potassium ion can be demonstrated in the pres ence of blocking levels of actinomycin D and is therefore independent of the synthesis of m-RNA. The low potassium ion levels found in ethionine poisoning are sufficient by themselves to cause a failure of lipoprotein secretion. The fatty liver of ethionine can thus be seen to arise in the se quence low ATP -- low cell K+ -* reduced lipoprotein secretion -* triglyceride accumulation. We can explain the experiments in which ad ministration of adenine in the presence of actino mycin D-reversed the effects of ethionine, since adenine elevates cell potassium ion within thirty minutes of administration. Present Status of the Problem. Previous work on triglyceride accumulation following CCL or ethio nine intoxication suggested the following sequence of events: (1) disaggregation of polyribosomes (?due to failure of m-RNA synthesis); (2) failure of protein synthesis; (3) failure to form apoprotein and hence lipoprotein, leading to (4) accumula tion of triglycerides (fatty liver). However, other observations indicate that this explanation is not completely adequate. For example: (1) actinomycin D (which blocks m-RNA synthesis) causes no fat accumulation; it also fails to stop reversal of the manifestations of ethionine intoxication by adenine; (2) not all inhibitors of protein synthesis cause fatty livers when administered to animals; (3) apoprotein may circulate for some time and can be re-utilized by the liver to form lipoprotein. New evidence suggests interference at dif ferent points in the process of accumulation of fat in liver cells at (1) the point of attachment of triglyceride to apoprotein and (2) the sites ot transport of lipoprotein (and other, proteins) across the cell membrane. The original cautionary remarks are therefore justified. When we study the mechanism of an abnormal process, we have to be aware oPthe spreading of secondary disturbances that follow ^ Volume 49, November 1970 &FG36126 611 BIOCHEMICAL MECHANISMS OF LIVER INJURY--JUOAH ET AL. the primary assault on the cell, and have to de cide which are crucial for the next step and which will lead nowhere. LIVER CELL NECROSIS If the present knowledge of the development of the fatty liver is still doubtful in many details, that of the mechanisms of cellular necrosis is in a worse state. This is not surprising in view of the many ways in which cells may be killed. In the process of fat accumulation, the fate of a single group of molecules, the triglycerides, together with their associated protein carriers, is being studied by excellent methods. When, however, the chemistry of cell death is considered, it is hard to know where to begin. Different lines of inquiry have to be pursued, until a common pat tern may emerge. A few of the main suggestions that have been made will be discussed, but it must be admitted that progress so far is relatively slight. The first difficulty is one of definition. Necrosis is a histolo gist's conception. The dead cell is recognized by changes that are the consequences of cell death, or autolysis, which are seen when the degradative enzymes of the dead cell attack its structures and destroy them. These signs take some time to develop, hence one is ignorant of the precise mo ment of cell death. Whole chains of secondary changes have to be distinguished. CCI, poisoning leads to death of the liver cell and has been much investigated. Its history proves instructive. Role of the Mitochondria. Mitochondriafdamage was early put forward as the initial cause of liver damage in CCI, poisoning. This theory was inade quate to explain all changes, but it is still just possible that cell death might follow from a pri mary attack on these organelles. Two possible mechanisms have been suggested: (1) Attack on the mitochondrial membrane by CCI, or a metabolite of CCI,: This possibility has never been ruled out. The earlier work of Christie and Judah [6] could explain cell necrosis, especially if newer methods of analysis were applied. The cru cial factor is time. More sensitive methods for determining changes in the mitochondrial mem brane might show that mitochondrial damage oc curs quite early. (2) Mitochondrial damage resulting from calcium accumulation: Mitochondria in vitro take up an enormous amount of Calcium (Ca:^) from the surrounding medium, and this Ca;- eventually destroys the mitochondria, causing irreversible changes in their membranes, disrupting the en ergy-conserving mechanisms of oxidative phos phorylation and allowing the loss of intramitochondrial components such as nucleotides, po tassium ion and Mg2+ [24,25]. The possibility that Ca-^- is a killing agent had been suggested by Thiers, Reynolds and Vallee [26], who showed that a very early rise in liver Ca2+ was a feature of CCI, poisoning. This is an attractive idea, and the nature of the Ca2+ accumulation has been studied extensively. But little firm information has resulted. It has been found by use of <r,Ca in vivo that the rate of Ca2-*- uptake early in CCI, poison ing (up to five hours) is normal; this can only mean that the rate of exit is impaired. Further investigation [27] shows that Ca2+ taken up by the poisoned liver cell exchanges very slowly with external Ca2^ and that this unex changeable fraction increases rapidly with time. Thus about 20 per cent of the normal cell Ca2+ is slowly exchanging, but within sixty minutes of CCI, poisoning this figure is 40 per cent, and it rises steadily thereafter. Some intracellular Ca2+ may migrate to an abnormal site and Ca-`- may flow in from without to compensate for this. Unfortunately, there is no evidence that shifts of Ca2* play any part in cell death from other toxic agents. There is no early shift of Ca24- after poisoning with thioacetamide. dimethylnitrosamine or by pyrrolizidine alkaloids, all of which kill liver cells. These changes in Ca-~ levels may, therefore, represent a curiosity of CCI, poisoning. Role of Peroxidative Changes. Recknagel [3] and Dianzani and associates [28] showed that lipid peroxidation accompanies CCI, intoxication. Since the first signs of peroxidation appear within a few minutes of CCI, administration, and since the endoplasmic reticulum appears to be the first site of the change, it might well be of crucial impor tance in initiating cell necrosis. The importance of lipid peroxidation is two fold: .(1) The phospholipids, with a high level of unsaturated fatty acids, are an obvious target for such reactions. This in turn might damage mem branous structures, which of course contain much phospholipids. (2) Peroxidation involves the forma tion of free radicals. This means that a small initiating reaction can rapidly spread, as the free radicals by their attack form others to initiate a chain reaction. The damage is amplified as more and more molecules are attacked and in turn form reactive substances which attack others. The chemistry of free radical formation as it affects cellular injury is discussed by Slater [29]. The significance of the suggested mechanism is 612 _ The American Journal of Medidne 24902004 BFG36127 increased by experiments which suggest that CCI4 itself has to be metabolized to a toxic intermediate. The most likely toxic substance formed from CCI4 is itself a free radical. CCIi. the CCI4 having under gone a homolytic split, losing a Cl. and leaving the residue with a highly reactive unpaired electron. The implication of this is clear. The CCIi, radical would attack unsaturated fatty acids, which would undergo peroxidative degradation, themselves forming further free radicals that amplify the damage, which would then spread through the cell. This hypothesis is unproved, for it has yet to be shown that the peroxidation reactions are the cause and not the result of the cell damage. Some of the normal pathways of cellular oxidation in volve free radical formation. Normally these are controlled by the intrinsic organization of the elec tron-transport chain, which is geared to promote a free flow of electrons in a given direction. Alter natively, the liver cell contains large amounts of antioxidants, such as reduced gfutathione, which will mop up free radicals. In damaged cells these protective mechanisms might be lost and a random chain reaction might starts Evidence against this hypothesis is derived from experiments in which antioxidants are ad ministered to rats. Phenothiazines [30] and alphatocopherol [31] have both been used to prevent necrosis due to CCI. However, McLean [32] finds but a small effect, and if the spread of liver cell necrosis in CCI4 intoxication depended upon the autocatalytic effect of lipid peroxidation, then anti oxidants should be protective, which has not been demonstrated in CCI4 poisoning. Promethazine, a potent antioxidant, is not much more effective than such nonantioxidant stabilizers as diphen hydramine [30]. Although the liver injury caused by choloroform is similar to that produced by CCI4, there is no - evidence of lipid peroxidation [33.34], ROLE OF DRUG-METABOLIZING ENZYMES IN CHEMICAL TOXICITY The drug-metabolizing enzymes in the endoplas mic reticulum of liver ceils are usually thought of as detoxicating enzymes. In some cases toxins may be inactivated but recent work has shown that these enzymes may in fact activate otherwise inert substances into toxic molecules [35.60]. When an animal is exposed to phenobarbital or DOT a massive-synthesis of the whole system, in cluding cytochrome P-450, takes place. This in duction requires DNA-dependent RNA synthesis TABLE I Effect of Nutritional State and Inducing Agents on Microsomal Hydroxylating Enzymes Activity ~ and on Lethal Action of Carbon Tetrachloride Diet Protein-free Protein-free Standard cube diet Standard cube diet Treatment __ _ D--DT Phenobarbital Pyramidon Mean Demethyl- ID* (ml ation CCI, kg 'VM gm body Seer, hr) weight) 0.1 14.7 0.5 4.3 0.7 6.4 2.6 0.5 NOTE; CCI, was given orally in liquid paraffin oil fo male rats, allowed food throughout the experiment. DDT was given as a single subcutaneous dose of 100 mg.kg one week be fore CCI,. Phenobarbital was given in the drinking water as a 1 mg/ml solution for one week, and plain drinking water was substituted at the time of CCI, dosage. and also protein synthesis [36.37]. When an animal is fed a purified low protein (3 per cent casein) diet, the activity of the enzyme system and the cytochrome P-450 content of the liver fall to a third, or less, of the normal level in four-days. Induction of enzyme synthesis by phenobarbital or DDT is also markedly inhibited [38,39], How ever, synthesis of the enzyme system can be in duced in protein-deficient animals to levels above those found in animals fed stock diets (Table I). The induction of microsomal enzymes by ex posure to DDT and phenobarbital is an extreme example of a common phenomenon. Many of the non-nutrient components of food are inducers of the enzyme system. Ethanol, oxidized fats and several anti-oxidants are also inducers. Carbon Tetrachloride and Chloroform. The feed ing of a low protein diet almost abolishes the lethal and hepatotoxic effects of CCt4 in rats (Table I). This effect is reversed by the administration of _ DDT or phenobarbital. The toxicity of CCI4 to any animal goes hand in hand with the activity of the microsomal hydroxylating enzymes, which me~ tabolize CCI4 [38,40,41]. In each situation the le thal and hepatoxic effects are proportional to the amount of CCI4 metabolized (as measured by con version of CCI4 to carbon dioxide both in vivo and in vitro). Thus the same amount of liver damage is pro- duced by a dose of 2.5 ml CCI4/kg body weight in * a rat fed stock diet as is found after a dose of 0.25 ml/kg body weight to a phenobarbital- fsj treated rat. The concentrations of CCI4 in the liver differ by a factor of 9. but the amount of CCI4 _ ( metabolized is 12 mg/kg body weight/six hours O in each case. Volume 49, November 1970 BFG36128 6i3 y| BIOCHEMICAL MECHANISMS OF LIVER INJURY--JUDAH ET AL. TABLE II Effect of Diet on Chloroform Poisoning DMN requires demethylation in a microsomal Diet Standard cube diet Standard cube diet + phenobarbitone Protein-free Protein-free and DDT ID,, Log (nil kg body weight) Plasma ICD 1.3 1.9 0.5 >2.9 1.1 1.4 0.4 2.4 enzyme system before it exerts' its toxic and carcinogenic effects. In protein-depleted animals DMN metabolism is greatly reduced in the liver but unaffected in the kidney. The decreased liver clearance of DMN in the protein-deficient animals -allows more DMN to come into contact with the kidneys [49]. NOTE: Piasma isocitric dehydrogenase (ICO) activity is ex pressed as log miiM/ml/min. In unpoisoned rats these If DMN is given as a single injection of 60 mg/kg body weight, normal rats almost all die in values are about 0.15. The rats were given oral doses of chloroform 0.75 ml/kg body weight, and killed twenty-four hours later for determination of ICD activity. a few days, with severe liver necrosis. In proteindeficient animals the severity of the liver lesions is much reduced, and most of the animals survive. All the survivors die with kidney tumors in twelve It seems likely that CCI4 combines with micro- months. Here the protein-deficient diet has somes at the P-450 site [42] and is there con changed the action spectrum of DMN. Instead of a verted. as already mentioned, to a highly reactive predominant hepatotoxic effect, the single dose CCI; free radical by the hydroxylation enzyme produces malignant renal tumors in the majority system [43.44]: this radical in turn attacks numer of animals. ous cell sites. This mechanism would explain the Aftatoxin. Madhavan and Gopalan [50] showed observations that only cells containing hydroxyla that the acute toxicity of aflatoxin was greatly tion enzymes are sensitive to CCI4, that low pro enhanced in protein-deficient rats. This effect can tein diets protect, and that inducers of micro be reversed by dosing with DDT [51]. It appears somal hydroxylation potentiate CCI4 toxicity. likely that aflatoxin B] and perhaps other aflatoxins The failure of vitamin E to protect against the are themselves the toxic and carcinogenic agents, hepatotoxic effects of CCI4 [32] suggests that the and that they are converted to nontoxic metabolites autocatalytic lipid peroxidation observed by Reck- in the liver by the hydroxylation system. In current nagel [3] may noHje relevant to' cell injury. experiments on the carcinogenic action of aflatoxin, Chloroform. Protein depletion does not materially rats were fed peanut meal containing aflatoxin for alter the toxicity of chloroform given as a single nine weeks, with simultaneous dosing with pheno- oral dose (Table II). However, enzyme induction renders rats more sensitive to the lethal and barbital to some groups. At the time of writing this paper, eighteen months after cessation of adminis hepatotoxic actions of chloroform [33]. Retrorsine. Pyrrolizidine alkaloids such as retrorsine cause diseases of animals and man tration of aflatoxin one of twenty of the aflatoxin group is alive, the remainder having succumbed with massive liver tumors. Of the group that re [35.45], Neither low protein diet or enzyme in duction alters the acute toxicity of retrorsine in ceived phenobarbital as well as aflatoxin, eleven of twenty survive. vivo [33]. However, the in vitro conversion of retrorsine to a toxic metabolite is dependent on microsomal hydroxylation and is altered by diet and enzyme induction in the same way as py-' ramidon demethylation or CCI4 metabolism [46]. It is possible that retrorsine is metabolized to a toxic product which in turn is further metabolized It is a sad irony that two toxins, aflatoxin and DMN [52], likely to occur in the diet of man in areas in which protein deficiency is common, should both have their carcinogenic action eru_. hanced by the suppression of hepatic microsomal hydroxylation attendant upon protein deficiency. to an inactive substance: Altering the total amount of hydroxylating enzyme may alter the rate of both reactions and need not change the amount of toxic substances reaching the sensitive site [47]. Dimethylnitrosamine. There is'a well marked COMMON MISCONCEPTION RELATING TO LIVER DAMAGE There is a widespread belief that protein deficiency and malnutrition increase the toxic effects of all poisons, especially in their actions on the liver. protection against acute dimethylnitrosamine (DMN) poisoning in rats fed protein-deficient diets. The LDj,, is almost-doubled, and the liver damage is greatly reduced [48]. This belief is incorrect but it is interesting how it came about. It rests upon three assumptions about which our views have changed in the past thirty years, and upon a fourth which was purely 614 The American Journal of Medicine BFG36129 24902006 IXUMl BIOCHEMICAL MECHANISMS OF LIVE* INJURY--JUDAH ET Al_ mythical but which has achieved the status of fact by transcription from one textbook to another. (1) In the late 1940's it was thought that protein deficiency, and especially deficiency of methio nine, caused liver necrosis. Schwarz [53] showed that dietary liver necrosis was caused not by pro tein deficiency but by a simultaneous deficiency of vitamin E and selenium. (2) About the same time it became clear_that there was widespread and serious protein de ficiency among children of many developing coun tries [54], In kwashiorkor, excessive fat accumu lates in the liver, and it was thought that the high incidence of cirrhosis and cancer of the liver in many tropical countries [55] was a sequel to kwashiorkor. Since then it has become clear that kwashiorkor alone does not lead to cirrhosis and hepatoma. Other factors, such as fungal toxins in foodstuffs, may account for these diseases. (3) Experiments have shown that dogs depleted of protein by plasmapheresis, and rats made protein- deficient with various diets, had more liver ne crosis, histologically assessed, after chloroform anesthesia [56]. These results conflict directly' with our own (Table II), and it is not easy to re solve this conflict. Factors in our work which might account for the difference include larger numbers of animals, quantitative.assessment of liver dam age, oral administration of chloroform instead of by inhalation, and highly purified diets. It is pos sible that when chloroform is inhaled the limiting factor is the amount of chloroform that remains in the liver, and this will depend on the amount of fat present [57]. (4) It has been assumed that protein deficiency sensitizes animals to acute CCI4 poisoning. There has never been any evidence for this view. Opie and Alford [58] and Campbell and Kosterlitz [59] showed that low protein-high carbohydrate diets protected against CCI4 and chloroform poisoning, when compared with high protein or high fat diets. For unknown reasons this work never achieved adequate recognition. REFERENCES I. McLean ACM. McLean E. Judah JD: Cellular necrosis in the liver induced and modified by. drugs. Int Rev Exp Path 4: 127, 1965. 2_ Magee PN: Toxic liver necrosis. 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J Exp Med 21:1.1915. 59. Campbell RM. Kostertitz HW: The effect of short term changes in dietary protein on the response of the liver to carbon tetrachloride injury. Brit J Exp Path 29:149,1948. 60. McLean AEM: Conversion of inactive to toxic mole cules by the liver. Mechanisms of Toxicity, Bio logical Council Symposium (Aldridge WN, ed) London, MacMillan & Co. 1970. 616 The American Journal of Medicine BFG36131 i \) f N V 0 03 i M :* mis e z..'9 > s> <5> t s ? t pjo I"' H M H 0 "OcC/D> as 0 rr-4 D * CrHl 0 O' U I o a: UJ o * H cr X o X o o 2 >0 Q fO UJ O' X K) M HO3 O UJ 3Q h. CD sh-C - J3 +-> z UJ a 3 { UJ Q 3 00a33s <X c: Ui > 0C/33 H 3 <X X Ui C3 M h d <c o M .. CD X Z UJ l/> Ct o i tr CM CM UJ c- CM 3 C- O' O' :> C1 H CD llj 03 O ^=> C<C3 C<CD **H* :1 :S UJ o * UJ o z <x -J > W> if) ** UJ X o *"D -C 11 >)))>)) ......... -- K) K) O' O' Z W W <t m in a) 3 Bf7G36l32 o tso