Meeting Proceedings 871

18 HYPERPLASTIC ALTERATIONS IN THE LIVERS OF RATS WITH A CONGENITAL PORTACAVAL SHUNT

F.-J. Vonnahme, L. Dubmsson* and M Grtint

Patholog~sches lnst~tut der Umversltat des Saarlandes. 6650 Homburg-Saar, Federal Repubhc of Germany, *Unlversltb de Bordeaux II, Bordeaux, France, and ~'St Vincent Krankenhaus, Malnz. Federal Republic of

Germany

Hyperplast~c focal and nodular alterations have been reported to occur m the hvers of rats subjected to portacaval anastomosis The dwers~on of the portal blood also leads to hver atrophy However, other authors could not find any hyperplast~c alterations m the hvers of rats after an experimental shunt. Therefore, it seems of interest to report here that focal and nodular hyperplastic changes also occur in the hvers of rats with a congenital portacaval shunt in which the organ is naturally deprived of portal venous blood Our findings in these animals strongly indicate the existence of 'hepatotrophlc factors' in the portal blood These factors seem to be essential for the control of the regeneration and d~fferentmt~on of liver cells

19 SELECTION OF PHENOTYPIC VARIANTS OF RAT HEPATOCYTES DERIVED FROM N-NITROSODIETHYLAMINE-TREATED RATS

D Paul

InstltUt fur Toxlkologle der Unxvers~tat Hamburg, Federal Republic of Germany

[Abstract not received]

20 EMERGENCE OF A POPULATION OF SMALL DIPLOID HEPATOCYTES DURING HEPATOCARCINOGENESIS

P E. Schwarze, E P Pettersen, M C Shoalb and P O. Seglen

Norsk Hydro's Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, Oslo 3, Norway

Isolated hepatocytes from normal and carcinogen-treated rats were analysed for their DNA and protein content using flow cytometry For the carcinogen treatment, 4-wk-old rats were subjected to a partial hepatectomy and 24 hr later were Injected with N-nltrosodlethylamlne (50 mg/kg body weight) After 1 wk on an ordinary &et the rats were fed a &et containing 0.02% 2-acetylamlnofluorene for 4 wk. After another 3 wk the rats were used in experiments The treatment caused the appearance of focl and nodull (and ultimately cancer) in all the ammals tested, as verified by hlstochemlcal staining of foci and noduh for ~.,-glutamyltransferase (posmve) and adenosme-5'-tnphosphatase (negatwe). Hepatocytes isolated by the collagenase perfuslon techmque were stained for ~,-glutamyltransferase and 35°0 of the cells from carcinogen- treated livers were found to stain positively for the enzyme as compared to 5°o of the cells from normal liver Isolated hepatocytes were also analysed for their DNA content. About 85°o of the hepatocytes from a normal hver were found to be tetraplo~d, about 10°o diploid and the remainder octaplold In contrast, about 75°o of the hepatocytes from carcinogen-treated livers seemed to have a diploid genome Furthermore, the protein content d~stnbutlon of ~solated hepatocytes showed a pattern slmdar to the DNA content, suggesting that the diploid hepatocytes are small cells about half the s~ze of tetraploid cells Measuring isolated nuclei of ~solated nodules, we found that about 90°o of the nucle~ were diploid The results indicate that the carcinogen treatment leads to the outgrowth of a population of small &plold hepatocytes, among which the preneoplastlc cells are presumably to be found

21-26 A TRIPHASIC PROTOCOL FOR CHEMICALLY INITIATED HEPATOCARCINOGENESIS

M Roberfrold, M Lans. V Pr6at, H S Taper, J de Gerlache and K O. Vo

Umt6 de Blochlmle tOXlCOloglque et canc&ologlque, Umverslte Cathohque de Louvaln, 1200 Bruxelles, Belgmm

21 hmoductton (MR & JdG)

The complexity and mult~phasic nature of experimental carcinogenesis demonstrate that th~s process ~s not due to a single causative event Since the pioneering work of I Berenblum, it has been proposed that chemical carcinogenesis may be divided into at least two steps, which can be reduced by exposure to molecules hawng

872 Meeting Proceedings

different chmmcal or bmlog~cal propemes Howe~er the bmlogxcal modxficatmns relevant to these steps have not been estabhshed Our research m that field has used hepatocarcmogenests as a tool to characterize the different phases of the process of transformation leading to the appearance of mahgnant tumours

A mphastc protocol has been developed (abstr 22), consisting of an mxtmtxon phase induced by a single dose of a chemical carcinogen (N-mtrosodmthylamme. NDEA) followed 2 wk later by a selection procedure for cell prohferatxon adapted from that described by Solt and Farber (Nature, Lomt 1976. 263, 701), and then a third step consisting of some treatment or the admmxstratmn of an agent to promote the development of mahgnant tumours

Morphological and hxstochemxcal analyses of the liner at different stages of the process (abstr 24) indicate that the promoting treatment acts both on neoplastic lesions and on surrounding parenchyma The effects of tumour promoters can be demonstrated b) comparing their effects on initiated and non-mmated tissues

The kinetics and incidence of mahgnant tumour development m the tnphasxc protocol have been compared with those obsmved in other models used under the same experimental condltmns The systemic analysts of the results allov~s some conclusmns to be drav~n concerning the characteristics of the process (abstr 22)' an', two-step protocol combining mttmtmn and promotmn xs always less "potent', xn terms of the number of mahgnant tumours and the latency period, than a long chromc exposure to the same chemical carcinogen Moreover, the selecnve effect of a carcinogen could be distract both from pure ruination and from promotion

Hepatocarcmogenesls xs thus a complex and progresswe process, which causes a d~sruptlon m tissue homeostasis and whmh xs not due to a single causatwe e~ent The distinction between m~txatlon and promotion remains purely conJectural, since ~t does not correspond to well-estabhshed biological events

22 Descrtptum and ~ompalt.son it tttl other models (M L & JdG)

The tnphasxc protocol de~eloped for experimental hepatocarcmogenesls consists of (a) the administration of a single dose of a carcinogen INDEA) followed by' the feeding of a normal diet for 2 wk, (b) a modified Solt & Farber (loc ctt ) selectmn procedme m~olvmg 2-wk feeding of a diet containing 0 03°0 2-acetyl- ammofluorene (2AAF) plus, after wk 1. one single necrogemc dose of carbon tetrachlorlde, and (c) chromc admmxstratmn of a promoter, usualb phenobarbital IPB)

Whereas m the Solt & Farber protocol, the percentage of the hver parench~ma occupied by areas positive to 7-glutamyltransferase (TGT+) decreases shortly after the selectmn procedure, xt reaches up to 40°0 m the tnphasm protocol after PB treatment for only l wk Furthermore, the hver presents a pseudo-nodular aspect After 5 months of chromc promotion with PB, 70°,, of the treated rats alread~ have hepatocellular carcinomas, compared w~th 5-10°. m the bxphasxc protocol In this case, a latency period of 9 12 months xs reqmred for most of the rats to have mahgnant hepatm tumours

We have apphed the Teebor & Becker (Cam et Re~ 1971, 31, I) protocol (4 x 3 ~ k of 0 0Y',, 2 AAF feeding) and the Peralno et al (thtd 1971, 31, 1506~ protocol (3 wk of 0 03°0 2AAF feeding followed b3 chromc admmxstranon of 0 0500 PBI to the same strata of rats In the first case, mahgnant tumours developed w~thm 10 12 months m most rats In the second case, only 65°0 of the ammals were bearing mahgnant tumours 19 months after the beginning of the experiment

By analysing the kmetms of the appearance of mahgnant turnouts induced either b2, the trlphasm protocol or by prolonged administration of a carcinogen 12AAF) or b.~ the bxphaslc protocols 12a, AF plus PB or NDEA plus selectlon), It can be concluded that (at the tnphasm model xs the most effectwe, (b) the Solt & Farber protocol (NDEA plus selectmn) leads essentmlly to the same results as the chromc administration of 2AAF, and (c) the Solt & Farber protocol xs more effectwe than the bxphasm protocol using 2AAF plus PB

Inmatxon plus selection, as recommended by Solt & Farber, xs thus sufficient to reduce efficmntly a complete carcinogenic process, whmh can however stdl be accelerated (a shortening of the latent period) and amphfied (an increased number of mahgnant tumoursl b.~ promoting factors

23. Modulation o/ the promotum phaw (VP)

In the trlphasm protocol for hepatocarcmogenesls, different promoters ha',e been investigated PB gwen after initiation and selection both accelerates and ampl,fies the carcinogenic process. Such an effect reqmres at least 3 months of PB exposure to be both complete and irreversible DDT gwen at the same concentration (0 05°0 in the dmt) acts sxmdarl 3 Both promoters reduce the appearance of a large number of 7 G T + lesions which occup~ up to 40"0 of the hver parench,,ma Hepatocarclnomas appear as early as 6 months after initiation in up to 70°° of the rats

With regard to preneoplastlc lesions, BHT has an effect similar to that of PB or DDT but the number of rats bearing hepatocarclnoma(s) remains much lower, being equivalent to the non-promoted animals ( _+ 10",,) at 6 months

When the rats are also treated ~uth Nafenopm ~0 1"o in the dmt) following the lnmatmn and selection, few 7 G T + lesmns are obsm~ed up to 3 months Thmr relative importance is even lower than m the control (mirrored selected) ammals Surprisingly, hinderer, after 5 months of Nafenopln administration, 95"o of the treated rat~ have ~everal carcinomas pet h~et This finding indicates the need for great caunon m us,ng preneoplastm lesions as an absolute sign of hxer cancer promotion

A surgical techmque (portacaval anastomosis) apphed after mmatmn-selectxon has been found to be a good promoting treatment, since 6 months after mltmtmn most of the rats have hepatocarcmomas When the surgery' follows ruination alone, however, no mahgnant tumours ale obserxed 6 months later

Meeting Proceedings 873

24. Histological and hlstochemtcal alterattons m the bt- and trtphastc models (HST)

Histological pattern and hlstochem~cal alterations (activities of alkahne and acid DNAases and glucose-6- phosphatase as well as nucleic acid and glycogen contents) were compared m the early stages of the bl- and tnphas~c models of rat hepatocarcmogenesls The mare differences between these two models occurred 6-7 wk after NDEA lnltlauon In the blphaslc model, sparsely &strlbuted small focl of hepatocytes with coarse granular cytoplasmic basophiha and decreased activity of glucose-6-phosphatase could be seen In the tnphas~c model at the same stage, numerous very large areas of phenotyplcally altered hepatocytes were observed We called them 'pseudonodules' They contained considerably enlarged and rounded up hepato- cytes with abundant filamentous eoslnophlhc cytoplasm. They were distinctly demarcated from the sur- rounding hver parenchyma containing numerous "oval cells" Typical neoplastic markers were homogeneously d~strlbuted (acid and alkahne DNAase and glucose-6-phosphatase deficiency and glycogen accumulatmn)

From wk 17 after the NDEA administration, the real neoplastic nodules appeared, the histological and h~stochem~cal patterns being the same m both experimental models These nodules contained a heterogeneous populauon of hepatocytes (variable histological patterns and irregularly distributed neoplastic markers)

25 Modulatton o f the tmttatmg phase (JdG)

The question was raised as to whether two nltrosammes NDEA and N-mtrosomorphohne (NMOR) differ quanutat~vely and/or quahtatlvely m their capacity to initiate hepatocarcmogenes~s The two carcinogens were given at three &fferent doses as the lmtmtor m the tnphas~c model for hepatocarclnogenesls Early stages were analysed by identifying and quantifying the ) ,GT+ area of the liver parenchyma The percentage of the hver tissue occupied by such lesions increased w~th the dose of e~ther carcinogen, but was always higher after NDEA than after NMOR Thls parameter increased as a function of t~me, as a result of an increase in s~ze rather than m the number of loci and nodules H~stologlcally the early lesions were more or less the same, containing mainly eosmophfl~c cells In some cases, pseudonodular structures were also observed, mainly after the highest dose of e~ther mtrosamlne

In order of verify whether the dose of m~tlator influences not only the preneoplastlc stages but also the appearance of mahgnancy, the number of hepatocarclnomas will be registered after 6 and 9 months of PB promotion

26 Opttmahzatton and charactertzatlon o f the selection procedure (KOV)

The selection procedure, which is probably due to a selective toxic effect of 2AAF towards non-initiated cells, is the second step m the trlphaslc protocol In order to reduce the heterogeneity usually observed m the number and size of neoplasuc hepatic les~ons, this step has been carefully defined by modifying the protocol in various ways and studying m particular the influence of the duration or route of 2AAF administration

The second objective of th~s study was to try to replace 2AAF by another selective agent Neither NDEA (a strong carclnogemc initiator) nor paracetamol (a toxic but non-carcinogenic aromatic am~de) can replace 2AAF, as judged by the relative importance of TGT+ lesions after 1 wk of PB promotion On the other hand, a 3-wk treatment with 2AAF (0 03°o m the diet), even without any stimulus of prohferatlon, ~s sufficient to induce the appearance of ; ,GT+ focl

Finally ~t has been found that co-admlmstratlon of 3-methylcholanthrene w~th 2AAF completely inhibits the selective effect of the arylamlde

Taken together w~th demonstrations that the actlwtles of the 2AAF-metabohzmg enzymes are dramatically reduced m preneoplast~c nodules, these observations seem to m&cate that the peculiar property of 2AAF to act as a selecuve agent ~s related to the pattern of ~ts metabohsm m premahgnant lmtxated cells

27 MECHANISM OF PROMOTING EFFECTS OF PHENOBARBITAL AND PARTIAL HEPATECTOMY IN N-NITROSODIETHYLAMINE

HEPATOCARCINOGENESIS CELL KINETICS

H Barbason

Anatomle Pathologlque, Umverslte Libge, Belgmm

N-Nltrosodlethylamlne (NDEA) was fed to rats for 2, 4 and 6 wk After the cessation of NDEA treatment, the rats were subjected either to partial hepatectomy or to phenobarbital administration. Partial hepatectomy did not promote neoplastic transformation except after the 6-wk NDEA treatment Phenobarbital given continuously after the carcinogen administration promoted neoplastic transformation even after the sub- carclnogemc NDEA treatment (2 wk) In the different experimental models, the promoting effect was associated with the induction of a chromc cell prohferatlon, inhibition of the rapid response to the two-thirds hepatectomy and disruption of the mitotic c~rcadmn rhythms normally present during hver regeneration. It is concluded that the promotion mechanism could revolve &sturbmg the mitotic control In order to maintain, for a long time, a chronic low level of cell prohferatlon permitting the selective growth of preneoplastlc cells