Growth stimulation following serum transfer from carcinogen-treated donors to normal rats: a new aspect of early carcinogen actions

Exp. Path., Ed. HI, S. 2;)

;\;-) (1\)78)

Friedrich-Schiller-University J ena, Institute of Pathology (Head: Prof. Dr. sc. med. F. BOLCK)

Gr owth stimulation following serum transfer from carcinogen-treated donors to normal rats: a new aspect of early carcinogen actions') By M. DANZ, H. URBAN, R. BRAUER and A. SCHMIDT Wit h 2 figures Received February 24, 1978) Address: Dr. med. M. DANZ, Pathologisches Institut der Friedrich-Schiller-Universitat, DDR· 69 Jena, Ziegelmuhlenweg 1

Key words: Carcinogen; growth inhibition; promoting activity; serum transfer; humoral stimuIation; cell division; liver; adrenal cortex; thymocytes

Summary Especially in their early phase of action carcinogens are strong inhibitors of cell proliferation' This is an apparent contradiction to the promoting activity of oncogens in the process of carcinogenesis. Because of obvious similarities between restorative and neoplastic growth processes on th e tissue level we have studied the possibility, whether such similarities do exist also with regard to stimulatory activities in the serum. To overcome the non-specific inhibitory effect of a carcinogen, the serum of 2-acetylaminofluorene (A AF) treated male Sprague-Dawley rats was transferred to normal recipients. The results demonstrate that the proliferation of the same tissues (hepatocytes, adrenocortical cells, thymocytes) as in liver regenerating animals were stimulated by the serum of the carcinogen-treated rats. Whether the observed short-term effect corresponds to the so-called promoting activity of the carcinogen(s) is discussed. Nature and origin of the humoral stimulator(s) are still unknown. Mainly from experimental studies in amphibians R. T. PREHN (1971) in an interesting paper concludes: "Admittedly, there are many differences between the blastema of regeneration and a neoplasm. However, as I have pointed out, some of these are not so great as might seem to be the case at first consideration. Since the neoplasm is pathologic, it may be wiser to discount the differ ences and emphasize the similarities." There is some evidence that such similarities do exist also in regeneration and carcinogenesis of mammalian tissues. In addition to the same biotechnical realization by enhanced as well as most accelerated cell proliferation in both regeneration and carcinogenesis comparable changes occur on the tissue level. Among them a prominent event is the reduced production of growth inhibitory substances (chalones), which are cosidered to be of general importance in local growth regulation (for lit. see BULLOUGH 1975). In the case of stimulated growth the liver is one of the most investigated tissues. Hepatocyte inhibitory factors were found in the liver itself (NADAL et al. 1976) as well as in the serum (ONDA and YOSHIKAWA 1973, NADAL 1975, ONDA 1977). Another phenomenon associated with stimulated cell division is a loss of functional capacity in restorative (WILSON and SPELSBERG 1976, for further lit. see BUCHER 1963) as well as in preneoplastic (SOLT and FARBER 1976) and neoplastic growth (VASILIEV and GUELSTEIN 1967, ABUL-HAJJ and MORRIS 1977). Also the appearance of alpha-fetoprotein is independent of the quality of the growth process, whether it is embryonic, restorative or neoplastic (SELL et al. 1976, SMUCKLER et al. 1976). Furthermore, with regard to the inhibitory substances already a single dose of a hepatocarcinogen is able 1) Dedicated to Prof. Dr. se. med. F. BOLCK on the occasion of his 60th birthday.

23

t o pro duce an early mirmcry of liver cell gro wt h (VOLM et al. 1969), alth ough espe cially carcinoge ns are effect ive inhi bitors of n ormal cell multiplication during their ac ute act ion (CIHAK and R ABES 1974, CRADDOCK 1976). Only during pre neop lastic lesions the re is a pr oliferati ve advantage of the initiate d cells over their norm al pr ogenitors ( KITAGAWA 1971, SOLT and FARBER 1976). But , t hese initiated cells h ave n ot yet acquire d pr operties id enti cal with the so-called " a uto nomy " (SOLT and :FARBER 1976) as was sho wn by the differ ent behaviour in the case of cult uring in vit ro (R ABES et al. 1970, 1972). Furthermore, preneoplasti c cells can b e stim ulated to gro w in a suit able environment, e.g. by partial h epatectom y (RABES et al. 1970, KITAGAWA 1971, SOLT and FARBE R 1976). At pr esent it is n ot clarified, what mak es the initiat ed cells divid e still b efore neoplasia is r ealiz ed and, whether it depends up on n ewly ac quire d propertie s only. Is it a mere lack in act ivat ing car cinogen s whi ch n orm al cells ar e ab le to do and by wha t they ar e inhibit ed (SOLT et al. 1976)? Th e latter could be a ma in ca use of preventi ng t he pr om ot or ac tivity of carci noge ns up t o a relatively long lasting peri od. Assuming t hat carcinoge ns " affect selecti vely the same cell structures t hat are esse ntia l for growth cont rol" (VASILIEV and GUE I,STEIN 1967) a growth resp onse cou ld be expecte d at the orga nismic level, t oo. Thi s was t he crit ical p oint whi ch induced our pr esent experiments. Our in te rest is focu sed on t he qu esti on, whether a single dose of a carcin ogen provokes gro wt h stimulat ory activity as r eported in the case of partial hepatectomy (FRIEDRICH-FREKSA and ZAKI 1954, WBRA and RABES 1967, RUTZKY et al. 1971). To overcome the inh ibition of cell proliferation in carcinogen-treat ed rats, we have transferred their serum t o n orm al recipients .

Materials and methods 1. Ani mals a n d t reat me nt Random-bred male Sprague-Dawley rats were maintain ed on sta ndard diet (pellets) and wa ter a d libitu m. They were housed 4 to 6 per cage at least 2 weeks before starting the experiment. 25 animals aged about three months received once by stomach t ube 120 mg 2-acetylaminofluo rene (AAF) per kg b.w. suspended in 2.5 ml sunflower oil. The treat ment was performed in slight e t her anesthesia at 11 p.m. The blood of th ese animals was obtained by cutting up the right carotide artery. In this way 9 animals in each case were killed 9 hrs. p. appI. and 14 hrs. p. appI., 7 animals 24 hrs. p. appI. Eight rats (11 weeks old) served as untreated cont rols. They were killed at 11 a.m, The bloo d of each group was pooled and stored overnight at 4 °C. Thereafter it was centrifuged and the serum st erilized by means of millipore filter s. The cr ude serum of the AAF-tr eat ed donors (groups D I, II and III) as well as th at of control donors (group DC) was transferred intraperit oneally (1.5 ml per 200 g h.w.) to normal recipients aged 9 to 10 weeks. All recipients were killed in slight ether anesthesia by decapitation 28 (2 p.m.) and 48 (10 a. m.) hours, respectively, following serum injection (at 10 a.m.). These time inter vals were chosen because of the results of the proliferati ve activity of the tissues investigated following partial h epat ect omy in animals of th e same strain. These results will be reported elsewhere.

2. Counting of th e mi t os es Tissueswereremoved immediat ely after death, fixed in Bouin's solution for 24 hours and embedded in paraffin. Counting of th e mitoses was performed in HE-stained sections of identical areas of the right anterior lobe of th e liver, in the equatorial zone of th e adrenals, in the upper esophagus and in the upper jejunum. The mode of evaluation of the adrenocortical mitoses was described earlier (AMLACHEIl et aI. 1974). The mitotic index of the livers refers to 10,000 nuclei and that of the jejunum t o 2,500 nuclei. In th e latter case only longitudinal cuts through the whole crypts were taken into consideration. In th e esophagus we determined the average mitoti c number of three cross-sections .per animal. 3. In v it r o st im ula ti on of t hy mocy te s To test t he effect of the seru m on th e DNA synthesis of lymphoid cells cult ured ill vitro, th e th ymus of a male rat (3 months old) was gently disintegrat ed in RPMI 1640 medium (GIBCO). The tissue was passed through nylon gaze and the th ymocytes were washed thr ee tim es in medium and th en resuspended in RPMI 1640 medium containing 350 mg/ml sodium bicarbonate, 10 mM: HEP ES buffer, 2 m ~I gluta mine, 100 U/ml penicillin, 100 " g/ml streptomycin, and 20 % t est ser um at a density of 2.0 x 106 viable cellsj rnl, Thymocyt es were cult ured in plastic tubes with lid in v ol of 0.2 ml and incubated for 24 h at 37°C. For the last 18 h th e cells were radiolabeled with 0.51lCi 3H-thy midine per t ube (UVVVR Prague). 0.1 ml aliquots of the cell cult ures were pipet ted on

24

filterpaper disks U.4 cm diameter) and precipitated in the cold with 10 % trichloracetic acid (TeA) for 30 minutes. After washing with 5% TeA (three times), ethanol, ethanol-ether (3: 1), and ether, the dried disks were counted with a toluene scintillator (4 g PPOjl, 0.2 g POPOPjl) in a Packard liquid scintillation counter. 4. Biostatistical evaluation All data given in the figures are expressed as means ± S.D. The intergroup values were compared using the parameterfree MANN-WHITNEY u-test. The p-values represent the one-sided level of error.

Results

1. Organ weight of livers and adrenals The liver wet weight of the AAF-treated grou ps D I and D II is within the range of normal rats of comparable age and body weight (b. w.), This agrees with the results obtained in former experiments 36 and 48 hours p. appl., respectively (DANZ et al. 1977). Only in group D II (14 hours p. appl.), the liver weight was somewhat smaller (p = 5 'Yo) than in D I and D III, although the b. w. did not differ significantly. The absolute adrenal weight was similar in the three carcinogen treated groups. In the recipients the liver weights are different in some cases. Compared to the control donors (DC) and control recipients (RC 1 and RC 2) an elevation (p = 1 %) was detected 48 hours and 28 hours p. inject. of D land D II-serum, respectively. The observed increase 48 hours following D III-serum was not significant. This shows, that the serum of carcinogen-treated animals affects the liver weight in recipients depending upon the time of serum gain and duration of its influence. In the adrenal weight there was the only difference between the two time groups of R II (p = 2 'Yo), but each of them was not different from that of control donors and that of the other carcinogen-serum injected rats. The forementioned different adrenal weight 28 hours and 48 hours after D II-serum parallels the finding in liver weight of these groups. 2. Histological examination In various experiments in male Sprague-Dawley rats different doses of AAF (50, 60, 100, 150, or 200 mg/kg b. w.) did not produce liver cell necroses following single oral application. But, analogous to the more toxic N-hydroxy-derivative which causes periportal liver cell death (THORGEIRSSON et al. 1977), in the present study a very mild inflammatory response in the portal fields occurs 24 hours p. appl. Beside this there were also a slight loss of basophilic material and a mild edematous alteration of the hepatocytes in the peripheral zones of the liver lobules. In the other organs (adrenals, jejunum, esophagus) we could not detect any deviations from normal by light microscopy. The same applies to all organs examined in the serum recipients. 3. Mitotic count In each tissue investigated we have registered all pro-, meta-, ana- and telophases. The results are summarized in fig. 1. Liver Following AAF-treatment the hepatic mitotic index declines significantly from the first to the third point of time of investigation (p < 1 'Yo). The mitotic number 9 hours p. appl. is already lower than in the untreated controls (p = 1 'Yo). This difference could be caused partially by differences in age and b. w. between the treated rats and normal controls. Nevertheless, an early inhibitory effect of the carcinogen is probable, since the sequential slope down of the mitoses is evidenced to be a non-specific effect in various other studies (cp. GRISHAM 1974, JACKSON and IRVING 1973, CIHAK and RABES 1974). The situation in the recipients is quite different. Whereas the control serum does not affect the mitotic index of the recipients (RC 1 and RC 2) in any way, a differential action occurs in the carcinogen-serum recipients. The hepatocyte stimulation 28 hours after the injection of the D l-serum is most striking. The mitotic index of these animals is elevated in comparison

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influence of a single dose of 120 mg AAF/kg b.w. on the mitotic number of various tissues in male Sprague-Dawley rats as well as serum following transfer to normal recipients. The shadded areas within the columns of the adrenal cortex represent the mitotic number glomerlliosa. [Statistical evaluation was done by means of the parameterfree MANN-WHITNEY u-test. All values (columns) indicate S. D. Only between the adjacent groups the level of error was given (p in per cent); for further information see text (results).

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t o all oth er groups in cludin g cont rols (p < 1 % t o P < 5 %). A further difference exists betwe en the 28 hours mit ot ic number following seru m D II and D III (p < 5 %). Wh eth er the latter indicates a second peak of st imulato ry activity which ma y result fr om a more prolon ged carcinogenic influ ence is t o be te sted. Th e pr esent results are not conclusive, becau se the 28 hour s-group in R HI eve n exceeds th e pret end ed level of error (p = 5 %) compare d t o RC 1 and nc 2. Adrenal s Th e results given in fig. 1 were taken partially from DANZ and BOLCK (in pr ep.). Like th e mit oti c behaviour in th e liv er also thi s epithelial ti ssue signals st imulat ory fa ct ors transferable with seru m obtain ed fr om AAF -t reat ed rats. But, the differences between adrenals and livers ar e obvious (see fig. 1). The mit oti c decrea se in the adrenals of the carcinogentreated donors is restrict ed to D II, i.e. 14 hours p. appl. Thereafter the values return t o th e level found in compara ble unt reat ed cont rols (8 ± 2; DANz et al. 1977). In th e re cipients the ser um of cont rol donor s is ineffecti ve regarding the mitotic number. Th erefore, a handling-induced effect should be exclud ed. An entire other response results from carcinogen-serum in R I , R II and R III. The most st imulat or y activity appears in th e serum 9 hours p. app\. of AAF, since the enhanced cell division is continued up t o 48 hours p. inject. of this seru m. The value at this tim e is significant ly larger than th e analogous one of R III (p < 0 %) and those of RC 2 as well as of DC, but the latter do not differ significantl y from each oth er. 48 hours after D 11- and D III-serum the stimulati on ha s disappeared. We hav e also exa mined, wheth er t he prolif eration of th e tw o out er zones of the adre nal cortex behaves alik e. Th e shadded ar eas at th e base of t he columns (fig. 1, 2nd diagr am ) demonstrat e the av era ge of t he absolute mit oti c number in the zona glomerulosa. KIRIJ,LOV (1973) reported, that t he mit oti c ind ex is relatively higher in thi s zone during the second half of t he da y. Alt hough t here are no significant differences betwe en the AAF -trea te d grou ps, an appropriate t end ency ma y be seen t oward t he night hours. But, alr ead y in th e early afte rn oon in the cont rol recipient s (RC 1) one can observe an enhancement compare d t o th e values at 10 a. m. two days p, inj ect. of th e cont rol serum (p = 3 'Yo) as well as t o th ose in DC (p < 1 '/,;) , which were killed at 11 a.m. It is less likely, that these differences ar e brou gh t ab out by the treatm ent it self. If thi s would be t he case, one should expect a similar sit uation in all carcinogen -seru m re cipient s, t oo. However, thi s is evid ent (p = 1 %) only in th e group R II receivi ng t he seru m obtained 14 hours (at 1 p.m. ) following AAF administ rat ion. Ex cept t he 28 hour s-group of R III, the glomerulosa mit oses of the an alogous subgroup in R II ar e significa ntly enlarged in compar ison with all ot her re cipients of carcinogen-serum. The cause of th ese differential effects remains open. The tw o subgroups in R II resemble the controls ( RC 1 and R C 2). On th e ot her hand, there is an inverse relati onship to the mitotic ind ex of th e hepatocytes, which is in creased at the time of lowered mit oses in the zona glom erul osa. PAYET and ISLER (1976) have reported, that growth hormone stimulates the zona glornerulosa cells. Therefore, it is possible that the handling (st omach tube in ether an esth esia) lead s to a delayed circadian in crease of the blood level of growt h hormone in D I by an act iva t ion of the pituitar y-adren al axis, which ma y be reflecte d by a dimini shed cell division in the out er adr enal la yer. Aft erwards the sit uat ion is widely normalized. Summarizing t hese findin gs we must emphasize, that th e stimulatory action of the carcinogen-seru m is not distributed throughout the adrenal cortex, but it is strikingly limited t o oute r and middl e la yers of t he zona fasciculata. E s opha gu s e p i t h el i u ru In the third gr oup of columns in fig. 1 the number of dividing ba sal cells is r epre sented. Th e mean ± S. D. is calculated fr om t he average number of each three sect ions per animal.

28

In the AAF-treated rats the mitoses decline continously to a minimum 24 hours p. appl. at 11 p. m., corresponding to normal diurnal changes. This is in agreement with the results obtained from other experiments of the same strain (unpublished). Comparisons of the groups investigated in the morning hours - i.e. D I, DC, 48 hours p. inject. of both, carcinogen- and control-serum - reveal identical values. This is paralleled by the similar proportion between D II and those rats, which received serum and were killed after 28 hours in the early afternoon at 1 p. m. (D II) and 2 p. m. (recipients), respectively. That the mitoses in the early afternoon are significantly exceeded in number by those of the ante meridiem killed rats is certainly set up by circardian differences. They consist in a rise of the esophageal cell division during the morning hours followed by a steady decrease in the afternoon to a minimum at about midnight (for lit. see BULLOUGH 1965). Therefore, the results appear to be independend of the actual treatment. The cause of the only difference (p < 2 %) between D I and the 48-hours-group of R I cannot be explained. However, the slight, but not significant difference to the mitoses in all other recipients as well as in DC, which were examined in the morning hours, could also refer to a diurnal increase occurring between 8 a. m. and 10 a. m. or 11 a. m. Apart from the different experimental arrangement, the results by LIOSNER and MARKELOVA (1975) in mice admit a, remarkable change of the mitotic index of esophageal epithelia within two hours. From these results one can conclude, that neither AAF inhibits the epithelial cell division of the upper esophagus, nor are these cells stimulated to divide by serum transferred from carcinogentreated to normal rats. Jejunal crypts AAF is known to be a very strong hepatocarcinogen opposite to its very weak carcinogenicity in the small bowel (MILLER et al. 1958). By the route of administration used (stomach tube) one has to expect direct toxic influences, but they are not pointed out in form of cell death. In the literature available we could not find experimental studies dealing with the metabolic activation of AAF in the intestine. Since we observed a slight decline in mitotic number following three doses once daily of 50 mg/kg b. w. (paper in preparation) the lowered mitoses also in the present experiment may indicate a direct toxic alteration by the parent substance or by metabolites excreted in the bile (IRVING et al. 1967). The counting of the mitoses in this tissue we have performed only 28 hours p. inject. of the serum. Fig. 1 shows unequivocally the serum of both carcinogen-treated and control rats to be without influence upon the mitotic index of the jejunal crypts. There are no significant differences between all serum recipients and control donors. In vitro stimulation of thymocytes Experimental investigations in partially hepatectomized animals have shown that their serum contains factors able to stimulate lymphocytes in vivo and in vitro (fee SAKAI and KOUNTZ, 1975). Therefore, we have examined, whether such factors emerge also in the serum of animals treated with a hepatocarcinogen. The results of the in vitro effect of the carcinogen-serum on thymocytes are represented in fig. 2. Depending upon the duration of the AAF-influence in the SErum donors, the stimulatory activity of the serum on thymocytes varies. The 24 hours serum is very effective concerning the incorporation of 3H-thymidine. A small but not significant increase is already produced by the serum 14 hours p. appl. of AAF (group D II) compared with the controls, but it is significantly different from that of the serum obtained 9 hours p. appl. It is striking, that the last mentioned serum opposite to the other ones has not any stimulatory effect. Although not significant in the present experiment, lowered values were observed repeatedly. In an analogous investigation of spleen cells under the same conditions no stimulation was found. But, in some cases the effect of the 9 hours-serum was similar to that in thymocytes. That the other two carcinogensera fail to stimulate spleen cells may be due to a larger heterogenicity of this cell population.

29

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Discussion As postulated in th e two-stage theory by BERENBLUM and SHUBIK (1947 ; for further lit. see PITOT 1977), carcinogens are known to be growth promotors beside their initiating action especially in th e so-called target tissues. In view of thi s, the effective inhibition of cell proliferation in the early phases of carcinogen action appears to be a contradiction at first glance. This inhibitory effect is not restricted to the target tissues, though pronoun ced there ("MARQUARDT and PIlILlPS 1970, KITAGAWA 1971, CRA DDOCK 1976). Furthermore, the inhibiti on is not specific and occurs also after other toxi c substances, antimetabolites, cyt ostatics and stress hormones (GRISHAM 1974, CIHAK and RABES 1974, MOOLTEN et al. 1970, HENDERSON and LOEB 1974), alth ough th e causativ e mechanisms may be different in each case. Under th e conditions of repeated small carcinogen doses the increasing proliferation of initiated cells is delayed for a more or less long period and emerges first ly in advanced stages within the preneoplast ic lesions (KITAGAWA 1971, RABES et al, 1972). Up to now it is not yet enlightened, in which manner in the case of enhanced cell multi plication th e event s on th e tissue level - e.g. the loss of functional capacity and of growth inhibitory substances - ar e linked with each other as well as with hu moral stimulators. Their existence and relevance are clearly evidenced in th e process of liver regeneration (FRIEDRICH-FREKSA and ZAKI 1954, WRBA and RABES 1967, VIROLAINEN 1967, MORLIW and KINGDOM 19n). The discussed findings and other data from the literature indicate that in any way changed (enhanced) cell proliferation is regulat ed both by a loss of inhibitory substances and by an active " positive" regulatin g principle. It appears very probable, th at the " switching on" of th e growt h process is st arted on the level of that tissue which was induced to grow, r estorativel y as well as neoplastically. That the proliferative response in regenerating livers is paralleled by a decreased cell renewal in various other tissues (VIROLAINEN 1967, STO CKER et al, 1969) may account for the tissue specificity of th e actual stimulat or (WRBA and RAB ES 1967, OGAWA and NOWINSKI 1958). But , it was shown in other experiments, th at lymph ocytes (KULBERGand EVNIN 1972, SAKAI and KOUNTZ 1975)

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and adrenocortical cells (DAC\'Z and BAl'MBACH 1972, and unpublished data) are induced to grow after partial hepatectomy. The present results indicate that beside the hepatocytes these tissues are stimulated also in the recipients by the serum of AAF-treated animals, although the different behaviour of the stimulated tissues in the carcinogen-serum recipients is obvious. The question arises, whether this is brought about by the emergence of qualitatively different stimulators or by a different sensitivity to both, stimulators and perhabs small amounts of circulating AAF-metaholites which could have heen transferred with the serum. A mitotic increase of the liver cells is only seen as a consequence of serum obtained 9 hours p. appl. of AAF. 'I'his means, that the hepatocyte stimulatory activity is already present in a phase which is followed by a mitotic decline in the AAF-treated animals (see fig. 1, liver). Thereafter the stimulation disappears or the hepatocytes become refractory by transferred carcinogenic metabolites with the serum. However, in contrast to the mitotic inhibition in the livers of the carcinogen-treated rats there is no comparable effect in the carcinogen-serum recipients. Also by reason of the findings of DECKERS et al. (1968) it is less probable, that metabolites of the carcinogen prevent the stimulatory action of the serum. Another explanation may results from the investigations of VOLM et aI. (196\)) and W AYSS et al. (1973). They found, that a single dose of hepatocarcinogens (diethylnitrosamine or AAF) produce a transient loss of liver "chalones" preferentially during the first 12 hours p. appI. If this event would be the trigger of the humoral stimulator, then its production or release into the blood stream, respectively, could be stopped together with the reappearranee of the growth inhibitors as may be reflected by the normalized mitotic index in the recipients of the later carcinogen-sera (groups R II and RIll). The behaviour of the liver and the adrenal cortex is different in two ways. Firstly, in agreement with former results (DAXZ et al. 1(76) we observed that AAF fails to inhibit adrenal cell division, but the mitotic number is elevated and outlives 48 hours in AAFtreated animals. The mitotic decline 14 hours after AAF in the present experiment is certainly caused by thehandling of the animals (DANZ and BOLeK, in prep.). Secondly, the stimulatory activity of the carcinogen-serum on the adrenocortical cell" is preserved in each group of the AAF-treated donors. If one sums up the time intervals of AAF-exposure of the rats and that to the carcinogen-serum of the recipients, a similar period of increased mitotic number results as shown in carcinogen-trated animals themselves (DANZ et aI. 1(76), i.e. in the case of R I 48 plus 9 hours and in the case of R III 24 plus 28 hours. From this we can conclude that the causative mechanism in stimulating the adrenocortical cells may be the same, both in AAF-treated rats and in normal recipients of their serum. Thus, a stimulation due to carcinogenic metabolites can scarcely be assumed, since 50 mg/kg b. w. were found to be the minimal dose able to provoke the adrenocortical response. Furthermore, this would imply that at any given moment an adequate concentration of metabolites had to be contained in the transferred serum amount, which is highly improbable. In the given context it is noteworthy, that carcinogens of various chemical structure behave like AAF in respect to the proliferation of the adrenal cortex (the results will be summarized in a further paper). Also in these cases one should expect the adrenocortical response to be a phenomenon associated with the growth induction anywhere in the organism and, therefore, independent of the histiotropism of a given substance. The advantage of the adrenal epithelia seems to be that the sensitivity to such humoral stimulation prevails over the inhibitory action of carcinogenic metabolites, if the latter occurs in the adrenals at all. For that reason this tissue could serve as a useful indicator for potential growth events, too. This must be emphasized insofar, as by the AAF-dose applied no real loss of liver tissue and, therefore, no reparative proliferation was induced in the carcinogen-treated animals. Thereby the situation is entirely different from that in partially hepatectomized rats and can justify the term "potential growth event". The question remains open, why the mitotic number is still enhanced 48 hours alone after D I-serum. The third cell type are thymoeytes which are stimulated by carcinogen-serum. A similar effect was demonstrated by post-hepatectomy-serum on antibody-forming spleen cells

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(SAKAI et al. 1970, K"ULBERG and EVNIN 1972), on lymphocytes from lymphnodes in vitro (SAKAI and KOUNTZ 1975) as well as on lymphocytes of various lymphoid tissues in vivo following partial hepatectomy (CRADDOCK et aI.1964). Whereas the lymphocyte transforming activity of the serum in liver regenerating animals appears already during the first postoperative hours (SAKAI and KOUNTZ 1975), we found it only in the serum of those rats which were exposed to AAF 24 hours. The delay in comparison with the proliferative response in the livers and in the adrenals is surprising in the same way as the lowered 3H-thymidine incorporation following the H-hours serum. The latter could be due to a still enhanced serum level of corticosteroids, which are known to be inhibitors of thymocyte growth (cp. MUNCK and LEUNG 1977). If this would be the case one should also expect a similar effect in the livers. However, the hepatocytes are stimulated by the same serum. Carcinogenic metabolites or the parent substance should be excluded as discussed above and, because only large doses of AAF in the medium had a toxic effect on the thymocytes (data not shown). An immunosuppressive action by alpha-fetoprotein is impossible because of the time intervals investigated (SMUCKLER et al. 1976). The temporal graduation of the stimulatory activities in the serum on liver, adrenal cortex and thymocytes necessitates the question for the nature of these humoral principles. It is far from proven, that they are identical in these tissues, but it is probable, that they resemble those emerging in liver regenerating animals. The modifying role of the anterior pituitary gland and of growth hormone, respectively, in liver regeneration has suggested that STH may be the first candidate for the humoral stimulation (RABES 1967, ECHAVE LLANOS et al. 1971, MOOLTEN et al. 1970). This is supported by the fact, that the growth of all three tissues (liver, adrenal cortex, thymocytes) can be stimulated by STH (ECHAVE LLANOS et al. 1971, CATER and STACK-DUNNE 1955, cp. HELPAI' et al. 1975). Except from the adrenal mitotic value after 48 hours in group R I, the number of mitoses is elevated only 28 hours after the carcinogen-serum. The enhanced 3H-thymidine incorporation into thymocytes is also restricted to 24 hours and has disappeared after two days. This speaks in favour of a short-term pulselike effect and perhaps a brief half life period of the stimulating activity as evidenced in the case of growth hormone (STROSSER and MIALHE 1(75). However, in the case of liver regeneration the possible stimulatory role of STH is doubtful, since its serum level declines postoperatively up to the third day (MORLEY et al. 1(75) and, because the serum level "reflects the rate of hormonal secretion of the pituitary gland" (STROSSER and MIALHE 1(75). In this respect further hints are provided by the present results. The thymocytes were stimulated for DNA-synthesis only by the carcinogen-serum obtained at about midnight. At this time the thymus DNA-synthesis (MISUROVA et al. 1977) and serum growth hormone (SIMON and GEORGE 1975) show low diurnal values. Vice versa the control donors (DC) are characterized by maximal values of these parameters, nevertheless the serum is ineffective in the control recipients at all. To return to our initial question, according to PREHN'S (1971) postulation, we can conclude, that similarities between restorative and the very early phase of neoplastic development may exist also on the level of humoral stimulation. Possibly the presumed gap between early growth inhibition and the well known promoting activity of chemical carcinogens may be closed partially. In this case "partially" must be emphasized, because we do not know, whether this phenomenon is still observable following continued carcinogenic influences and whether it is brought about by other carcinogens, too. In view of the two-stage theory, which is applicable also in liver carcinogenesis (FARBER et al. 1977, PITOT 1977), this should be expected. Apart from the initiating carcinogenic alterations within the target cells, a promoting activity is needed to eventuate the process of carcinogenesis in malignant neoplastic growth via progression (FOULDS 1969, TEEBOR and BECKER 1(71). It is suitable that also the promoting component of a hepatocarcinogenic action can be replaced by some non-carcinogenic agents (POUND et al. 1973, cp. PITOT 1(77) as well as by other hepatocarcinogens (syncarcinogenesis). In this connection the recent results by SOLT et al. (1976) are very instructive. The authors found, that following a severe liver damage by diethylnitrosamine small daily doses of AAF in combination with a partial hepatectomy lead to

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the appearance of basophilic foci already after four weeks. Because of the different zonal action of these two carcinogens in the liver lobules an interpretation of the findings by SOLT et al. (1976) might be possible in the following way. As a result of the reparative response to the nitrosamine intoxication a pool of hepatocytes develops which is able to divide, especially in the damaged central zones of the lobules. Thereafter, the preferentially peripheral action of AAF may trigger off a humoral stimulation which is strengthened by the additional hepatectomy. Under these conditions the initiated cells could be restricted in their ability to metabolize AAF and thereby become refractory to its inhibitory action. On the other side this is a prerequisite to the humoral stimulation to become effective in dividing of the initiated hepatocytes by diethylnitrosamine. This may correspond to the diminished capacity of hepatocytes - in only liver regenerating animals - to metabolize dimethylnitrosamine (LAWSON 1977). Referring to the findings of SOLT et al. (1976), FARBER et al. (1977) postulate in their working hypothesis: "Chemical carcinogens do not seem to induce cancer in the liver, but rather trigger a chain reaction which ultimately may eventuate in the development of cancer." One of these events can also consist in the induction of humoral stimulators on the organismic level, which was the starting point of the present experiments.

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