endogenous liver carcinogenesis in the rat *

Review Article

Endogenous liver carcinogenesis in the rat*

Dai Nakae

Department of Oncological Pathology, Cancer Center, Nara Medical University, Nara, Japan

develop in a background of chronic liver damage withoutexposure to exogenous carcinogens; for example in LongEvans Cinnamon rats,6 in mice infected with Helicobacterhepaticus7 and in rats subjected to chronic dietary cholinedeficiency (discussed below). Because HCC are induced inthe absence of chemical carcinogen administration in thesecases, they can serve as the experimental models to as-sess possible mechanisms of endogenous carcinogenesis.Choline deficiency is particularly advantageous, because it does not require a special animal strain or bacterial infec-tion. The present paper reviews the accumulated findingsand current understanding for hepatocarcinogenesis in ratscaused by this dietary regimen.

HISTORICAL BACKGROUND OF DIETARYCHOLINE DEFICIENCY

Choline is an amine which must be consumed in the diet, even though small amounts can be biosynthesized.8 As avitamin, it serves various biological functions as a precur-sor for structural phospholipids, phosphatidylcholine andsphingomyelin, as well as signaling phospholipids, platelet-activating factor and sphingosylphosphorylcholine, themethyl donor, betaine, and a neurotransmitter, acetyl-choline.8,9 The availability of choline thus widely affects physi-ological conditions, and its deficiency causes a variety ofdisorders in man and other animals.9,10 During a historicalevent in the discovery of insulin,11 depancreatization of dogswas found to cause liver dysfunction associated with fat accu-mulation, which disturbed complete restoration of normalhealth with insulin treatment.12,13 Lecithin, however, was effec-tive for this purpose.13 Dietary lecithin deficiency was found tocause fatty liver in rats,14 and this was attributed to the lack ofthe component, choline,15 serving as a methyl group donoritself or by way of methionine.9,10 Dietary or parenteral defi-ciency of various other methyl group donors such as vitaminB12,16 threonine,17 and histidine18 also results in fatty liver inanimals. The bioavailability of methyl group donors is thusessential for normal liver fat metabolism, by maintaining a suf-ficient supply of very low-density lipoprotein (VLDL), leading

Pathology International 1999; 49: 1028–1042

Carcinogenesis may be effected not only through exposureto exogenous stimuli but also by genetic and epigeneticinfluences derived from endogenous factors. In the lattercase, the mechanisms are still largely obscure because ofthe limited availability of appropriate in vivo experimentalmodels. However, continuous feeding of a diet deficient incholine and methionine is well known to cause hepatocellu-lar carcinomas (HCC) in rats in the absence of any knownexogenous carcinogens and can serve as a good researchmodel. A semi-synthetic, choline-deficient, L-amino acid-defined (CDAA) diet, containing practically no choline andlow methionine, induces HCC with a background of fattyliver and hepatocyte death, subsequent regeneration andfibrosis resulting in cirrhosis. Using the CDAA diet, we haverevealed the participation of oxidative injury to DNA andother subcellular components and of alteration in intrahep-atic signal transduction pathways in the mechanismsunderlying this rat liver carcinogenesis model. In the pre-sent paper, the current understanding of endogenous ratliver carcinogenesis, due to dietary choline deficiency, isreviewed.

Key words: dietary choline deficiency, endogenous carcino-genesis, gene methylation, oxidative stress, rat liver, signaltransduction

Carcinogenesis involves a series of qualitatively differentstages that occur as a result of genetic and epigeneticchanges, induced mainly by exposure to a variety of exoge-nous substances present in the environment.1–3 However,since such exogenous factors cannot explain all carcinogeniccases, the roles of endogenous factors have recently at-tracted attention.3–5 The mechanisms underlying endogenouscarcinogenesis, however, are still largely obscure, as appro-priate in vivo experimental models are limited. There aresome situations in which hepatocelluar carcinomas (HCC)

Correspondence: Dai Nakae, MD, Department of OncologicalPathology, Cancer Center, Nara Medical University, 840 Shijocho, Kashihara, Nara 634-8521, Japan.Email: [email protected]

*The content of this paper was presented as the A Address at the44th Autumn Meeting of the Japanese Society of Pathology held inNara, Japan, in 1998.

Received 3 June 1999. Accepted for publication 11 August 1999.

Endogenous rat liver carcinogenesis 1029

to their being termed lipotropes.10 Deficiencies of methylgroup donors also causes liver cirrhosis,19,20 and in 1946,Copeland and Salmon reported the development of HCC inAES rats subjected to a dietary deficiency of multiple methylgroup donors including choline.21 However, this discovery wasnot considered convincing mainly because of the later findingthat their results might have been influenced by contamina-tion of aflatoxin in peanut meal within the diet.22 In the 1980s,however, it was rediscovered that dietary choline or multiplemethyl group donors deficiency can indeed induce HCC inrats in the absence of any carcinogens under conditions thatprecluded contamination of the semi-purified or semi-synthe-sized, amino acid-defined diet used.23–26 Since then, numer-ous studies have been performed, and there is presently aconsensus that deficiencies of choline, as well as methionineto a lesser degree, are conditions inducing HCC in rats andthat the lack of other methyl group donors such as vitamin B12

and folic acid can induce non-neoplastic liver lesions andenhance liver carcinogenesis but does not possess carcino-genic potency per se towards the liver.27

DEVELOPMENT OF A CHOLINE-DEFICIENT, L-AMINOACID-DEFINED DIET

In the late 1980s, we decided to assess the effects of ma-nipulation of contents of various dietary components on ratliver carcinogenesis due to dietary choline deficiency. Forsuch studies, proteins had to be replaced by pure aminoacids because of the unavoidable and unpredictable contam-ination of target nutrients in the protein sources of semi-purified diets. We chose the option of a novel semi-syntheticdiet instead using the then available amino acid-defineddiets, because we wanted to apply a widely used, semi-purified choline-deficient (CD) diet25–27 as a positive controland thus to make our diet have as similar composition as pos-sible. As a result of cooperative work between ourselves andthe CD diet manufacturer (Dyets Inc., Bethlehem, PA, USA),a CD, L-amino acid-defined (CDAA) diet was developed byreplacing the proteins by pure amino acids to give an equiva-lent amino acid profile.28 The CDAA diet as well as its control,a choline-supplemented, L-amino acid-defined (CSAA) dietare now commercially available. Table 1 documents the compositions of the two diets. Despite an overall similaritybetween the CDAA and CD diets,28 the amino acid definitionresulted in the substantial decline of the content of choline, 0(below the detectable limit) and 630 pmol/kg for the CDAAand CD diets, respectively, and phosphatidylcholine, 11 and891 pmol/kg, respectively (information provided by Dyets).While the methionine content is also lowered in the CDAAdiet as it is in the CD diet, this is not too low, because no par-ticular disorders have been noted in rats with long-termfeeding of the CSAA diet with the same low methionine

content.29,30 The contents of other methyl group donors likevitamin B12, folic acid and lipotropic amino acids are all ade-quate. Unless otherwise specified, our experiments havebeen conducted using male Fischer 344 rats aged 6 weeks atthe commencement. Rats can grow healthy on the CDAAdiet, but bodyweight gain begins to be retarded from week 4,while relative liver weight increases from day 3.30,31

EARLY CHANGES INDUCED IN RATS FED THE CDAA DIET

One of the earliest changes in the livers of rats fed the CDAAdiet is the formation of subcellular injury due to reactiveoxygen species (ROS)-derived oxidative stress. Nuclear DNAis the early target, because 8-hydroxydeoxyguanosine (8-OHdG), the most abundant oxidative DNA damage32 capableof forming specific mutations from GC to TA transversions,33

is significantly detectable after only day 1 and progressivelyaccumulates at least up to day 84 (week 12) (Fig. 1a)30,31 inthe absence of mitochondrial DNA or DNA from dead cells in

Table 1 Compositions of the choline-deficient, L-amino acid-defined diet and choline-supplemented, L-amino acid-defined diet28

CDAA diet CSAA dietIngredients (g/kg) (g/kg)

L-Arginine 12.70 12.70L-Histidine 3.40 3.40L-Lysine hydrochloride 9.10 9.10L-Tyrosine 5.70 5.70L-Tryptophan 1.80 1.80L-Phenylalanine 7.30 7.30L-Methionine 1.70 1.70L-Cystine 3.70 3.70L-Threonine 4.60 4.60L-Leucine 10.50 10.50L-Isoleucine 6.10 6.10L-Valine 6.30 6.30Glycine 6.20 6.20L-Proline 7.60 7.60L-Glutamic acid 28.90 28.90L-Alanine 5.10 5.10L-Aspartic acid 15.80 15.80L-Serine 7.20 7.20

Corn starch 100.00 100.00Dextrin 100.00 100.00Cellulose 50.00 50.00Sucrose 406.67 392.19Sodium bicarbonate 4.30 4.30Corn oil 50.00 50.00Primex 100.00 100.00Modified AIN-76 salt mixture without iron 35.00 35.00AIN-76A vitamin mixture 10.00 10.00Ferric citrate 0.33 0.33

Choline bitartrate 0.00 14.48

CDAA, choline-deficient, L-amino acid-defined diet; CSAA, choline-supplemented, L-amino acid-defined diet.

1030 D. Nakae

the assayed materials.34 This formation of 8-OHdG in hepato-cellullar nuclei could be confirmed immunohistochemicallyusing a monoclonal antibody recognizing 8-hydroxyguaninemoieties in DNA (unpublished results). Oxidative damage toliver DNA by dietary choline deficiency is supported by theinduction of single-strand breakage35 but not alkylating or aro-matic type adducts.36 Extra-DNA subhepatocellular compo-nents are also oxidatively injured a little later than DNA, beingdetected as the generation of 2-thiobarbituric acid-reactingsubstances (TBARS) from 3 days after the commencement ofthe CDAA diet feeding (Fig. 1b).30,31 Serum alanine amino-transferase (ALT) activity and liver content of triglyceride are

also significantly elevated from day 3 with a closely similartime course to that of the TBARS generation.30,37,38 Oxidativeextra-DNA injuries are thus suggested to play some roles inthe hepatotoxic mechanisms with the CDAA diet feeding.While the exact nature of these TBARS is not clear, we havepreliminarily detected significant production of carbonyl de-rivatives of amino acids and various by-products of lipid peroxidation such as malondialdehyde (MDA), 4-hydroxy-alkenals and lipid hydroperoxide in the cytoplasm of hepato-cytes obtained from the CDAA diet-fed rats. Thus, it isconceivable that peroxidative products of proteins and lipidsare involved. The sources of ROS in the livers of rats under

Figure 1 Sequential increase of thelevels of (a) 8-hydroxydeoxyguanosine(8-OHdG) and (b) 2-thiobarbituric acid-reacting substances (TBARS) on feedingof the choline-deficient, L-amino acid-defined (CDAA) diet.30,31 Black columnsare significantly different from the day 0value. dG, deoxyguanosine; MDA, malondialdehyde.


dietary choline deficiency have not been elucidated so far, butwe very recently found that NADH-dependent production ofhydrogen peroxide at the Complex I site of the mitochondrialelectron transport of rat hepatocytes was markedly enhancedafter 3 days feeding of the CDAA diet, suggesting that themitochondrion may be a good candidate. Altered arachido-nate metabolism by the induction of inducible cyclo-oxygenase (COX2) 39 and possibly products of activated non-parenchymal liver cells could also contribute.

SEQUENTIAL HISTOPATHOLOGICAL CHANGESINDUCED IN THE LIVERS OF RATS FED THE CDAA DIET

Fat deposition is first observed at zones 2 and 3 of hepatocytelobuli and quickly expands so that a diffuse fatty liver resultsby week 1 (Fig. 2a).30 Hepatocyte death is randomly observedfrom week 4 by the presence of shunken cells, no longer incontact with the surrounding parenchyma, with condensednuclei and found in association with regenerative hepatocyteproliferation (Fig. 2b).30 These histological characteristics arein accordance with criteria for apoptosis, and their apoptoticnature has indeed been confirmed by immunohistochemistryusing anti-single-stranded DNA (anti-ssDNA) antibody, a sen-sitive method to detect apoptotic cells,40,41 (Fig. 2c) and elec-tron microscopic observation (Fig. 2d). No morphologicalevidence of inflammation or any discernible form of diffusenecrosis was observed in the livers of rats fed the CDAAdiet.29–31,37,38,42 It would be premature, however, to deny thepossible induction of necrosis by dietary choline deficiency,because of the early increase of serum ALT activity.30 The his-tologically observed regenerative hepatocellular proliferationin rats fed the CDAA diet is evidenced by a significant incre-ment in bromodeoxyuridine (BrdU) or proliferating cell nuclearantigen labeling indices after week 2.37,38,42 Along with thedeath and regeneration of hepatocytes, oval cell proliferationstarts from Glisson’s sheaths and progresses strictly in theperiportal area from week 4.30 Collagen fibers then begin to beextended, also from Glisson’s sheaths, and connective tissuedeposition progresses, at first in the periportal area and lateraround central veins to form bridging fibrosis destroying theliver lobular architecture.30 Borderline cirrhosis results byweek 12.28,30,37–39,42–46 Sakaida et al. reported immunohisto-chemically detected extensive accumulation of type III colla-gen at week 16,47 when the stellate cells are activated tomyofibroblast-like cells, a critical event in fibrogenesis/cirrho-genesis,48 as demonstrated by both α-smooth muscle actinimmunohistochemistry and electron microscopy.47 Frank cir-rhosis eventually develops by week 30 (Fig. 2e,f).29,30,43,46

With this sequential occurrence of non-neoplastic lesionsas the background, altered hepatocyte foci consisting ofeosinophilic cells containing less fat begin to be detected atweek eight and subsequently grow into large-sized neoplas-

tic nodules.43–46 These focal lesions are considered to be putatively preneoplastic49 because of their histologicalcharacteristics50 and immunohistochemically detected glu-tathione S-transferase placental form (GST-P)-positive phe-notype (Fig. 3a,b),30,31,37–39,42–46 one of the best markers for ratliver neoplastic lesions.50,51 The numbers of the GST-P-positive lesions increase in a time-dependent manner at leastup to day 84 (week 12) with significant induction obtainedfrom day 28 (week 4) (Fig. 4a), while significant increase insize of lesions from day 3 (Fig. 4b). The hepatocytes in theGST-P-positive foci demonstrate elevated BrdU labeling andlower anti-ssDNA-positive indices than those in the surround-ing tissue (Fig. 5), indicating enhanced proliferating activityand resistance to apoptosis of the altered cells within theselesions. Hepatocellular carcinomas eventually develop in thelivers of rats fed the CDAA diet from about week 30, reachingan almost 100% incidence by week 52 (Fig. 3c,d).29,30 Someof the induced HCC metastasize to the lungs (Fig. 3e).29,30

GENETIC CHANGES IN HCC DEVELOPING IN RATS FEDTHE CDAA DIET

Clear gene mutations have not been detected in HCC in ratsfed the CDAA diet. For example, no mutations were found inexons 5–7 of p53, or in exons 1–2 of p21 or p16 in HCC afterthe CDAA diet for 70 weeks, while they were rare in exon 1 ofKi-ras (1 out of 7 samples) and in glycogen synthase kinase-3� phosphorylation consensus motif of �-catenin (2 out of 15 samples, without amino acid alteration).52,53 In the livers ofrats fed the CD diet, frequent p53 mutations were found in both preneoplastic nodules and HCC in association with c-myc amplification.54–56 While it is not clear why there is adiscrepancy in terms of the p53 mutations between the CDAAand CD cases, it has been suggested that the p53 mutationsmay not be an obligatory step for hepatocarcinogenesis dueto dietary choline deficiency.55 Therefore, it is possible thatthe non-obligatory p53 mutations may be skipped in theCDAA case in which hepatocarcinogenesis is progressedmore rapidly than in the CD case.28–30 There must be,however, critical gene mutations for hepatocarcinogenesis inrats fed the CDAA diet or due to dietary choline deficiency ingeneral. Further exploration of such critical gene mutations isthus required.

SIMILARITIES AND DIFFERENCES WITH CHANGESOBSERVED IN THE LIVERS OF RATS FED THE CDAA

AND CD DIETS

The above spectrum of acute and chronic changes in the livers of rats fed the CDAA diet is highly repro-ducible28–31,37–39,42–47,57–59 and basically the same as seen in

1032 D. Nakae

rats fed the CD diet.10,19,20,27,28,30,60–62 There is, however, a cleardifference in that the CDAA diet causes these alterationsmore rapidly and to a greater extent.28–30 This is particularlyapparent when the diets are administered to female Fischer344 rats, which are largely resistant, for as yet undefinedreasons, to the acute hepatic effects of the CD diet and in turndo not develop HCC.63–66 During a 12-week feeding of the

CDAA diet, in contrast, diffuse fatty liver with the increase of liver content of triglyceride, hepatocyte deaths with theincrease of serum ALT activity and regenerative hepatocyteproliferation are induced in such rats.66 In addition, we re-cently observed HCC development in the cirrhotic livers offemale Fischer 344 rats fed the CDAA diet for over 1 year,albeit with a lower incidence than obtained in the male case.

Figure 2 Non-neoplastic lesions induced in the livers of rats fed the choline-deficient, L-amino acid-defined (CDAA) diet.28–30,37–39,42–46 (a)Fatty liver (week 1, HE). (b) Death (arrows) and mitosis (arrowhead) of hepatocytes (week 4, HE). (c) Apoptosis detected by anti-single-stranded DNA immunohistochemistry (arrows) (week 4). (d) Ultrastructural appearance of an apoptotic body (week 4). (e) Macroscopic view ofcirrhosis (week 32). (f) Cirrhosis at the microscope level (week 32, Azan–Mallory, loupe).


The question as to why the CDAA diet exerts greater effectsthan the CD diet remains unanswered. One possibility issimply that there is less choline in the CDAA diet, but thisseems unlikely since we have preliminary data that shows the addition of choline bitartrate to the CDAA diet to give acholine amount identical to that in the CD diet does not at-tenuate its effects. An alternative explanation concerns thespeculation that the supply of methionine and other methylgroup donors as free amino acids in the CDAA case mayallow easier and greater utilization by the intestinal flora,resulting in less efficient absorption and, in turn, more severe

deficiency in the liver than with the CD diet, where they areprovided as components of proteins.67,68 Further studies areneeded to resolve this issue for a more detailed understand-ing of the liver carcinogenicity of dietary choline deficiency.

PREVENTION OF LIVER CARCINOGENESIS IN RATSFED THE CDAA DIET

2-O-Octadecyl ascorbic acid (CV-3611), a lipophilic and lesspro-oxidant derivative of vitamin C, reduces the numbers and

Figure 3 Focal lesions induced in the livers of rats fed the choline-deficient, L-amino acid-defined (CDAA) diet.29–31,37–39,42–46,57 (a) Glu-tathione S-transferase placental form (GST-P)-positive lesion (week12). (b) GST-P-positive lesion (week 12). (c) Hepatocellular carcino-mas (HCC) evident as gross tumors (week 70). (d) HCC microscopicappearance (week 70, HE). (e) Lung metastasis of an HCC (week 70,HE).

1034 D. Nakae

sizes of the GST-P-positive lesions and the levels of 8-OHdGand TBARS in the livers of rats fed the CDAA diet for 12weeks but does not affect the non-neoplastic morphologicalchanges (Table 2).69 Its parent L-ascorbic acid, α-tocopheroland 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid(Trolox), a water-soluble derivative of α-tocopherol, exertsimilar effects, but to a lesser extent (Table 2).69 Effects ofdietary deficiency of iron, a major catalyst in the generation ofROS,70 are similar to those of vitamin C and E derivatives(Table 2).31 According to Sakaida et al., chronic administra-tion of a ferric iron chelator, deferoxamine, also inhibits thedevelopment of preneoplastic lesions and TBARS generationin the livers of CDAA diet-fed rats, but the continuous ironchelation is also preventive for the elevation of serum ALTactivity and fibrogenesis.59 N-(4-Hydroxyphenyl)retinamide(4-HPR), a synthetic retinoid, effectively inhibits the preneo-

plastic lesion development and 8-OHdG formation, and theadministration of this agent substantially protected the liversof the CDAA diet-fed rats from hepatocyte death and subse-quent regeneration as well as connective tissue depositionbut not fat accumulation (Table 2).37 While 4-HPR possessesactions as a retinoid, such growth inhibition and induction ofdifferentiation and apoptosis71 is without dependence onclassical retinoic acid and retinoid X receptors, and it is thusmore efficient than conventional retinoids.72 It also inhibitscyclo-oxygenases and prostaglandin synthesis.73 Similarly,non-steroidal anti-inflammatory drugs (NSAID) having the potential to inhibit COX2, acetylsalicylic acid (ASA) and piroxicam (PXC), exert almost the same effects as 4-HPR(Table 2).43–46 In transgenic male Wistar rats harboring anadditional rat GST-P gene, the development of preneoplasticlesions, hepatocyte death with subsequent regeneration,

Figure 4 Sequential increase of the(a) numbers and (b) sizes of gluta-thione S-transferase placental form-positive lesions. Black columns are sig-nificantly different from the day 0 value.CDAA, choline-deficient, L-amino acid-defined.


connective tissue deposition and 8-OHdG formation by theCDAA diet feeding are all inhibited, but a fatty liver still results(Table 2).42 While phenyl N-tert-butyl nitrone (PBN), a radicaltrapper, exerts similar effects (Table 2), it also inhibits nuclearfactor (NF)-kB activation, COX2 activity elevation and GSTactivity depletion in the livers of male Wistar rats fed the

CDAA diet.39 Sakaida et al. reported that a proryl 4-hydroxylase inhibitor, HOE 077,58 and a herbal medicine,Sho-saiko-to,47 both antifibrogenic agents, inhibit not onlyfibrosis through the disturbance of stellate cell activation butalso the preneoplastic lesion development in the livers of ratsfed the CDAA diet. Taken together, the data indicate thatoxidative subhepatocellular injury and altered signal trans-duction pathways are relevant to the genesis of morphologi-cal alterations, both neoplastic and non-neoplastic, in thelivers of rats fed the CDAA diet. In addition, 1�-acetoxychavi-col acetate (ACA), a natural compound contained in seeds orrhizomes of Languas galanga, reduces the numbers of theGST-P-positive lesions and the 8-OHdG levels but not thelesion sizes or the TBARS levels,38 whereas N,N�,-diphenyl-p-phenylenediamine (DPPD), a highly lipophilic anti-oxidant,in contrast reduces the sizes of GST-P-positive lesions andthe TBARS levels but not the lesion numbers or the 8-OHdGlevels (Fig. 6).57 This suggests that oxidative DNA and extra-DNA injuries are preferentially involved in the induction andthe subsequent growth of the preneoplastic lesions, respec-tively, in the livers of CDAA diet-fed rats. Conversely, there isa clear and common characteristic of the above-describedmodulators, the absence of effects on the fatty liver induction.Intrahepatocellular fat accumulation is a distinct feature ofdietary deficiency of choline or multiple methyl group donorsbecause of their lipotropic nature.19,20 This might, however, bean epiphenomenon caused by an early deterioration of VLDLsupply due to decreased availability of phosphatidylcholine inplasma membranes but not directly related to liver carcino-genesis as previously assumed.60 It may be necessary to

Table 2 Effects of various chemicals and manipulations on alterations induced in the livers of rats fed the choline-deficient, L-amino acid-defined diet

GST-P-positive lesions

Inhibitory effects

Chemicals and manipulations (% Non-neoplastic changes

Dose inhibition) Fatty Hepatocyte Hepatocyte Oxidative injury

Category Chemical (%) Route No. Size liver death proliferation Fibrosis 8-OHdG TBARS Ref.

Vitamin CV-3611 0.10 In diet 72 76 – – – – � � 69derivatives L-Ascorbic acid 0.10 In diet 31 32 – – – – � � 69

α-Tocopherol 0.10 In diet 24 40 – – – – � � 69Trolox 0.10 In diet 35 49 – – – – � � 69

Dietary iron 55 15 – – – – � � 31deficiency (� 5%)

Retinoid 4-HPR 0.16 In diet 91 98 – � � � � Not assessed 37NSAID ASA 0.20 In diet 71 20 – � � � � Not assessed 43, 44, 46

PXC 0.04 In diet 72 63 – � � � � Not assessed 45, 46Insertion of a 50 93 – � � � � Not assessed 42

rat GST-Ptransgene

Radical PBN 0.13 In water 25 95 – � � � � Not assessed 39trapping agent

8-OHdG, 8-hydroxydeoxyguanosine formation; TBARS, 2-thiobarbituric acid-reacting substances; ASA, acetylsalicylic acid; PXC, piroxicam; PBN, phenyl-N-tert-butyl nitrone; 4-HPR, N-(4-hydroxyphenyl)retinamide; GST-P, glutathione S-transferase placental form-positive; NSAID, non-steroidal anti-inflammatory drug; �, inhibited; –, not inhibited.

Figure 5 Bromodeoxyuridine (BrdU) labeling and anti-single-stranded DNA (anti-ssDNA)-positive indices in the glutathione S-transferase placental form (GST-P)-positive lesions (�), surroundingareas ( ) of the livers of rats fed the choline-deficient, L-amino acid-defined diet for 8 weeks and their week 0 values (�). The week 8values for the GST-P-positive lesions and the surrounding areas aresignificantly higher than the week 0 value either in the cases of BrdUlabeling or anti-ssDNA-positive indices. The BrdU labeling and anti-ssDNA-positive indices for the GST-P-positive lesions are signi-ficantly higher and lower than those for the surrounding areas,respectively.

1036 D. Nakae

confirm the effects of the modulators using development of HCC as the end-point, and such a study is now under way in our laboratory, employing PBN as a representativemodulator.

POSSIBLE MECHANISMS UNDERLYING LIVERCARCINOGENESIS IN RATS FED THE CDAA DIET

The mechanisms underlying rat liver carcinogenesis bydietary choline deficiency have not been fully elucidated, but repeated hepatocyte death with subsequent regenera-tion,27,60,65,74–77 oxidative stress,27,78,79 altered signal transduc-tion,9,60,61,76,80–83 and DNA hypomethylation26,84–87 are known toplay their role. In the case of the CDAA diet feeding, oxidativestress is reflected in induction of typical subhepatocellularinjuries.28,30,31,37–39,42–46,57 While our data suggest a contribution

of oxidative DNA injury to the induction of preneoplastic cellpopulations,38,57 there is a long-standing controversy regard-ing the initiating activity of dietary choline deficiency. Duringthe growth of the induced GST-P-positive lesions, new singlepositive cells and small foci are continuously induced as longas the CDAA diet is fed, suggesting an initiating activity of theCDAA diet per se, as also proposed for the CD diet.35,88 It isthus unlikely that dietary choline defiency only exerts a pro-moting activity on pre-existing, ‘spontaneously initiated’hepatocytes.89,90 The fact of irreversible changes is indicatedby the finding that preneoplastic lesions persist when rats arefed the CDAA diet for 24 weeks and then a control diet for 28weeks.29 It is necessary, however, to assess the initiatingmechanisms caused by dietary choline deficiency in moredetail. It will then be addressed whether and, if so, how theinitiating processes are different from those associated withclassical exogenous carcinogens. Lipid peroxidation in thelivers of CD diet-fed rats has been another controversialissue. While Perera et al. reported lipid peroxidation in themicrosomal membranes,78 Rushmore et al. detected lipidperoxidation in the nuclear and mitochondrial, but not in themicrosomal membranes.79 Furthermore, Bani et al. claimedthat the CD diet did not induce lipid peroxidation in any sub-hepatocellular membranes and that trans fatty acids withconjugated dienes, present in a partially hydrogenated fat,were absorbed and assimilated in hepatic lipids in rats.91,92

The nature of the generated TBARS in the livers of CDAAdiet-fed rats must therefore be carefully examined.

Alteration of the ROS status and, in turn, intracellular redoxstate can exert various effects on signal transduction path-ways responsible for the maintenance and regulation of cel-lular functions.93–96 For instance, interleukin (IL)-1α, IL-1�,tumor necrosis factor (TNF)-α, interferon (IFN)-γ and arachi-donates are all involved in the regulation of mitochondrialROS production and NF-kB activation,97–102 and NF-kB thenregulates COX2 induction.103 Reactive-oxygen species, IL-1�

and TNF-α are related to the progress of apoptosis by regu-lating the functions of key enzyme-like caspases and c-JunN-terminal kinases.104–106 Furthermore, ROS, IL-1α, IL-1�, IL-6, TNF-α, IFN-γ, transforming growth factor (TGF)-� andplatelet-derived growth factor are involved in the activation,proliferation and functioning of stellate cells and thus fibroge-nesis/cirrhogenesis.48 Assuming extensive oxidative stress,the status of such signal transduction pathways must be sub-stantially altered, and this may play a role in the genesis ofthe various changes occurring in the livers of rats sufferingdietary choline deficiency. In fact, our preliminary study oflivers of rats fed the CDAA diet indicated NF-kB to be activated from week 1, with multiple changes in mRNAexpression of cytokines detected at week 12, includingincrease for the IL-1�, IL-5, IL-6 and TNF-α genes as well asdecrease for the IL-1α gene. mRNA overexpression of theCOX2 gene and overproduction of COX2 protein are appar-

(% in

hibi

tion)

(% in

hibi

tion)

Figure 6 Inhibitory effects of (a) 1�-acetoxychavicol acetate(ACA)38 and (b) N,N�,-diphenyl-p-phenylenediamine (DPPD) on theof glutathione S-transferase placental form positive lesions, 8-hydroxydeoxyguanosine (8-OHdG) formation and 2-thiobarbituricacid-reacting substances (TBARS) generation (% inhibition). (a) (�)Lesion number, ( ) 8-OHdG, (�) TBARS. (b) ( ) Lesion size, ( )8-OHdG, (�) TBARS.


ent in the livers of rats fed the CDAA diet for 12 weeks, and itsenzymatic activity is also increased as determined byenhanced production of prostaglandin E2 (PGE2).39 Pro-nounced PGE2 production has also been shown for the CD case.107 Zeisel’s group and others demonstrated the par-ticipation of alteration of various factors regarding signaltransduction pathways in the mechanisms underlying thechanges that occurred in the livers of rats fed the CDdiet.76,81–83,104 In addition, despite a lack of clear evidence for acontribution of inducible nitric oxide synthase (iNOS) andreactive nitrogen species in rat liver carcinogenesis due todietary choline deficiency, they are also strongly suggested totake part because of the fact that ROS, IL-1�, TNF-α andIFN-γ all positively regulate iNOS induction via NF-kB activa-tion.102,108–110 Furthermore, iNOS and COX2 are coregu-lated,111 and nitrosative stress appears to be involved in avariety of carcinogenic processes.110,112 Moreover, in rats fedthe CD diet, levels of TGF-α, TGF-�1 and hepatocyte growthfactor (HGF) are increased, and numbers of hepatocellularsurface receptors for insulin and epidermal growth factor aredecreased, while those of glucagon receptor and c-Met forHGF are well maintained.61 In contrast to non-neoplastichepatocytes, cells in the CD diet-induced HCC demonstrateup-regulation of c-Met, and HGF is produced at high levels incells surrounding tumors,61 indicating a different profile of thesignal transduction systems between non-neoplastic andneoplastic hepatocytes. This might account, for instance, forthe increased telomerase activity in both preneoplastic liverlesions and HCC induced in rats fed the CDAA diet.113

DNA hypomethylation has been considered to play a sig-nificant role in hepatocarcinogenesis through acceleratedand prolonged cell proliferation by sustained switching-on of growth-related genes.86,87 Short-term feeding of a multiplemethyl group donor-deficient diet can cause irreversiblehypomethylation of genes like c-fos, c-myc and c-Ha-ras, andreversible overexpression of such genes.84,85 We have alsodemonstrated c-myc and c-Ha-ras overexpression in thelivers of rats fed the CDAA diet for 2 to 11 days but could notdetect hypomethylation of such genes on a whole liverbasis.114 Although the observed gene overexpression maysimply be secondary to the presence of regenerative cell pro-liferation, it might alternatively be explained by continuouspressure to hypomethylate specific genes for maintenance ofan active status in as yet non-neoplastic cells during the earlyperiod. The latter may be supported by the indication that theintervention of certain transcription factor(s) is suggested inthe hypomethylation and overexpression of the genes,87 andthat ROS are mediators of such phenomena.115 For instance,ROS and altered redox state activate the expression of geneslike c-fos, c-jun and c-myc transcriptionally using factors suchas NF-kB and activating protein-1 under the mediation ofvarious signaling pathways.115–121 In addition, substitution ofguanines at specific site of synthetic oligonucleotides with 8-

OHdG inhibits DNA methylation.122,123 As is suggested in thecase of signal transduction pathways in general, regulation ofgene hypomethylation and thereby overexpression may alsobehave differently in neoplastic cells induced in the long termin CD rat livers. To assess this possibility, detailed studies onliver preneoplastic lesions and HCC induced in rats fed theCDAA diet are now underway in our laboratory.

Taking the available data into consideration, possiblemechanisms underlying hepatocarcinogenesis in rats fed theCDAA diet can be speculated as illustrated in Fig. 7. Investi-gations are now required to test the included speculations.

RELEVANCE OF ENDOGENOUS LIVERCARCINOGENESIS IN RATS DUE TO DIETARY CHOLINE

DEFICIENCY TO THE HUMAN SITUATION

Serum choline deficiency is observed with various humanconditions (Table 3).80,124–130 Some of these are associatedwith liver functional and morphological disorders, such asfatty liver and cirrhosis.80,126,127 Furthermore, choline adminis-

Figure 7 Schematic illustration of possible mechanisms underlyinghepatocarcinogenesis in the livers of rats fed the choline-deficient, L-amino acid-defined (CDAA) diet. VLDL, very low-densitylipoprotein; HCC, hepatocellular carinoma.

1038 D. Nakae

tration exerts therapeutic effects.125,127 While the relevance ofcholine deficiency to the development of human HCC isobscure, there is some potential for involvement.

CONCLUSIONS

Chronic viral hepatitis is one of the major factors for humanHCC development, but there are cases of liver tumors arisingin the absence of viral infection or apparent carcinogen expo-sure.131,132 Not all human malignancies can be explained by preceding encounters with carcinogens in the environ-ment.3–5 Furthermore, endogenous factors may be majorcontributors to carcinogenic processes triggered by exoge-nous stimuli.133 Endogenous mechanisms of carcinogenesisare thus critical and need to be clarified as a high priority.Because of the limited information on this type of carcino-genesis when compared with that induced by chemical car-cinogens, it is difficult to make conclusive statements on itsmechanisms. Nevertheless, the results of investigation of rat hepatocarcinogenesis due to dietary choline deficiencyprovide very good basis to build a more complete understanding.

ACKNOWLEDGMENTS

I would like to express my gratitude to Professor YoichiKonishi (Department of Oncological Pathology, CancerCenter, Nara Medical University), for his dedicated supervi-sion. I also wish to thank the faculty members of the Depart-ment of Oncological Pathology, Drs Ayumi Denda, MasahiroTsutsumi and Toshifumi Tsujiuchi, and other associated indi-viduals for their contributions to the work described in thispaper, scientific and technical assistance, and approval touse their data. Similarly, the results described here arelargely the fruit of a collaborative effort with generous supplyof important materials and helpful discussion from numerousinvestigators. For these and also kind permission for me to cite preliminary data, I would like to especially thank Drs

Robert A. Floyd, Kenneth L. Hensley, Yashige Kotake (Okla-homa Medical Research Foundation), Akira Murakami (KinkiUniversity), Benito Lombardi (University of Pittsburgh),Masami Muramatsu (Saitama Medical School), MinakoNagao (Tokyo University of Agriculture), Hajime Ohigashi(Kyoto University), Regina M. Santella (Columbia University),Toshihiro Sugiyama (Akita University), Toshiya Suzuki(Saitama Medical School), and Keiji Wakabayashi (NationalCancer Center Research Institute). The research was sup-ported by a variety of grants from the Ministry of Health andWelfare and the Ministry of Education, Science, Sports andCulture of Japan.

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endogenous liver carcinogenesis in the rat *

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