Significant Biochemical Effects of Hepatocarcinogens in the Rat: a Review* E

Significant Biochemical Effects of Hepatocarcinogens in the Rat: A Review* E. REID

(Chester Beally Research Instthde, institute of Cancer Research: Royal Cancer Hospital, London, S.W. 3, England)

CONTENTS INTRODUCTION Introduction In precancerous liver, as obtained by giving Validity of comparing â€œhepatomasâ€•with nor rats a hepatocarcinogen for several weeks, there ma! liver are diverse biochemical abnormalities. These are Validity of comparing precancerous liver with now surveyed and appraised with respect to their normal liver or with hepatomas possible significance for neoplasia. Consideration Bases for expressing results is also given to primary hepatomas, but only Present Approach briefly to transplanted hepatomas. In the vast Rate-limiting steps literature on hepatocarcinogenesis, there have been Trial of various hepatocarcinogens and of in diverse speculations, but only a few attempts active analogs (e.g., 53, 83, 149, @10)to collate and weigh the Trial of treatments that potentiate or retard evidence. hepatocarcinogenesis In the study of neoplastic changes by biochemi Survey of Biochemical Data cal as distinct from histochemical methods, the Does precancerous liver show biochemical ab use of liver has obvious advantagesâ€”notably the normalities similar to those in primary hepa predominance of one type of cell. Parenchymal tomas? cells comprise ca. 90 per cent of the weight of â€œScore-cardâ€•for significance of observed ab the liver in the rat, although only about 65 per normalities cent of the actual number of cells (46, 80, 196). Might any of the abnormalities entail rate Nevertheless, there are certain assumptions and changes in metabolic processes? pitfalls that have not always been recognized. The reversibility criterion Validity of comparing â€œhepatomasâ€•withnormal Search for hepatomas with minima! deviations liver.â€”Uncritical comparisons have often been from normal liver. Dramatic deviations made between normal liver tissue and â€œhepa Conclusions tomas,â€• repeatedly transplanted and conceivably Theories of hepatocarcinogenesis unlike the original tumor (83, 149, 151), or primary Future work liver tumors which may have been bile-duct tu References mors, or even hyperplastic nodules. Transplanted hepatomas, or hepatomas cultured in intro or * The experiments in the reviewer's laboratory' were sup @ by grants to the Chester Beatty Research Institute (In rendered ascitic, can of course give valuable in stitute of Cancer Research: Royal Cancer Hospital) from the formation (149); but neglect to study primary Medical Research Council, the British Empire Cancer Cam hepatomas is hardly excusable merely because paign, the Anna Fuller Fund, and the National Cancer Insti of the time and work needed to induce the tumor tute of the National Institutes of Health, U.S. Public Health Service. Biochemical differences among transplanted The following abbreviations are used: AAF, @-acetylamino hepatomas, or between transplants and primary fluorene (@-fiuorenylacetamide); AAT, o-aminoazotoluene; hepatomas, are briefly considered later. In at AB, aminoazobenzene (not azobenzene); MAE, 4-methyl-AB; least one study (@)where transplanted hepatomas DAB, 4-dimethyl-AB (N,N-dimethyl-p-phenylazoaniline); DMN, dimethylnitrosamine; DNA, deoxyribonucleic acid; differed biochemically from primary tumors, the RNA, ribonucleic acid. Conventional abbreviations are used for latter may have been of bile-duct origin (â€œcholi nucleotidesâ€”e.g., AMP, adenosine-5'-monophosphate; GDP, angiomas,â€• sometimes termed â€œcholiangiocarci guanosine-5'-diphosphate; UTP, uridine-5'-triphosphate; d (as nomasâ€•). In comparison with primary hepatomas, prefix), deoxy. primary choliangiomas may be lower in alkaline Received for publication September @1,1961. phosphatase (69), in rhodanese (17@), in xanthine 398

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1962 American Association for Cancer Research. REIDâ€”BiOChemiCal Effects of Hepatocarcinogens 399 oxidase ([1@], but see [164]), and in the capacity fair agreement on the following categories (69, to synthesize serum albumin (3@); but higher in 153, 170, 195) : (a) trabecular hepatoma, small the capacity to synthesize phospholipide (80). celled or large-celled (cf. 146) ; (b) adenocarcinoma; No marked differences have been found in gly (c)anaplasticcarcinoma;(d)mixedhepatomaand cÃ§dysisrate (@6), in drug-metabolizing reactions choliangioma. Trabecular hepatomas are less ab (1), or in the content of ribonucleoprotein particles normal than other types with respect to $-hy (144). Hyperplastic nodules (â€œhyperplastomas,â€• droxybutyrate oxidation (61), although not with â€œregenerative fociâ€•), perhaps premalignant (115, respect to the content in the supernatant fraction 119), resemble normal liver at least with respect of RNA and of acid ribonuclease.' In the latter to the oxidative capacity of the mitochondria respects small-celled hepatomas differ little from (60, 61). large-celled hepatomas,' but their glucose-6-phos Primary hepatomas themselves vary in histolo phatase and tryptophan peroxidase content may gy (69, 84). With 3'-Me-DAB, although not with be lower, and they may be morphologically more DAB, the proportion of â€œparenchymalâ€•cellstends cognate to biliary epithelium (146). to be lower than that in normal liver (50, 53). It is then, important, that tumors as used for Lymphocyte infiltration, or changes such as re biochemical comparison with normal liver should generation, cirrhosis, fibrosis, necrosis, or cyst indeed be malignant and be hepatomas in the formation (53), may complicate biochemical stud sense of having histological affinities to liver pa ies and be irreve!ant to the process of carcinogene renchyma. The latter stipulation implies a com sis (69, 105, 119). Cirrhosis may contribute to the promise between two extreme points of view. It low catalase activity of hepatomas (1@) and has been suggested that even supposedly liver-cell necrosis to the high free ATPase (166) and low tumors originate from bile-duct cells, proliferation rhodanese (7@) activitiesâ€”although not to the of which may indeed occur in precancerous liver low xanthine oxidase activity (164) or the ap (153). On the other hand it has been suggested parently high glycolysis (@6).However, acetanilide that even choliangiomas are of parenchymal origin acy!ase is low in hepatomas but somewhat in (105, 195) or, less improbably, that choliangiomas creased with cirrhosis (106), and nucleases are and liver-cell tumors have a common origin in a low in intact hepatoma cells but high in necrotic primitive hepatic cell (119). areas (53). Isotopic studies indicate that the tissue Male rats fed a 20 per cent protein diet con lying deep within a hepatoma nodule differs from taming 3'-Me-DAB (88) eventually develop highly that in an outer layer 5 mm. deep, probably be malignant tumors, which in our experience are cause of impaired circulation associated with ne mainly trabecular hepatomas or adenocarcinomas, crosis (215). It is, then, advisable to reject the and which are in the form of discrete nodules central part of the nodule. It is also useful to such that there is no excuse for including adjacent pass the tissue through a disk with perforations nonmalignant tissue in the tumor sample (cf. of under 1 mm. in diameter, thereby reducing the 68). 4'-F-DAB, in common with AAF (which content of fibrous and other nonhepatoma ele is rather slow-acting, especially in old rats [115]), ments (53, 156). is somewhat toxic. Dimethylaminobenzene-1-azo The study of metastases, from sites such as the 1-naphthalene (69) was only slightly carcinogenic lungs and diaphragm, may be advantageous on in our hands. DAB has been widely used but often histological grounds (170); but the rat may die with neglect of the possibility that the tumors before the metastases are large enough for bio were choliangiomas. With supplementary B vita chemical examination. In a comparison of primary mins (80), or with a low-protein diet (53,2)â€”which, hepatomas with metastases, the depression in cat however, could give protein-deprivation effects alase and N-demethylase activities was much less in the controls (110)â€”DAB can indeed produce marked with the metastases (122). hepatomas. Hepatomas used for biochemical studies should, Firminger (69) draws attention to the finding then, be routinely checked histologically for non of Opie that, in the absence of cirrhosis, the right hepatoma elements as a minimum precaution, lobe of the liver is particularly prone to develop and the presence of metastases should be noted tumors, presumably because most of the portal as one criterion of malignancy. To facilitate com blood goes to this lobe. Such a localization of parison of results from different laboratories, a tumors, although not found by the Millers, has description should be given of the gross and micro been seen in other laboratories; but biochemical scopic appearance, perhaps with a dassification such as medical pathologists employ. There is 1E. Reid, unpublished experiments.

changes have not been found earlier in the right showed an increase in bile-duct tissue to 5 per lobe than in the other 23 cent at 2 weeks and to 30 per cent at 4 weeks. It seems self-evident that controls should be Similar changes, expressed in terms of cell number, used with rats given no carcinogen but otherwise have been reported with 3'-Me-DAB (53), and comparable with the carcinogen-fed rats. Yet some also with DAB given under conditions that ulti workers (e.g., 45, 80) have used animals given no mately gave both pure hepatomas and hepatoma carcinogen but also different in important respects choliangioma tumors (46). At 30 days the propor (110) such as nutritional history, age, or even tion of parenchymal cells fell to 50 per cent with species; and others (e.g., 61, 134) have had no 3'-Me-DAB and to 42 per cent with DAB. With control tissue other than nonmalignant liver from 3'-Me-DAB the proportion was 42 per cent at hepatoma-bearing rats. The hypothesis that re 42 days; with DAB it was 36 per cent at 60 days generating liver is the ideal control for hepatomas and subsequently rose again. Data have also been (149) is considered later. reported for thioacetamide, which induces cho Validity of comparing precancerous liver with liangiocarcinomas (80). normal liver or with hepatomas.â€”One objection The various biochemical analyses reported from to the biochemical study of precancerous liver, Millers' laboratory were made with pulp expressed from rats given a hepatocarcinogen for a short from a tissue squeezer. It is striking that, in period, is that hepatocarcinogens may cause wide recent studies by the Montreal group (53), the spread cell damage but that relatively few cells proportion of the liver mass retained in a squeezer become malignant (113, 115). However, malig was Ca. 12 per cent for normal liver and up to nancy may be the end-product of a series of 20 per cent for liver from DAB-fed rats; the biochemical events (16, 115, 149), the earlier of numerical proportion of parenchymal cells in the which occur in the majority of liver cells. As will pulp was normal (90 per cent) with 4 weeks be shown, biochemical abnormalities in precan of DAB feeding and still high (80 per cent) at cerous liver usually anticipate those in hepatomas 7 weeks. (125). It is argued below that, for early biochemical In precancerous liver there may be histological changes to be significant, it is not essential that changes which are merely incidental to carcino they be shown to persist if the carcinogen treat genesis but which can complicate the interpreta ment is stopped. It is, however, advisable that tion of biochemical changes. Kupifer cells rich in at least a day should elapse between the last acid phosphatase may proliferate (137). Cells such ingestion of carcinogen and autopsy, since, when as lymphocytes may infiltrate (196). Cirrhosis the liver is permeated with carcinogen, there might may occur (119) and may cause altered utiliza be transient effects irrelevant to carcinogenesis. tion of amino acids (28). Marked proliferation There is, in fact, no need for carcinogen treatment of bile-duct tissue may occur after a few weeks to be continued beyond a certain period. This of treatment. This was found with 3'-Me-DAB critical period is of the order of 5â€”8weeks for if given for over 3 weeks, but not with 4'-F- 3'-Me-DAB (6, 76,@), for DAB (45), and for AAF DAB even after many weeks (153); the prolifera (212). The common practice of continuing treat tion was more marked with 24 per cent casein ment until tumors appear offers no advantage in the diet than with 12 per cent. Other authors except perhaps some shortening of the latent pe agree that bile-duct proliferation is considerable riod (57, 76). This critical period may reflect with 3'-Me-DAB (189,@)and less with 4'-F-DAB.8 the onset of rapid cell proliferation following sup Proliferation in or near bile-duct tissue has also posed cell damage (113, 115). been found with DAB (53, 84, 119), but may be Obviously, the study of precancerous changes less with a diet rich in B vitamins (80). There may be invalid if it is uncertain that the conditions may be nonparenchymal cell proliferation with used lead to the appearance, in high incidence, AAF (115). of malignant hepatomas. One report (80) suggests With 3'-Me-DAB given for 28 days (but not that mitochondrial protein is low in pre-cholian with the inactive analog 2-Me-DAB) the amount gioma liver but not in pre-hepatoma liver (see of parenchymal tissue in the liver falls to 55 per also 196). cent, with increases in bile-duct tissue (to 16 Bases for expressing results.â€”The results of bio per cent) and in unclassified tissue (to 29 per chemical determinations are here expressed on cent) (196). Another study with 3'-Me-DAB (189) a commonly used basis, as amount per gram of fresh tissueâ€”or, for determinations in Millers' @5.Doak, L. A. Elson, and A. Lewis, unpublished experi ments. and Cantero's laboratories, per gram of a pulp 8 J@ T. Nodes and E. Reid, unpublished experiments. prepared with a tissue squeezer. Some authors

(e.g., 46, 53, 154 ; cf. [661) express their results as are usually employed, retard growth (inhibited on the basis of the number of cells in the sample, growth-hormone secretion?) even in comparison as estimated by counting nuclei or measuring with controls restricted to the same food intake DNA. The per-cell basis is felt to be more open (Table 1). They may even cause an absolute fall to criticism. If, for example, a constituent present in body weight in growing ratsâ€”perhaps particu in parenchymal cells but absent from bile-duct larly so in strains which are not robust. Liver cells was unaffected by carcinogen treatment, and weight may also be affected, although to a propor if the number of parenchymal cells per gram tionately less and rather variable extent (Table of liver were little decreased but the number of 1, and [66]). Obviously, then, a small change in bile-duct cells (which are smaller than parenchy the amount of a liver constituent expressed per ma! cells) markedly increased, there would appear gram of liver does not imply that the amount to be a fall in the amount of the constituent per per liver or per 100 gm. body weight is likewise cell. The per-gram basis may, then, be preferable changed. However, if there is marked growth unless there is information on cytology and on stasis or actual weight loss, perhaps accompanied the intercellular distribution of the constituent by fatalities as mentioned by some authors, the studied. There is no evidence that constituents biochemical findings may be toxicologic in nature to be considered below are largely nonparenchy and hardly gain validity by being recalculated ma!, with the possible exception (147) of dCMP on a different basis. The real remedy is to choose deaminase. A similar argument in favor of the a rat strain (not necessarily â€œpureâ€•)andfeeding per-gram basis applies to hepatomas, which com conditions such that the problem does not arise monly have more DNA per gram than does normal (cf. 90, 115). liver, although the actual hepatoma cells are not Table 1 also gives data for large particles dis consistently smaller than normal parenchymal cerned in the mitochondrial fraction and assumed, cells. If they were smaller and if each had a normal perhaps unwisely, to consist almost solely of mito complement of most cytoplasmic constituents, chondria. From such data, some authors have there would be an absurd lack of room for water. concluded that the amount of certain mitochon Should it be desired to appraise the present drial constituents may change per gram of liver data on the basis of cells or DNA, requisite data (or per cell)but not per averagemitochondrion. will be found in Table 1, not only for potent Still another basis that has been used for ex hepatocarcinogens but also for analogs which are pressing results is tissue nitrogen or dry weight. slightly carcinogenic (2'-Me-DAB, 3-Me-MAB) Relative to tissue wet weight, nitrogen is typically or virtually noncarcinogenic (2-Me-DAB, 4'-Me decreased by about 15 per cent in precancerous DAB). Table 1 shows that 3'-Me-DAB has given liver and 30 per cent in hepatomas (4, 48). A in several laboratories (but cf. [66]) a striking constituent that is high in amount on a wet-weight rise in nuclei and DNA per gram at 3â€”5weeks basis would evidently be even higher on a nitrogen and that hepatomas likewise show high but vari basis, although it might be low on a cell basis. able values. The values tend to be high in pre The ratio of nitrogen to DNA usually falls in cancerous liver with other carcinogens such as precancerous liver and hepatomas, but exceptions AAF, but variations have been found even by the to this trend have been noted (176). same investigator (113, 114). High values found with 4'-F-DAB can hardly be due to bile-duct PRESENT APPROACH proliferation. Certain topics are deliberately omitted, notably In precancerous liver the number of nuclei the classical phenomenon of binding of carcino serves as a rough measure of cell number (196), gens or metabolites thereof (cf. 202) to liver pro as does the amount of DNAâ€”the average amount teins (66, 122, 125). That this binding is crucial of DNA per nucleus being unaffected by AAF for hepatocarcinogenesis is suggested by several (43, 114, 209), DAB (43, 209), 4'-F-DAB (158) lines of evidence. There is usually a good correla or 3'-Me-DAB (43, 158). However, the average tion between the carcinogenicity of azo dyes and DNA content of the nucleus may rise in hepa the extent of binding to protein; moreover, with tomas induced by DAB (+50 per cent; [209, cf. a given dye the extent of binding in different 154]) or8'-Me-DAB (+30 per cent; [43]), although species is correlated with the liability to develop not in AAF hepatomas (43, 114). liver tumors. Aminofluorene derivatives likewise There may be grounds for expressing data for bind to protein (210). However, â€œWhilethe data precancerous liver as the amount per rat, per 100 available indicate that proteins may be the site gm. body weight or per liver (45, 90). Hepato of attack in carcinogenesis by the aminoazo dyes carcinogens or analogs thereof, in dosages such and the hydrocarbons, it seems reasonable to

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Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1962 American Association for Cancer Research. REIDâ€”Biochemical Effects of Hepatocarc'inogens 403 expect that nucleic acids will be found to be the being rate-limiting, breakdown of ATP to ADP locus of action in other instancesâ€• (125). Alkyla by virtue of functional activity of the cell will, tion of liver nucleic acids has in fact been found in the presence of sufficient orthophosphate, cause after injection of DMN (65) or ethionine (65, enhanced respiration (or glycolysis) and hence 193) ; but there is no reason to suppose that the rephosphorylation of the ADP (150, 184). For changes underlying neoplasia solely concern pro processes such as fatty acid synthesis, reductive teins or nucleic acids. carboxylations and ascorbic acid synthesis, the For every biochemical effect of a hepatocarcino availability of TPNH may influence the rate; gen that is a significant step toward neoplasia, the amount of TPNH may in turn be determined there are probably many that are irrelevant. Three by the activity of transhydrogenase, of TPNH criteria, hardly original, are now used: cytochrome c reductase, or of the hexose mono Criterion (1) : Key changes in metabolism will phosphate shunt. The latter may be stimulated, lie at steps which are rate-limiting in normal liver; and glycolysis retarded, by a rise in the TPN Criterion (2) : Any effect that is produced by level (150). For processes which require ATP diverse hepatocarcinogens, but not by non-carcino as a phosphorylating agent, a limiting factor may genic analogs thereof, is likely to be a key change; well be the ATP levelâ€”perhaps the level in a Criterion (3) : If treatment@ that potentiate or particular cell compartment (184). retard hepatocarc-inogenesis themselves have an effect In the biosynthesis of nucleotides and nucleic respectively similar to, or converse to, one produced acids, there again appear to be homeostatic mech by a he'patocarcinogen, that effect is likely to be a anisms (149) . For example, an amidotransferase key change. required for purine synthesis can be inhibited Rate-limiting steps.â€”Cell metabolism is gov by purine nucleotides, especially AMP, and reac erned by various multi-enzyme systems in which tions centering on IMP and leading to adenosine there are certain bottlenecks, especially at points or guanosine nucleotides may be governed by the where different systems ramify or converge. Such levels of ATP and GMP. In the biosynthesis of points in particular may be sites of action for pyrimidine nucleotides there appears to be regula factors or pacemakers (112) which regulate cell tion by uracil (repression of de novo synthesis), metabolismâ€”these factors including hormones, CMP (aspartic transcarbamylase, negative feed and also metabolites arising intracellularly and back), and UMP (orotidylic decarboxylase, nega acting by feedback or repression mechanisms, tive feedback), at least in microorganisms. perhaps thereby controffing their own formation With enzymic reactions which appear to be (149). Metabolism may also be influenced by readily reversible it is difficult to visualize how changes in the state of barriers which normally a change in enzyme level could alter the reaction limit the access of an enzyme to its substrate rate in a particular direction. For example, hor (149, 184); one example, not necessarilya normal mona! activation of phosphorylase, which can regulatory mechanism, is the appearance in the either form or break down glycogen in intro, would supernatant fraction of hydrolases which are nor hardly account for hormonally induced depletion mally bound in lysosomes (49, 163). of glycogen. Here the dilemma appears to have It is obvious that effects of hepatocarcinogens been resolved by evidence that glycogen synthesis can hardly be significant for neoplasia if they lie in vivo is mediated by an enzyme distinct from at steps which are neither rate-limiting in normal phosphorylase. This illustrates a common finding liver nor potentially rate-limiting if the amount â€”that synthetic reactions and the corresponding of the enzyme or substrate in question decreases. catabolic reactions may proceed through different So far there is only meager information con enzymes in vivo. cerning the identification of rate-limiting enzymes In the study of metabolic changes in hepato or substrates (112). Glycolysis and oxidative proc carcinogenesis, too little attention has been paid esses appear to be limited more by substrates to changes in substrate and product levels as than by enzymes, with the possible exceptions of distinct from enzyme levels. Evidence that the hexokinase and triosephosphate dehydrogenase; enzyme in question is rate-limiting is usually lack in other words, â€œthesubstrates of the intermedi ing. One approach is to determine the optimal ary enzymes are removed as rapidly as they are rates, in normal liver, of different steps in a formedâ€• (112). Hexokinase can itself be inhibited reaction chain; steps which show high rates in by glucose-6-phosphate. Other substances which vitro (say > 1 mole of substrate decomposed per may, by a change in their concentration, influence mm. per gm. of tissue) are hardly likely to be energy metabolism include DPN, oxalacetate, or rate-limiting. This approach is admittedly crude, thophosphate, and particularly ADP. The latter since an optimum concentration of substrate as

used for the in intro assay may be lacking in the hepatomas (with few choliangiomas) found even animalâ€”particularly in the region of the cell con with a low daily dose (118) ; however, the latent cerned. period is long, and the tumors are not confined Trial of various hepatocarcinogens and of inactive to the liver (19, 115, 118, 210, 212). ana!ogs.â€”This criterion implies that there is a Among the other agents listed in the tables, common mechanism underlying the induction of MAB is as active as DAB and is in fact formed hepatomas in rats by different agentsâ€”a view from it in invo (125) ; ethionine, tannic acid, and for which there is some support from histology thioacetamide are moderately active (125), and (64, 69, but see [16]) and from synergism experi DMN is active but has a long latent period (65). ments (118). By this view it is reasonable to In the rat, as distinct from the mouse, AAT suppose that, when a key biochemical effect has is only feebly active and CC!4 is inactive (84, become manifest with a powerful hepatocarcino 125). Since the extensive biochemical literature gen, the effect will be smaller with a weak car on CC!4 is of ambiguous interpretation here, no cinogen and absent with noncarcinogenic analogs. attempt has been made to survey it. Failure of a particular effect to meet this cri Trio] of treatments that potentiate or retard hepa tenon does not, of course, disprove the possibility tocarcinogenesis.â€”It is reasonable to suppose that that it is a key step toward neoplasia. Thus, treatments which modify hepatocarcinogenesis act if neoplasia is due to successive biochemical events by influencing a key step in the latter process, some of which result not from the preceding event although not necessarily every step. In applying but from further intervention by the carcinogen, this criterion to detect key steps, it must usually only one of the events need be of a magnitude suffice to compare biochemical effects of hepato reflecting the potency of the carcinogen. There carcinogens with effects produced in normal rats might be differences among carcinogens in time by the treatment in question, since there have of onset of their effects, rather than in ultimate been few such studies in carcinogen-treated rats. magnitude. Moreover, accurate assays of carcino Azo-dye carcinogenesis is enhanced by ribo gens for relative potency would necessitate not flavin deficiency and retarded by riboflavin sup only the laborious determination of dose-response plementation, although with 3'-Me-DAB these curves, perhaps complicated by anorexia, but also influences are not prominent (75, 104) . Since â€œ... a decision on whether the response should take riboflavin is a constituent of coenzymes in sys account of time as well as of incidence. Taking tems which metabolically cleave and inactivate as the response the ultimate tumor incidence, these compounds,â€• evidently â€œriboflavin acts in Millers' laboratory this was almost 100 per mainly by modifying the effective dose of car cent with 3'-Me-DAB at a dietary concentration cinogen in the liverâ€• (198; cf. 126). In AAF car of 0.054 per cent but barely 10 per cent at a cinogenesis riboflavin is apparently without in concentration of 0.027 per centâ€”which suggests fluence (92). that the dose must be over a certain threshold With severe hyperthyroidism there is an aced for carcinogenesis (8, 118, 212). This view has been eration of carcinogenesis by DAB (127) or 3'- disputed (57) and is indeed hard to prove because Me-DAB (127,'), perhaps due to impaired destruc of the limited life span of the rat and of the known tion of the carcinogen (127). Hepatocarcinogenesis prolongation of the tumor induction period if by AAF is inhibited by complete ablation of the the daily dose is lowered ([57]â€”incidence un thyroid if prior to giving the AM?, although not stated; 8, 212). The dose level may also govern if after 14 weeks of feeding (18) . With AAF tumor size and number (195). (34), although not with azo dyes (88, 127), in The following values give an arbitrary but hibition has likewise been achieved by antithyroid useful indication of the relative potencies of azo drugs; but biochemical effects of the latter are dyes (157, 158): 3'-Me-DAB, 10â€”12;4'-F-DAB, often not clear-cut and are therefore excluded 10; DAB, 6; 2'-Me-DAB, 2â€”3;3-Me-MAB or from consideration. Inhibition of carcinogenesis 4'-Me-DAB, <1; 2-Me-DAB, or AB, 0. However, has been produced by alloxan diabetes with 3'- 2-Me-DAB may be slightly carcinogenic on cer Me-DAB (88) and, less convincingly, with AAF tain diets (188), and 4'-Me-DAB, although itself (19). virtually noncarcinogenic3 and without influence Adrenalectomy or hypophysectomy may inhibit on the action of the powerful hepatocarcinogen hepatocarcinogenesis by azo dyes or AAF in male AAF (118), can potentiate the action of DAB rats (18, 47, 143, 159, 210; cf. [88]). The effect (118). Similarly, 2-Me-DAB can potentiate 3'-Me of hypophysectomy was shown to be not merely DAB (8). The assignation of a high carcinogenic due to lowered intake of food and thereby of potency to AAF rests on the high incidence of dye; the level of bound dye in the liver was

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1962 American Association for Cancer Research. REInâ€”BiochemicalEffects of Hepatocarcinogens 405 undiminished (88), as was the capacity for enzymic even within a given sub-cellular fraction; individu destruction of the dye (125). Although androgens a! protein components have of course been studied, (18, 210) or thyroxine may favor hepatocarcino but only one such study (188) is considered here. genesis, the inhibiting effect of hypophysectomy Scant attention is given to enzymes that me may be due to ensuing lack not of these but of tabolize carcinogens or drugs (cf. 1, 122, 125), ACTH or growth hormone (18, 88) ; however, and none to trace metals (55, 80, 82, 83), to certain the evidence is too inconclusive to warrant con â€œdesaminasesâ€• (83, 106), or to Ca@-activated sideration of individual pituitary hormones. ATPase or alkaline phosphatase (4)â€”the physi Regenerating liver has been widely used as a ological significance of which is dubious (110). control tissue in biochemical studies on hepato Assays with cell-free preparations are quoted in carcinogenesis, but few authors have posed the preference to assays with tissue slices. question whether one would expect each of a series The emphasis is on changes found after 3â€”5 of events underlying carcinogenesisâ€”as distinct weeks of carcinogen treatment, rather than after from a key event causing unrestrained cell pro a few days or after isolated doses. In intro tests liferation (149)â€”to be lacking in regenerating of carcinogens or metabolites thereof for a possible liver. Hepatocarcinogenesis by AAF was uninflu influence on enzymes (e.g., 48, 55) are not con enced by partial hepatectomy, if performed re sidered. The changes as tabulated may differ from peatedly subsequent to 3 months of AM? feeding those stated in the reference cited, because of (187), but was accelerated if the operation was re-calculation on a tissue weight basis or of dis performed at the outset of the feeding period agreement with the conclusion reached in the as compared with before feeding or after 3 months actual paper. of feeding (1 15). Partial hepatectomy does not In Tables 6â€”8the changes given for regenerat affect the tumor incidence in DAB carcinogenesis ing liver are usually based on comparison with (76, 138) but was found to accelerate the appear sham-operated controls, since supposed effects of ance of tumors especially when older rats showing liver regeneration may be due merely to the only moderate lesions after 185 days of dye feeding operative manipulations (e.g., 4, 207). Effects of were hepatectomized (76). Partial hepatectomy hormones on protein synthesis (e.g., 63, 163) are done 9 days after commencement of 3'-Me-DAB not considered, since the evidence on changes feeding produced no striking acceleration of car with neoplasia is scanty and conflicting (Table cinogenesis.3 Evidently, it would be unwise at 3). For nucleic-acid synthesis there is likewise present to assume that the finding of a particular uncertainty, in part because phosphate and gly biochemical change both in precancerous liver cine as used in some in vivo experiments (excluded and in regenerating liver argues for its importance from Table 7) are rather unsatisfactory precursors. in the neoplastic process. Does precancerous liver show biochemical abnor malities similar to those in primary hepaknmas?â€”It SURVEY OF BIOCHEMICAL DATA may first be asked whether primary hepatomas Tables 2â€”4give data for short periods of treat induced by different agents differ in biochemical ment (3â€”12weeks) with different hepatocarcino pattern. Such differences are in fact exceptional gens and with inactive analogs thereof, together (Tables 2â€”5)and might be due to inclusion of with data for the tumors ultimately obtained. choliangiomas (see â€œIntroductionâ€•);but AM? hep Where precancerous liver showed no change in atomas and 3'-Me-DAB hepatomas may differ his the constituent or where diverse agents were not tologically (84). Decreases in the protein and RNA tested, the data are in Table 5. Tables 6â€”8give of mitochondrial and microsomal fractions are data for changes effected by treatments that may less consistently found with AAF hepatomas than modify hepatocarcinogenesis, in comparison with with azo-dye hepatomasâ€”hardly because of dif changes in precancerous liver or, failing that, in ferences in biochemical technic, since one com hepatomas. The format according to the constitu parison was made in a single laboratory. AAF ents or activities studied is as follows: Tables 2 hepatomas may differ from azo-dye hepatomas in and 6 deal with constituents estimated by chemi glutaminase, but not in glycogen, riboflavin, gua cal technics, beginning with nucleic acids (for nase, uricase, esterases, and mitochondrial swell DNA see Table 1) and protein. Tables 3 and 7 ing. With 3'-Me-DAB, DAB, and ethionine, the deal with nucleic-acid and protein metabolism, data for primary tumors usually show at least and Tables 4 and 8 with other enzymic activities. qualitative accord. Because of histological hetero In Table 5 the order of listing is similar. geneity, exceptions to the general trend of similari Data for protein or RNA in whole liver are ty among different primary tumors may be more deliberately excluded, since there is heterogeneity apparent than real. For nuclear constituents no

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Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1962 American Association for Cancer Research. TABLE 5â€”EFFECT OF HEPATOCARCINOGENS ON THE LEvELs OF VARious CONSTITUENTS AND ENZYMES IN RAT LiVER

Constituent or enzyme hepatomaConstituentsPrecancerous liver Primary

enzymesVitamins other than

and co-factors Ascorbic: i (55, cf. 45); Pyridoxine: Ascorbic: 0 (4o). DAB: Thiamine, Nia D (155) cia: D (@1);Pyridoxine: D (@1,155);Panto thenic: D (@1,95); Biotin: D (@1,8@);Vit B12:D (191); Ascorbic: 0 (@1,45), d (8@; Tocopherols: D (fi); Co. A: D (@1,t95 Cyt. c: D (8@). AAT: Vit. A: D (8@)

Phosphorylated compounds, glyco DAB: Phosphorcreatine: D, Glucose-1-phos lytic intermediates phate: I, Glucose-6-phosphate: i, Fructose 6-phosphate and -1,6-diphosphate: 0, Phosphoglyceric: d, Lactic: I (117)

Phospholipide (see also citation in DAB: 0 (80). Thioacetamide: d (mit. or DAB: d in mit. + mic., D in sup. (@*) @05) whole liver) (8O@)

Incorporation of phosphate into phospholipides in vise (see also DAB:80)Enzymes 0 (80). Thioacetamide: 0 (80*)AAF: DAB: d (Q, citation in Q05)DAB:

metabolismAspartic concerned in nucleic acid and protein

transcarbamylase or 3'.Me-DAB: ?d I (31 and t) Orotic acid formation from @. bamylaspartate in euro 8'-Me-DAB: Of 3'-Me-DAB: it Uridine nucleotide formation from orotic acid in viLro 3'-Me-DAB: 0@ 3'-Me-DAB: D@ UDPglucose pyrophosphorylase 8'-Me-DAB: Ot 3'-Me-DAB: dt UDPglucose dehydrogenase AAF: transiently i (@0@).S'-Me-DAB: 0@. 3'-Me-DAB: 0@ DAB: 0 (@0@) Deoxyribose phosphate aldolase DAB: I (@0) dCMP de*minase 8'-Me.DAB: I (147) 3'-Me-DAB or ethionine: I (151) Deoxynueleotidase 3'-Me-DAB: 0 (67) S'-Me-DAB: D (67) â€œ5'-AMPdeaminaseâ€•(ef. 68) 3'-Me-DAB: I (85) S'-Me-DAB: I (8S@) Inosine phosphorylase 8'-Me-DAB: 0 (164) 3'-Me-DAB: D (164) Uridine phosphorylase 8'-Me-DAB: 0@ S'-Me-DAB: It Guanase 3'-Me-DAB (or @.Me-DAB): 0 (50) AAF or DAB: 0 (197@). S'-Me-DAB: D (50). Glutamic dehydrogenase S'-Me-DAB: 0 (i-Me-DAB: I) (50) 8'-Me-DAB: D (50), 0 or D (146). Ethio. nine: d (146) Histidase DAB: D (8@, @04;cf. @8) Arginase @04)Other AAF: D (197). DAB: D (82, D-amino acid oxidase4'-F.DAB DAB: D (140)8'-Me-DAB:

processesHexosamine enzymes and

synthetase d (109) I (109) Glycogen synthetase S'-Me-DAB: 0@# 3'.Me.DAB: Dt Phosphorylase DAB: D (91) Glucose.6-phosphate dehydro. genase AAF: 0 (%0@) DAB: I (1, 61) Anaerobic glycolysis 8'-Me-DAB: 0 (66). DAB: I (57). Thioacet Various: I (@6,and citations in 1@4) amide: 0 (@*) Various: 0 or I (citations in 124) Tricarboxylic acid cycle oxidases 8'.Me-DAB: 0 (152). DAB or thioacetam 3'-Me-DAB: D (136, 152, 181) (various) ide*: 0 (80) TPNH-cytochrome c reductase DAB: d (141) DAB: D (61) Cytochrome oxidase DAB: 0 (97). Thioacetamide: 0 (80*) DAB: D (82, 182) Diamine oxidase (cadaverine) DAB: D (10) DAB: 0 (10) Acetate utilization (for acetoace tate and fatty acids; slices) DAB: 0 (124) DAB: D (124) Esterase (tributyrin) AAF: d (100). DAB: d (100), D (106, 204) Cholinesterases AAF or DAB: 0 (106) AAF: I (106). DAB: I (106, 204) Acylases DAB: 0 (acetanilide), i (diacetyltyrosine), DAB: i or(acetylmethionine) 0 (106) d (acetylmethionine) (106) DAB: 0 (48) DAB: d (48, 129) 5-Hydroxytryptophan decarboxyl. ase 3'.MeD@: D (107) 3'-Me-DAB: D (107) Ithodanese S'-Me-DAB: 0, but D at 2 weeks (anorexia S'-Me-DAB: D (72). DAB: D (172) effect?) (72) ATP-ase, M@+- fmit., â€œtotalâ€• 8'-Me-DAB: d (166) S'-Me-DAB: d (166). DAB: D (61) activated )mit., â€œfreeâ€•S'-Me-DAB:S'.Me-DAB: i (166)3'-Me-DAB: S'-Me-DAB: 0 or (if necrosis) i (166)

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414

REIDâ€”Biochemical Effects of Hepatocarcinogens 415

4- 5 conclusions can be drawn; the diversity of the t@ 0 a S0 C findings for nuclear protein and RNA (Table 2,

S and 176) may reflect variable contamination of the â€” b â€˜â€”â€˜C â€” â€”@â€˜ â€” nuclei with cytoplasmic elements and perhaps CV â€˜,â€”, V â€” = =@,=C S â€˜@ paucity of replicate experiments. There may, how [email protected]__ r@â€¢6-Zâ€”â€˜ ever, be inherent variability, as shown for nuclear

C S protein in a careful study with DAB hepatomas C- C 0 3' â€” S (138)â€”although, relative to DNA, the average - 0 ,@ â€” â€˜-.-- S C value for nuclear protein was normal, as was that @ V 4+ .- for nuclear RNA or phospholipide (variation not VS.V stated) = = S The use of nuclei of varying purity may likewise help explain the diversity of the findings for the :s@@- --se C levels of nuclear RNA and protein in precancerous 2_@_@ @â€” â€˜@â€˜ I@n@se @V G1V -..@ liver (Table 2). With the mitochondrial and micro @-S.a@ @,5 somal fractions, RNA and protein tend to fall in precancerous liver as in hepatomas ; differences between laboratories may reflect differences in technics. Precancerous liver has shown in some @9 co @a studies a rise in supernatant-fraction RNA, as in e4'.2 â€”4-- :2'a@@I@ hepatomas; but precancerous liver and hepatomas show a rise and a fall, respectively, in slow h2 proteins which are known to bind azo dye. Other @% constituents which show converse changes in pre 9 â€” C .â€˜.0 C.@ â€˜@@â€˜ @, cancerous liver and in hepatomas include ascorbic C V V @ @:9â€˜;@â€˜ 2 acid, UDP-glucose and UDP-glucuronic acid (and @ zS.-z possibly AMP; see footnote to Table 2), hexosa I mine synthetase, and acid deoxyribonuclease other â€˜5 tâ€” than that in the supernatant fraction (see also

@ C SC 24, 53). Microsomal protein (202), TPN,3 glycogen S. â€˜C ,â€”@---@ 409 5'-.-' .@ 4) 5' 40,@5 -- synthetase, and tryptophan peroxidase are other ,_@S te@@ â€˜5I@;:@ C @, examples; but here the early precancerous change @â€”â€¢0@ @ V5 @, V @ S.â€” â€” â€” â€” S (an increase) persists for only 1â€”2weeksof dye @S. â€˜@â€˜5'@ â€˜@ C @@ z:@ z @a feedingâ€”a phenomenon of which other examples are known (202) and which may well be irrelevant

9; to carcinogenesis. C ,â€” @ I- C â€” C @ S.C â€˜5 04 â€” @â€˜ There are, however, examples of alterations @a @, @a- @- @ @.-â€˜@@â€˜ that persist during several weeks of carcinogen S S S S @ -@0* -@ -@ feeding, only to regress and finally reappear in the @ hepatoma. Evidence of such biphasic alteration .@ CS 0l @4) â€” I hasbeenfoundformitochondrialandmicrosomal C@ = @0-- S a swelling (6â€”8), acetanilide acylase (106), 5-hy @ Ã§z4S.@u.@% @:hydraseandcystathioninesynthetase(108),anddroxytryptophan decarboxylase (107), serine de â€˜@ possibly nucleases (48), catalase and N-demethyl 0 S @! ase (122), and rhodanese (72). A reported biphasic C s@__C 9 change in nuclear protein and RNA relative to @ DNA (114) has not been confirmed (176). Other

@- evidence, hardly clear-cut, comes from the finding @ that nonmalignant (but premalignant?) liver ad @ joining hepatomas is more like normal liver than 4, @ 8 6 5 like early precancerous liver or hepatomas in some @; biochemical respects (e.g., 1, 26, 36, 72, 106, 0 .@a 0@, :9 @4 @ V 107, 165, 204), as in cell composition (50). The

@ V a _9, fall in xanthine oxidase, if calculated on a cell @ @C) V .@ basis, shows transient interruptions with either

S .Ffl:@ .@. I 9@ 3'-Me-DAB or the noncarcinogenic analog 2-Me

.@ S h .@ -@. 9 ::i: DAB (50). @ to 5. @o a S. â€˜3 S. â€” S. S 4I@ @E-@ c@ E-' There are other reports which lack data for

C -@S â€˜54 C @ â€œ5 C â€”CC C @ Sâ€” V S â€” = S C @ z

S z -.â€”- S.

CS â€˜@- â€˜6-'@' â€˜5@ @ to â€˜54 C C â€œ5 C C @ â€œ5 â€” C - â€” â€” C â€” â€” @ @- C ,â€”@ â€” @ C â€” V V V@@' â€”-â€˜ VC V VVV @ z S S = = SSS z zzz0 0 0 CS 4,3 0 S C â€˜54 S. C â€˜-â€˜ â€˜5 C) a. S

@to

F-to Q toZ

-Q CS Q ZO toF 11 â€˜S â€˜54 S CS â€˜OQ'IS. -04 â€˜5 â€˜IS. 154 C C G@ â€˜5 â€”.- C â€”â€˜5 â€˜5 C@ @to VV V â€” V â€˜-â€” V SS â€” S 5â€” S zz0 0 z@ toto

to S â€˜54 S.

C @ .â€” â€” â€˜5 @â€” â€” â€œ5 Câ€” Câ€” t'- @â€” @- t@ â€” â€” â€”

-@ @ @4 z

C 9 S V 0) S @ to0 â€” 0 .;@â€˜3-@ S â€˜0 S â€˜54 .- V 5.@ F- S .â€”@ .4@ â€˜5@ â€˜54 â€œ5 C to â€” â€˜5@â€” â€˜54 â€˜54 V â€˜â€”â€˜ .8C -@S @ = =5 == C) S.@ @ z z S. C o4-, S F- 5.4) to 4) â€˜S S â€˜S â€˜S S. S.5.

F- Q to @â€˜,

@ S-@ -@ S.6 S.6 995 @ S -@4@ ,@ SC) V VC) S S@a 55 @J .9-@ @@ -@ â€˜S'S 0 9 8 S @ SV @, ,@;@-,â€˜;;-.@ C)a S.d.; 0 S.C 5 â€˜5 5 @@@@ â€˜@ S 0 0 5 0 â€˜S _ â€˜S SS@ @@@ .5@ cj â€˜5:@ t's@ @@â€˜s-@ 90) â€˜Sâ€˜S S0@ E-'E@ @ .9 @â€˜0,@ h -@--.@- @S-@a 0 Za @.â€œ5@â€˜5â€˜S0 Za â€˜-@ 9 * 4â€”4+ @ c,@ E-@ Q F-.'

REIDâ€”Biochemical Effects of Hepatocarc-inogens 417

hepatomas but which suggest a biphasic or even 4) â€˜@â€˜ #@@% a triphasic change in precancerous liver. For â€”SH

tS..@Vâ€”tS. â€”.,tS. â€”.5, levels (29), for DNA and glucose-6-phosphatase V (66), and for amino acid incorporation into protein zz@ in invo (116), the data are inconclusive. Two transient increases have been found in glucuronide

4-- synthetase (with AM' but not with DAB) and )@ a 4).; possibly in cortisone reductase (202). Glutathione C)

.@ reductase shows a protracted increase which ulti C mately regresses (202). -@â€˜54 â€˜5â€” V V VV â€” S S SS In general, where assays have been made at -.@ 0 0 0 C zz>.0 @zz intervals throughout the period of cancer induc tion (e.g., 4, 48, 53, 175, 177, 206), it has usually

@ S appeared that biochemical abnormalities, once mi C.â€”- .@ [email protected] S @ @â€˜C S. tiated, develop progressively; but there are some :S@ interesting exceptions. Moreover, there are evi dently a few exceptions to the generalization (125) .5â€” that, where biochemical abnormalities exist in @; @ C5).2 V precancerous liver, these resemble those found SSâ€¢@ @ @1:;:: in @ â€œScore-cardâ€•forsignificance of observed abnor @; m,alities.â€”Biochemicaleffectsof carbontetrachlo C') M@ @ -_â€˜C_@ ride or of partial hepatectomy have been dis 02-a -@o regarded in arriving at the â€œscoresâ€• (Table 9).

.@ The great majority of the biochemical abnormali @ ties score poorly, usually owing to scanty or equiv â€˜@.. :- ocal rather than adverse evidence. Abnormalities 4-) I -.-t@ -5-- to which this pessimistic conclusion applies in S @ .5 elude glycogen depletion, mitochondrial-fraction changes such as lowering of RNA and succin ic dehydrogenase, microsomal-fraction RNA and @T.0QIiIC protein depletion, and increased supernatant-frac tion activity of acid deoxyribonuclease and acid a phosphatase. The rises in supernatant-fraction -u acid ribonuclease4 and cathepsin score better, as do the decreases in catalase and certain oxidases

.@ (octanoic, choline, monoamine) located in the mi 4@22C .@ tochondrial fraction, and the changes in the levels â€˜@ of TPN, GMP, and nucleoside diphosphates. Evi @ dently the precancerous state may entail decreases ,@ in purine nucleotides and increases in uridine

@ -S 2 nucleotides, the change in the latter being asso @V CSS @. ciated with a rise in uridine kinase activity. The

.@ rise in glucuronide synthetase activity scores poor

.@ ly, but it is striking that this activity is enhanced : bytestosteroneandishigherinmalesthanin

LI females (101)â€”the carcinogenicity of 3'-Me-DAB 4)5) 9;:V@ being higher in male rats and being enhanced by androgens. Among the changes seen in hepa tomas as distinct from precancerous liver, the * . * rise in glucose-6-phosphate dehydrogenase scores S well. 8 j Might any of the abnormalities entail rate changes @5 C 4 Data for ribonucleases in this review refer to attack on RNA; the activity of liver mitochondrial and supernatant @ a -â€˜9S 6 @ 3' S V â€˜S@5 0 9 fractions toward cyclic mononucleotides (e.g., conversion of @ S S 2 a S+ to @ to .9 a .@ @I@5 S adenosine 2' :Sâ€•-phosphatetoadenosine 2'-phosphate) may de pend on different enzymes, and has been shown by J. T. Nodes c c@ -u,@ .s@ SD to be little altered in precancerous liver or hepatomas.

in metabolic processes?â€”With hepatocarcinogene cogen synthetase reaction but also for glycolysis. sis, in intro assays may show increased activity Since, however, glycolytic and respiratory rates of glycolytic enzymes and reduced activity of depend particularly on the level of ADP, the fall oxidative enzymesâ€”the fall in succinic dehydro in ADP in precancerous liver and hepatomas genase being a dubious example (140, 152, cf. [194]). (165) suggests that these rates are in fact sub Energy-yielding reactions may, however, be lim normal, although adequate to prevent any drastic ited in invo not by enzyme levels, but by other fall in ATP. It would be of interest to know the factors such as substrate supply. Hepatomas usu turnover rate of ATP; at least this rate is unlikely ally lack glycogenâ€”probably because of loss of to be enhanced, since one would then expect glycogen synthetase rather than of phosphorylase an increased steady-state level of ADP. â€”but they have undiminished amounts of glucose These speculations concerning energy-yielding 1-phosphate and glucose-6-phosphate (117), these reactions do not take account of complications compounds being requisite not only for the gly such as the existence of compartments within

TABLE 9 â€œSc0RE.CtRDâ€•FOR BIOCHEMICAL ABNORMALITIES IN PRECANCEROUS LivER (DAB OR8'-Me-DAB) OR IN PRIMARYHEPATOMAS Abnormalities are shown for hepatomas (in italics) only if lacking or uninvestigated in precancerous liver. Some of the enzymes listed under cell fractions were actually assayed with whole liver or unfractionated cytoplasm, but are known to reside mainly in the cell fraction indicated. C = comparative evidence, for precancerous liver, from â€˜@mpp@@gevidence scored as +, or ++ if strong; ad @ trial of different agents (Tables 1â€”4) verse evidence scored as â€”,or â€”â€”ifstrong; equivocal M = evidence from study of treatments that modify hepatocarcinogenesis (Tables 6â€”8) J evidence scored as 0

CM C M SVnoi,z LIVER:ACIDSOLUBLENuc@zoTmzs WROLR LIYZR: VABIOTI8 CONSTITUENTS Fall in IMP + 0 Rise in DNA 0 Fall in AMP + 0 Rise in -SH groups 0 + Fall in ADP +++ Fall in glycogen 0 0 Fall in Al? 0 + Fall in riboflavin + 0 Fall in GMP and GDP ++O Fall in Vitamin B12 + Rise in UMP â€” ++ Fall in cytochnome e Rise in UDP ++0 Fall in coenzyme A Risein UTP 0 0 Rise in glucose-i. and -6-phosphates + Rise in UbPacetylglucosamine 0 0 Rise in aspartic transcarbamylase 0 Rise in UDPglucose Rise in earbamylaspartateâ€”+orotate 0 Rise in UDPglucuronic acid â€”â€” 0 Fall in arginase Fall in DPN 0 Fall in asparaginase 0 + Fall in TPN ++â€” Fall in serine dehydrase 0 Foil in acetate utilization in euro + MITOcRONDRLLLTRACTION

Fallin no.oflargepartides 0 0 MZCROSOMAL TRACTION Fall in RNA 0 â€” Fall in RNA + 0 Fall in protein ++â€”â€” Fall in protein 0 + Fallin adenosinenucleotides ++â€” Fall in swelling in vifro Fall in alkaline RNase + 0 Fall in unease Fall in glycogen synthetase + Fall in glutamic dehydrogenase Fall in glucose-6-phosphatase Fall in D-aminoacid oxidase Fall in TPNH.cytochrome c reductase + Fall in glutamie-oxalacetic transaminase Rise in glucuronide synthetase 0 + Fall in octanoic oxidase Fall in succinicdehydrogenase 0 0 SUPERNATANT FRACTION Fall in various tnicarboxylic acid cyde Rise in RNA 0 oxidases + Rise in slow h2 proteins + Fall in cytochnome oxidase Fall in orotateâ€”+unidine nudeotides 0 Fall in DPNH-cytochrome c reductase + 0 Rise in unidine kinase ++â€” Fall in choline oxidase + + Rise in unidine phosphorylase + 0 Fall in monoamine oxidase + + Rise in adenosine deaminase + 0 Fall in catalase + ++ Fall in inosine phosphorylase 0 Rise in free ATPase 0 Fall in xanthine oxidase 0 + Fall in total ATPase 0 Fall in tryptophan peroxidase 0 Fall in glycogen phosphorylase Mi@c@onrJsu@LSUB-TRACTIONCONTAINING Rise in glucose.6-phosphate dehydrogenase ++ LYSOSOMXS Rise in acid DNase + 0 Rise in acid DNase â€” 0 Rise in acid RNase â€” + Rise in acid RNase ++0 Rise in cathepsin 0 ++ Rise in cathepsin + + Fall in acid phosphatase 0 + Rise in acid phosphatase 0 + Fall in fl-glucuronidase

RErnâ€”Biochemical Effects of Hepatocarcinogens 419

the cell, or the possible effect of hepatocarcino synthesis. Although UDP-glucuronic acid as well genesis in uncoupling respiration from phosphoryl as TPNH is decreased, there appears to be a ation. The finding of biochemical damage in iso normal supply of ascorbic acid. The fall in UDP lated mitochondria may signify impairment of the glucuronic acid in hepatomas does not, moreover, rate or efficiency of oxidative phosphorylation imply decreased glucuronide formation, since the but could be due merely to enhanced fragility latter is probably limited rather by the enzyme (62)â€”although the depletion of nucleotides is level (which is undiminished) or by the supply found even if sucrose solution is replaced by a dex of phenolic substrates. In precancerous liver the tran-raffinose medium which, with normal liver, level of the enzyme, and also of UDP-acetyl reduces morphological damage.' glucosamine (an activator) and of UDP-glucuronic Decreased levels of TPN and TPNH, as in acid, may be increased; but increased glucuronide hepatomas, may imply depression of the hexose formation is unlikely to be a key step in neoplasia. monophosphate shunt (despite the rise in glucose In the field of nucleic acid and nucleotide me 6-phosphate dehydrogenase) and also, in accord tabolism (Chart 1), again the discussion must with tissue-slice experiments (124), of fatty-acid be speculative, particularly with regard to nucleic

â€”S @Yvs, S I -@ .@ :@5' 2'AMPâ€”@â€”*Adsnoslnsâ€”*Inosins @i!Hypoxanthinsâ€”@-4Xanthins, @ ,,q ra@ @â€˜@Uricacid

@ RNA â€” GHP â€”@Guanosine Guanins @ 0 â€˜@â€˜@AIIantoin @ 3'UtiPâ€”sâ€•Urldine Uracil @Â±DihydrouraciII@

vs otic4-@--Carbamyl4â€”Carbamyl @ -j@: acid aspartate00phosphate

S. S. [email protected]@S AlP-*DPN Ip I@ UTP' @ ,UDPacetylqlucosamlns*â€”J :, vs,, UDP-qlucuronic GIucuronidesâ€•@ Thymidine @ â€˜@curonic acid

4 Mucopotysaccharides DNA 3'- fliP Ascorbicacid S VS@ 3LdCMP :.@ mit sup.

CHART 1.â€”Nucleic acid and nucleotide metabolism, as particularly fast (>2 @@moles/gm/min), slow (<0.3 @.tmoles/ affected by hepatocarcinogenesis. gm/mm) or very slow (<0.05 pmoles/gm/min) when as Changes in the level of a constituent per gm. of tissue are sayed with normal liver in t'ifro with optimal substrate con shown abovethe name of the constituent, and changes in centrations. enzyme activity per gin. of tissue are shown below the arrow Certain multi-stage reactions are shown as single-stage re for the reaction. Changes in precancerous liver produced by actions. No account is taken of the formation or catabolism of feeding with DAB or S'-Me-DAB for 3â€”12weeks are denoted the cytidylic acid in RNA and of the purine deoxyribonucleo .@- (increase), â€˜@ (decrease), or s@ (little or no change). tides in DNA. The conversion of UDP to dCMP may proceed Changes in prima!I, hepatoma compared with normal liver are via 5'-CDP. The relationship of 5'-IMP to inosine is not @ similarly denoted @, or @.For nucleases in different cell shown; moreover, it is uncertain that 5'-AMP can be converted fractions (see heading to Table 2 for abbreviations) data are to o'-IMP and thence to inosine (Ref. 68). Note that &â€˜-IMP given only where a change occurred. For ATPase, only â€œtotalâ€•and 5'-UMP occupy key positions in nucleotide synthesis. activity (in mitochondria) as distinct from â€œfreeâ€•activity is The participation of ATP in phosphorylation steps is not considered. shown. Late steps in nucleic acid synthesis are shown - â€”â€”@,and For references see Tables 1â€”S.The increased conversion of â€œsalvageâ€•reactions leading to 5'-nucleotides are shown thymidine to TMP and TTP was shown in an unpublished . __ . Ã· S F, S and VS denote reactions that are, respectively, experiment by P. A. Bianchi.

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1962 American Association for Cancer Research. 420 Cancer Research Vol. 292, May 1962 acid synthesis. RNA synthesis may be increased nucleotides is decreased, but aspartic transcar in hepatomas, although not in precancerous liver bamylase (which is unlikely to be rate-limiting) (161). The fall in the level of microsomal RNA is increased, and the conversion of carbamylas may be due to increased catabolism: the synthetic partic acid to orotic acid (probably the bottleneck) pathways for RNA in cytoplasmic loci are poorly is usually increased, at least in intro. Preliminary understood, but at least there appears to be un experiments to find if orotic acid, which is unde impaired translocation of soluble RNA to micro tectable in normal liver, accumulates in hepato somes in primary hepatomas.6 The rise in the acid mas have given negative results.' ribonuclease activity of â€¢thesupernatant fraction For purine nucleotide synthesis there is clearly (as distinct from the bound activity of the mito a lack of data. The de novo pathway may be chondrial fraction) suggests enhanced RNA break impaired, since IMP and other acid-soluble purine down both in precancerous liver and in hepatomas. nucleotides are diminished in precancerous liver In the latter there may, then, be faster turnover and hepatomas.3 There may be increased catabo of RNA. lism, since there is increased activity of 5'-nucleo With regard to the further catabolism of the tidase, and possibly of â€œ5'-AMPdeaminaseâ€•â€”for ribonucleotides formed by breakdown of RNA, which, however, there are conflicting reports from the changes in nucleoside phosphorylase activities the same laboratory (85) and whose very existence are probably irrelevant, because the reactions are is dubious (68). However, the fall in purine nucleo readily reversible and because the normal activity tide levels occurs much earlier during azo-dye of inosine phosphorylase is notably high (as signi feeding than the rise in nucleotidase (50) and, fled by F in Chart 1). The fall in xanthine oxidase moreover, at a time when there is no evidence may be more meaningful, because of its low ac of faster utilization of nucleotides for RNA syn tivity in normal liver and of evidence that it thesis in invo. limits catabolism in intro (53). However, the en With deoxyribonuclease, as with acid ribonu zyme may not be a bottleneck in the animal: clease, the rise in supernatant-fraction activity the fall in its activity after adrenalectomy does in precancerous liver (for which, however, the not lead to accumulation of hypoxanthine or xan â€œscoreâ€•waspoor) may signify increased catabo thine in the liver (168), and the fall produced lism of DNA, perhaps as a concomitantâ€”or even by azo-dye feeding does not lead to reduced excre a cause (24, 194)â€”of faster DNA synthesis. Little tion of uric acid or allantoin (14). is known about the behavior of de novo and Whether or not the ribonucleotides or ribo salvage pathways for synthesis of 5'-deoxyribo nucleosides produced by RNA breakdown are nucleotides, and the levels of the latter in liver degraded more slowly in hepatocarcinogenesis, are barely measurable. Potter (147, 149) has specu their re-utilization by certain salvage pathways lated that bile-duct cells are the locus of the rise (Chart 1) may be enhanced. Some evidence for in the normally low activity of dCMP deaminase, such re-utilization, which is low in normal liver, but this is unproved. comes from the data on uridine nucleotide levels, The above discussion has dealt mainly with on uridine kinase, and on the incorporation of changes in enzyme activity assayed under con injected uracil or adenine (Table 8). However, ditions designed to show optimal activity. For the experiments on uracil incorporation are in processes such as dephosphorylation of 5'-nucleo conclusive, because those in which the dose was tides and conversion of carbamylaspartic acid to high merely reflect the capacity of the liver to orotic acid, this optimal activity may greatly utilize uracil, and those in which the dose was exceed the free activity found in fresh homog low lack data for pool size which would enable enates. Moreover, no regard has been paid to the utilization of endogenous uracil to be cal possible changes in the endogenous level of regula culated. Of the uridine nucleotides in precancerous tory factors. For example, the rise in 5'-UMP liver (8'-Me-DAB), UMP shows the most striking might in fact â€œdampdownâ€•the de novo pathway increase;3 the fall in ATP may explain why the by which it is normally synthesized, provided extra UMP is not fully converted to phosphoryl that such feedback regulation is not impaired ated derivatives. The rise in uridine kinase is a by carcinogenesis and that such an influence of notably early effect and is evidently not a conse 5'-UMP is not offset by changes in the uracil quence of impairment of the normal (de novo) or CMP level (concerning which no data are pathway of synthesis. The latter pathway may available). Among the acid-soluble purine ribo be enhanced rather than depressed in hepatomas. nucleotides, AMP is exceptional in showing a rise The capacity to convert orotic acid into uridine in precancerous liver : this rise, although transient,

I C. J. Smith and E. Reid, unpublished experiments. might help depress de novo synthesis. The fall in

the level of GMP would normally lead to increased hepatomas may be irrelevant to the search transformation of IMP to GMP, but the fall in for changes underlying hepatocarcinogenesis. ATP would favor the converse transformation. It is futile to study only transplanted hepa The reversibilitycriterion.â€”Inassessingthe sig tomas of a single type, particularly since there nificance of biochemical abnormalities in precan may be inherent variability, as found, for example, cerous liver, a few authors have used a criterion in the Novikoff tumor with respect to alkaline not discussed above: if on withdrawing the hepato ribonuclease (4) and glutamic dehydrogenase (ci carcinogen the abnormality persists, it is likely tations in [61]). However, the recent trend toward to be a key step toward neoplasia. One of the systematic comparison of various types (60, 61, few examples of such persistent effects is the fall 149) is welcome, particularly if primary hepatomas in xanthine oxidase; unlike 3'-Me-DAB, the non are routinely included and if bile-secreting trans carcinogenic azo dye 2-Me-DAB does not produce plants (169) are studied. The hope is to demon an irreversible fall (50). Succinic dehydrogenase strate a lack of, rather than the presence of, abnor (152) and the capacity to form acid deoxyribo malities; â€œWewant to know what are the minimal nuclease after partial hepatectomy may be other deviations that must be effected in a normal cell examples (24). to make it a malignant cellâ€•(149). This criterion The following constituents show decreases which is analogous to criterion (2) as used above for are reversed on stoppage of the hepatocarcinogen: precancerous liver. Such systematic studies have asparaginase (93), acid phosphatase (4, 48), cata so far hardly disproved the view that â€œThevalues lase (131), rhodanese (172), and riboflavin (57; of the various components in the primary tumor note misquotation). The following activities show do not appear to be discontinuous between the increases which are reversible : acid ribonuclease normal tissue and the transplant, but approach assayed with whole homogenates (4), adenosine those of either one or the otherâ€• (83). deaminase (68), dCMP deaminase (147), and gly The Morris 5123 hepatoma is notably close colysisâ€”especially anaerobic (57; note misquota to normal liver in its enzymology, unlike the tion). In two other instancesâ€”the decrease in Novikoff hepatoma which may be of bile-duct glycogen (190) and in 5-hydroxytryptophan de origin (146, 147, 149, 151). Among the enzymes carboxylase (107)â€”where the change was found stated to be present in Hepatoma 5123 are glucose to be reversible, no conclusion can be drawn 6-phosphate dehydrogenase (this being increased, because the azo dye was given for so short a likewise in primary hepatomas), succinoxidase, period that few tumors would have resulted. catalase, unease, thymine reductase, arginase, glu The failure of a particular abnormality to per cose-6-phosphatase, glutamic dehydrogenase, cho sist if the carcinogen is withdrawn is not a strong line oxidase, and tryptophan peroxidase (149). argument against its importance for neoplasia. As Certain of these enzymes, such as the last four, already pointed out, certain biochemical changes are likewise present in appreciable amount in appear to temporarily regress at an intermediate some primary hepatomas (146, and Tables 3â€”5). stage between early precancerous liver and tumor, Some changes that are now tabulated as decreases even if carcinogen treatment is continued; and are relatively small in magnitude, such that the moreover the presumably precancerous liver dis enzyme would be treated as present in Potter's tant from hepatomas may resemble normal liver compilation (149) ; tryptophan peroxidase activity rather than hepatomas. Perhaps early precancer is in fact only 20 per cent of normal in Hepatoma ous changes persist in only a minority of the cells 5123 (149). in late precancerous liver, especially if carcinogen The present tables do not, then, emphasize treatment is stopped. Biochemical technics (as the point that certain constituents are not merely distinct from histochemical technics) might fail decreased, but virtually absent, in primary hep to disclose persistence of the changes only in atomas. For such abolitions it hardly matters cells destined to become cancerous. Even in early whether the basis for expressing results is per gm. precancerous liver, changes in a minority of the tissue or per cell. A decrease (calculated on the parenchymal cells may account for gross biochemi former basis) to below 10 per cent of normal can cal findings such as glycogen depletion (190). reasonably be considered an abolition, although Moreover, some enzymes are normally distributed few authors state the sensitivity of their assays. nonuniformly through the liver lobule (137). Constituents which are virtually abolished in Search for hepatomas with minimal deviations primary hepatomas, at least with azo dyes if from normal liver. Dramatic deviations.â€”For rea not with ethionine, include slow h2 proteins (188), sons such as histological heterogeneity, many of acetanilide hydroxylase (1 ; low even in Hepatoma the biochemical abnormalities found in primary 5123), glutamic dehydrogenase, and glutamic-py

ruvic transaminase (50), histidase (82, 204), un are no longer evident in late-precancerous liver, case (130), and possibly aspanaginase (93), N- despite continued carcinogen treatment, they are demethylase (122) and xanthine oxidase (53, cf. usually again evident in the hepatoma ultimately [164]). Glucose-6-phosphatase activity (206) and obtained. Only for a few constituents do hepa the endogenous activity of tryptophan peroxidase tomas show a change converse to that in precan (36) may be almost lacking in azo-dye hepatomas cerous liver. The biochemical evidence is compati although not in Hepatoma 5123 (146), but the ble with the view that many, if not all, of the evidence is not unanimous. Mitochondria show parenchymal cells are affected at an early stage abolitions such as loss of nucleotides' and impair of carcinogen treatment, but that beyond the ment of certain oxidative processes (136, 204). critical period certain of the changes may persist Impairment of the latter is less evident in certain in only a minority of the cellsâ€”in which case transplants (60, 61; control values lacking!) and the changes may escape biochemical detection might be due in part to loss of cofactors rather in late-precancerous liver or in the nonmalignant than enzymes. Primary hepatomas do, in fact, regions of liver with hepatomas. lack coenzyme A and pantothenic acid (21, cf. Theories of hepatocarcinogeneth.â€”Among the [95]), and also phosphocreatine (117) although many theories advanced to explain neoplasia, not ATP (165). An example of a synthetic activity many have only a weak experimental foundation. that is abolished is the incorporation of acetate Particular favor has rightly been given to the into lipide by tissue slices (124). â€œdeletionhypothesisâ€• (125, 149, 152, 188), despite The tables do not, moreover, show that in a the difficulty of thereby explaining ultimate ge few instances there are dramatic increases such netic changes. In hepatocarcinogenesis it is ob that the amount per cell is increased. Constituents vious that, with some striking exceptions, the which are increased by more than threefold in observed biochemical changes are in the direction primary hepatomas include esterifled cholesterol of loss rather than gain. Whether or not such (82), cholinesterase (106, 204), dCMP deaminase losses are compatible with the â€œconvergence hy (151), and glycolytic systems (26). Early pre pothesisâ€• of Greenstein (83), they do accord with cancerous liver seldom shows dramatic increases the opinion that â€œtheliver tumor cell preserves or decreases. the enzymes which are needed for its life and reproduction but can spare those related to a CONCLUSIONS specialized functionâ€• (4), and with the suggestion The advantages and pitfalls of using the liver (149) that there is a depression of catabolic reac to study biochemical aspects of carcinogenesis tions and an enhancement of anabolic reactions have been pointed out. Literature concerning bio with emphasis on â€œalternate pathwaysâ€• which chemical changes in precancerous liver and in have only minor importance in normal tissues. primary hepatomas has now been tabulated, with The latter idea lacks support in the case of a â€œtissueweightâ€• basis not only for convenience protein metabolismâ€”indeed, there may be in of comparison but also because the view that a creased protein catabolism as judged by the rise â€œpercellâ€•basis is preferable is disputed, at least in cathepsin, notably that in the supernatant for cytoplasmic constituents. The basis is, of fractionâ€”but some support comes from observa course, immaterial if the changes are dramatic. tions on uridine nucleotide metabolism that have The tabulation serves not only to reveal gaps been discussed above. The rise in unidine kinase and contradictions in the literature, but also to can hardly be attributed to a deletion occurring lay the groundwork for a â€œscore-cardâ€•appraisal in related pathways (de novo or catabolic). The rise may, nevertheless, be compatible with Pot of the significance of the various changes in neo ter's revised deletion hypothesis (149) : â€œThesim plasia. Consideration has been given to criteria ple deletion of catabolic enzymes such as thymine for such an appraisal. Little attention has been reductase may be preceded by deletions that open paid to transplanted as distinct from primary ate in a manner that will dc-repress the enzymes hepatomas, the literature being so vast that it associated with DNA synthesis and cell division.â€• warrants a review on its own. Possibly the salvage pathway for unidine nucleo Neoplasia may be the consequence of a series tide synthesis is de-repressed by hepatocarcino of changes, the latest of which might be caused gens, eventually irreversibly, and only subsequent either by the carcinogen itself or by the earlier ly is there deletion of catabolic enzymes such as changes. Particular attention has been given here xanthine oxidase. However, enzymes concerned to changes evident within a few weeks of starting in the initial stages of nucleic acid catabolism carcinogen administration. Even if such changes may be increased rather than deleted ; the increase

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1962 American Association for Cancer Research. REIrâ€”Biochemical Effects of Hepatocardnogens 423 in supernatant-fraction acid ribonuclease is a no what increased per gram in hepatomas and in tably early change that may be important. regenerating liver, whereas the membranes are In connection with his postulate that key decreased in hepatomas. In precancerous liver, mi changes in neoplasia lie at enzyme-forming sys crosomes show essentially normal behavior when tems, Potter (149, cf. [44]) points out that certain biochemically â€œdissectedâ€•(66,'). transplanted hepatomas, unlike normal liver, do Among other reported changes, those in gly not show increased tryptophan peroxidase activity cogen storage (cf. 148) and in respiration and gly when tryptophan is given, but that the fault colysis (cf. 66, 149) are of doubtful relevance may lie elsewhere than in the enzyme-forming to the search for key events in neoplasia. Even system (impaired catalase activity?). In primary the fall in catalase may be irrelevant (122). The hepatomas too, the capacity for this adaptation suggestion (132) that loss of the DPN-synthesizing is often lacking ([46]â€”note inaccurate citation of system (located in the nucleus) underlies carcino findings in Ichii's laboratory; also 146), and it genesis has not been tested in the rat. DPN is, is impaired in regenerating liver and, more dubi in fact, undiminished in the early stages of hepato ously, in precancerous liver (36, 44, 66). carcinogenesis. There is only poor evidence for Induction of glucose-6-phosphatase by cortisone the suggested importance (29, 66) of â€”SH groups is impaired in late precancerous liver (66). The in the precancerous state. process of liver regeneration as a whole (24, 115), Future work.â€”Hexokinase, triose phosphate de and the associated rise in acid deoxyribonuclease hydrogenase, and transhydrogenase deserve atten (24), are impaired in precancerousliver; the im tion, as do various enzymes (cf. Chart 1) concerned paired rise in deoxyribonuclease, with 60 days with nucleic acid and nucleotide metabolism and of DAB feeding, was attributed to damage to the with the formation of TPN, ascorbic acid, muco enzyme-forming system. With only 25 days of polysaccharides, and phospholipides. Moreover, DAB, the response to partial hepatectomy was actual isolation of enzymes is desirable, to test actually greater than normal. the idea that properties of enzymes may alter The latter example of a nonprogressive biochem with carcinogenesis (149)â€”as might be the case ical change, together with examples already cited with guanase (197) and xanthine oxidase (141). and with the biochemical resemblance of hepato The levels and turnover of endogenous substrates mas to early precancerous liver rather than to ad need more study, as one approach to the integrated joining nonmalignant liver, suggests the following study of enzymes and over-all metabolic processes broad hypothesis (cf. 122) : Some cells in precan in hepatocarcinogenesis. The literature on lipides cerous liver repair or even over-repair bio is notably poor and scanty. chemical damage caused by the carcinogen, and Changes in protein synthesis call for closer thereby survive without becoming malignant; it study, particularly since ethionine is carcinogenic is other cells which fail to repair the damage, but and is known to block protein synthesis. With develop â€œalternatepathways,â€• that become malig proteins, as with RNA, the heterogeneity of the nant. This hypothesis is akin to earlier hypothe polymeric products is such that it is unrewarding ses (e.g., 7, 113, 115, 148, 202) which emphasize merely to study the content in whole liver or that some cells become malignant because of bio even in the classical subcellular fractions. The chemical damage but not that other cells might latter warrant study by immunological technics overcome the damage and survive with an almost (cf. 32), and also enzymologically with attention normal metabolic pattern. to the distribution and functional availability of Possibly, then, â€œcarcinogens induce neoplasia enzymes such as acid deoxyribonuclease (cf. 51) by interference with rather specific mechanisms and TPNH-cytochrome c reductase. Although the that are required for the re-synthesis of certain cytoplasm may be the site of key steps in neo enzymes essential for the maintenance of a normal plasia, the nucleus (and constituents thereof, such cellâ€•(202). The nature and the intracellular locus as the nucleolus) has been rather neglected of these mechanisms are uncertain. Several work perhaps because of technical difficulties. ers (e.g., 1, 7, 15, 61, 66, 148, 202) emphasize Renewed attention, albeit judicious, should be the biochemical and morphological evidence of given both to primary hepatomas (if only to damage to mitochondria and microsomes (the supplement work on transplants) and to precan latter being essentially the endoplasmic reticulum, cerous liver, following 10 years of comparative from which the Palade particles may become neglect. It is advantageous to use not only DAB detached during carcinogenesis). One report (144), or, preferably, 3'-Me-DAB, but also certain other confusing in terminology, suggests that ribonucleo agents such as 4'-F-DAB-â€”which is highly carcino protein particles of types â€œCâ€•andâ€œEâ€•aresome genie but causes little bile-duct proliferation (153)

â€”ethionine (cf. 146), and a-naphthyl-isothiocya fluence of the Feeding of Aminoazo Dyes on the Swelling nateâ€”which is said to be noncarcinogenic but and Solubilization of Rat-Liver Microsomes. Biochim. et Biophys. Acts, 28:9â€”@0,1958. to cause bile-duct proliferation (80). In the ex 7. Aucos, J. C.; GoacH, H. H.; and ZICKAFOOSE, D. Fine ploration of new aspects of neoplastic metabolism, Structural Alterations in Cell Particles during Chemical difficulties due to disagreements between labora Carcinogenesis. III. Selective Action of Hepatic Carcino tories would be lessened if certain â€œmarkers,â€• gens Other Than 3'-Methyl-4-dimethylaminoazobenzene such as glucose-6-phosphatase, were routinely de on Different Types of Mitochondrial Sweffing. Effect of Stimulated Liver Growth. J. Biophys. Biochem. CytoL, termined (149), and if tumor material were ade 1O:@3â€”36,1961. quately described as recommended above. Extra 8. Ancos, J. C., and GiurrrrH, G. W. Ratio Dependent Syn work thus called for could be offset by discontinu ergism in Azo-Dye Carcinogenesis. Brit. J. Cancer, 15: ing one uninformative practiceâ€”the study of non @91â€”98,1961. 9. ASANO, B.-!. Choline Oxidase Activity in the Hepatic malignant liver adjoining hepatomas. Tissues of Rats Fed on Carcinogens. Gann, 46:41-46, Remarks have been made above about dubious 1955. observations (sometimes poorly controlled) and 10. ASHIK.AWA,K. Studies on the Amine Oxidase in the Liver inaccurate citations. The field of cancer biochemis and Other Tissues of Rats Fed with 4-Dimethylamino try is notable for, although not unique in, such azobenzene. Gann, 50:367â€”73, 1959. 11. AXELROD, A. E., and ELVERJEM, C. A. The Xanthine faults, and also for bad presentation. â€œIfthe Oxidase Content of Rat Liver in Riboflavin Deficiency.

cap fits, wear itâ€•â€”aproverb which may well J. Biol. Chem., 14O:7@5â€”38, 1941. apply to the present reviewer. There are, however, 1@. AXELEOD, A. E.; PorrEn, V. R.; and ELVEHJEM, C. A. many observations which are sound and clear Succinoxidase System in Riboflavin-deficient Rats. J. Biol. Chem., 142:85â€”87, 194g. in themselves but which are too piecemeal for 13. B@um, G. F. Changes in Liver Cell Elements during Stim their potential value to be appraised. A fable ulated Protein Synthesis. Acta Radiol. (SuppL), 147:1â€” quoted in another context (63) is apposite here: 111,1957. it concerns four blind men who came upon an 14. [email protected], A. D. ; TEPPERMAN, J. ; RICHERT, D. A. ; and Wra elephant. â€œOnefelt the side of the elephant and TERFELD, W. W. Excretion of Uric Acid and Allantoin by Rats Depleted of Liver Xanthine Oxidase. Proc. Soc. Exp. announced that it was a wall; another felt a leg Biol.& Med., 73:687â€”89,1950. and interpreted it as a tree; the third held the 15. BELOUSOVA, A. K. Change of the Intracellular Distribu tail and said it was a rope, while the fourth tion of Nucleic Acids and Protein in the Course of Malig said they were all wrong, that it was a large snake nization of the Liver. Cited in Biological Abstracts, 31: 11114,1957. as would be obvious from what he was handling, 16. BERENBLUM, I. Circumstantial Evidence Pointing to which was the trunk.â€• Differences between Cancers in Terms of Etiologic Fac toN. Cancer Research, 16:675â€”80, 1956. ACKNOWLEDGMENTS 17. BERTOLINI, A. M., and VOLTA, A. Comportamento dell' Attivita' Arginasica del Fegato di Ratto Tiroidectomiz I am grateful to Professor A. Haddow, F.R.S., for his inter zati 0 Surrenectomizzati. Boll. Soc. ItaL Biol. Sper., 29: est, to other members of the Institute staff (too many to men 1354â€”57,1953. tion) for help and comments, and to several investigators for 18. BIELCHOWSKY,F. The Role of Hormonal Factors in the allowing citation of unpublished data. Development of Tumours Induced by @-Acetylamino fluorene and Related Compounds. Acta Unio Internat. REFERENCES contra cancrum, 17:1@1â€”30,1961. 1. ADAMSON,R. H., and Form, J. R. The Metabolism of 19. BIELCHOWSKY,F., and BIELCHOWSKY,B. M. Carcino Drugs by Hepatic Tumors. Cancer Research, 21:667â€”7@, genesis in Alloxan-Diabetic Rats. In: G. E. W. WOLSTEN 1961. HOLME and C. M. O'CONNOR (eds.), Carcinogenesis, Ciba

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Cancer Res 1962;22:398-430.

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