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Serum alpha-fetoprotein in experimental hepatocellular carcinomas in rats
Europ. J. CancerVol. 13. pp. 295-301. Pergamon Press 1978. Printed in Great Britain
Serum Alpha-fetoprotein in Experimental Hepatocellular Carcinomas m Rats* N. R. GOPAL NAIDU,t B. K. AIKAT,t R. J. DASH~ and S. SEHGAL~ ~Department of Immunopathology, +Department of Endocrinology, Postgraduate Institute of Medical Education and Research, Chandigarh, India
Abstract-- The present study has beencarriedout mainly to study the sequentialchangesof serum alpha-fetoprotein (AFP) levels with the evolution o.1"experimental hepatocellular carcinoma (HCC) and ~ocorrelate with the histology of the tumors. Ratsfed with 0.06% of azodyes developedHCC by 8-24 months and on the basis of their capacity to synthesizeAFP, the animals can be divided into two distinct major group~ " qukk responders" and "slow responders". In thejqrsl group the animals developed tumors by 8-10 months and mostly of poorly differentiated type with high serum AFP levels. The slow respondersdevelopedtumors only after 16 months and they weremostly of well differentiated with comparatively low AFP values. Both the groups showed biphasic response of AFP during the evolution of HCC. The animals in aJlatoxin B1 group did not show AFP levels more than 2#g/ml.
The present investigation was undertaken to study the serum AFP levels using R I A during the evolution of liver cancer using different types of chemical carcinogens and to correlate the morphological type of lesions with AFP synthesis.
INTRODUCTION ALPHA-FETOPROTEIN is a feto specific serum globulin synthesised by fetal liver and secreted into the serum during embryonal life of mammalian species. Abelev et al. [1], for the first time reported the presence of AFP in blood of mice bearing transplantable hepatomas. Reappearance of fetal antigens is now recognised as common phenomenon during oncogenesis. Elevated levels of serum AFP during hepatic carcinogenesis has subsequently been reported in different species of animals including man [2, 3]. Besides its appearance in rats with chemically-induced H C C its early elevation has also been demonsLrated within weeks after the administration of" chemical carcinogens [4]. However, certain chemical carcinogens like aflatoxin induced '.HCC which are not associated with significant rise of AFP [5]. The techniqtle of agar gel immunodiffusion has been used fi)r the estimation of serum AFP, but the sensildvity is relatively low. Among the more sophisticated methods for the early detection of HCC, radioimmunoassay (RIA) happens to be most sensitive one. In fact the use of R I A has revealed that even normal individuals and animals have a minimum quantity of AFP in circulation [6].
MATERIAL A N D M E T H O D S Wistar strain rats of both sexes weighing 90-120 g were kept in groups of four in wire mesh cages and maintained on laboratory diet. The carcinogens used were as follows: Batch 1 : ( 5 0 animals) was fed with 0.06% of 4-dimethyl-aminoazobenzene (DAB). Batch 2 (52 animals) with same dose of 3-methyl-4dimethylaminoazobenzene (3-Me-DAB) and batch 3 (10 animals) was administered aflaxtoxin B1 (gift from E. K. Weisburger, National Cancer Institute, Bethesda, Maryland, U.S.A.) orally 25 ~ug in normal saline for 5 consecutive days for 8 weeks [7]. Control animals were maintained for each group. The azodye containing diet was fed to the rats for 6 months and then they were kept on normal diet. Due to lack of azodyes during the experimental period for some time, feeding with the experimental diet was discontinued after 10 weeks in 15 animals in each group and they were observed for 24 months for the development of HCC. The animals were sacrificed after the end of the experiment. The animals were either sacrificed or biopsied at monthly intervals and tissues processed after
Accepted 16 September 1977. *This study was supported in part by Indian Council of Agricultural Research, New Delhi, India. Reprint requests to B. K. Aikat. 295
296
N. R. Gopal Naidu, B. K. Aikat, R. J. Dash and S. Sehgal
fixing in t)esh Carnoy's fluid for 24 hr. Sections were cut at 4/~m, stained by H & E and Toluidine blue. Blood was collected at monthly interval by puncturing the occular sinus, serum was separated and stored at - 2 0 ° C until further use. Radioimmunoassay. Quantitative estimation o f serum AFP was done by RIA using double antibody technique [8]. Pure rat AFP was kindly supplied by International Agency for Research on Cancer, Lyon, France. Monospecific antibody raised in sheep was kindly supplied by Dr. Helena Phiko.* Antisheep gamma-globulin was raised in rabbit in our laboratory. Purified rat AFP was iodinated by chloramine-T method [9]. The radioimmunoassay was carried out as shown below:--
PBS, 0.05M, p.H. 7.0 (0.1% gel) Serum or standard Labeled AFP (10,000 counts per 100 sec) Sheep antirat AFP (1 in 4000 dilution) Normal sheep serum (1 in 100 dilution) Mixed, incubated at 4°C for 24 hr Rabbit antisheep gamma-globulin (i in 3 dilution) Mixed, incubated at 4°C for 24 hr 2000 rev/min for 30 min Counted the precipitate
0.4 ml 0.1 ml 0.1 ml 0.1 ml 0.1 ml
0.1 ml
The antibody dilution binding at least 20% of the precipitable counts was selected for the inhibition test. Standard inhibition curve was obtained by diluting pure AFP by two fold dilution ranging from 1000 to 1 ng and standard curve was plotted. Serum samples were tested in duplicate and the values were read against the standard curve (Fig. 1). IO0[
Standardcurve of RIA Of AFP
\ o 40
113 k~
0
I
L
i
I0
I00
I000
Standard AFP in n9
Fig. 1.
Standard curve of radioimmunoassay of rat AFP.
*Department of Serology and Bacteriology, University of Helsinki, Finland.
From the standard curve obtained under the condition of the assay used, quantitation of purified rat AFP above 1 ng/0.t ml could be reproducibly demonstrated. Hence a quantity of 10 ng of AFP per ml of serum sample could be detected with confidence. A set of dilution of known serum sample containing AFP and a set of standard AFP were used for calibration. The curves plotted on the same graph showed parallelism indicating specificity. The assay precision as determined by the coefficient of variation of inter and intra assay of reference sample did not exceed 10°/o. Synthesis of AFP in the tumor tissue was demonstrated by immunoperoxidase technique [10] and the tissues were processed according to the Sainte-Marie's method [1 !]" RESULTS
The serum AFP levels of normal rats were 10-80 ng/ml, the average being 49 ng/ml. Males showed higher values (57+9.4 ng/ml) than females (41+14.1 ng/ml) and also the incidence of HCC in males was significantly higher. A biphasic reaction of serum AFP levels was observed in most of the animals during the evolution of HCC with azodye carcinogens. The rats that were fed with diet containing 0.06% ofazodyes for 6 months developed HCC during 8-24 months of observation. The serum AFP levels began to rise in the first month and increased to attain a peak between second and third month with the mean of 4/~g/ml. By the third month a clear suppression of AFP synthesis could be appreciated. From the fourth month onwards a majority of the animals showed subsequent progressive rise in AFP with the appearance of HCC. The sequential changes in AFP levels are depicted in Fig. 2. Animals that showed the maximum AFP levels during the first 3 months were the earliest to develop HCC by 8-10 months (Group A). The subsequent serum AFP levels of these animals ranged from 250 to 1100 #g/ml. Two animals, which showed the serum AFP levels 7/~g/ml. during the first peak period, developed HCC as early as 8th month, showing a maximu'm AFP levels of 1100 /~g/ml. Animals which showed gradual rise of AFP levels from fifth month onwards developed HCC by 16-24 months and the leVels were less than 15 #g/ml (Group B). Three animals (Group C) which failed to show any rise of serum AFP during the first peak period, yet developed HCC by 16-24 months and the AFP levels were low as
Serum Alpha-fetoprotein
Fig. 3. Fig. 4. Fig. 5.
Hepatocellular carcinoma of grade H in DAB carcinogenesis. H
297
and E x 110.
Hepatocellular carcinoma of grade IV (anaplastic type) showing spindle shaped cells with numerous giant cells.
H and E x 110.
(a) Tumor showing lack of AFP in highly ill differentiated cells (Top right). The adjoining poorly differentiated cells show intense reaction. Immunoperoxidase x 440. (b) Random distribution of AFP synthesis by tumor cells lying singly or in clusters with intermixing AFP negative cells. Immunoperoxidase x 440.
299
Serum Alpha-fetoprotein IO00
•
f , , ~
incidence of AFP levels were observed in grade III where the values were 250-1110#g/ml. Grade II HCC showed levels between 7 and 15 #g/ml. The synthesis of AFP was comparatively less marked in grades I and IV. Two tumors were recorded in aflatoxin B1 group: one is HCC of grade III with serum AFP levels less than 2 #g/ml and the other is a cholangiocarcinoma with no rise of AFP levels. The findings were correlated using immunoperoxidase staining for AFP synthesis in these tumor tissues (Fig. 5). The poorly differentiated HCC showed intensive staining reaction indicating that all the cells synthesize AFP. Whereas in the tumors of grades ! and II the AFP ceils were distributed either singly or in clusters with intermixing AFP negative cells.
A
100
.E [3-
i ';'2.:5::.-. IO
~
7"--
i-- - --~,~x--x--~-E
5
I0
15
20
Months
DISCUSSION Fig. 2. Serum AFP levels in 3-Me-DAB carcinogenesis. Group A quick responders: groups B and C slow responders. Group D showed only primary reaction a~d group E no rise of serum AFP levels. No tumors d,;veloped i n groups D and E.
compared to animals which showed biphasic response. Four animals (Group D) which showed initial vise of AFP during the first three months did not develop HCC and there was no subsequent rise of AFP. Four animals (Group E) did not show any rise of AFP during the whole period of experiments. Since the animals were being sacrificed at regular intervals to study sequential changes, it was not possible to document either the percentage of animals that developed HCC nor the precise percentage of animals showing absence of a second peak of AFP synthesis.. However, the results of AFP levels of some of the animals have been depicted in the graph. Fig. 2. Twelve animals (7 in 3-Me-DAB and 5 in 4-DAB group), which were fed with experimental diet for 2½ months only, also developed HCC after 20 months. None of the animals in aflatoxin B1 group showed rise of AFP levels more than 12 #g/ml and 2 animals developed tumors after 26 months. Histology Tumors were classified into four grades. Grade I, well differentiated; grade II (Fig. 3), moderately differentiated; grade III, poorly differentiated and grade IV, undifferentiated (Fig. 4) according to Edmonson [12]. Animals which developed HCC by 8-10 months were mostly of grade III and some of grade IV. High
Early appearance of serum AFP in experimental rats has been designated as "Primary reaction" [13]. This is characterized by proliferation of the controversial "oval cells" and small hepatocytes in the liver [14]. The sequential changes observed in the rat liver during oncogenesis are as follows: injury, regeneration, proliferation of neoplastic nodule and ultimately to frank HCC [15]. In this study the synthesis of AFP started from first month onwards ranging 1-4/~g/ml (Fig. 2) presumably associated with the onset of regeneration. Sell et al. [16] observed a marked rise of AFP synthesis after partial hepatectomy in rats during the period of regeneration and serum AFP levels were six times the normal values. The observed rise in AFP during the "primary reaction" is most likely due to regeneration. In the present study 12/30 animals were fed the carcinogenic diet for 10 weeks only yet developed HCC. These observations are in accordance with those made by Hirai et al. [13], in which the DAB containing diet was replaced by a normal diet before the 7th week: none of the animals developed HCC; however, when DAB feed was replaced on the 12th week, HCC developed in most of the animals. It possibly indicated that the carcinogen has to act on the regenerating cells to cause mafignant changes. Mice given 0.15% thioacetamide (TAA) after partial hepatectomy developed HCC earlier than those with TAA diet alone [17]. It may thus be speculated that the transitory AFP elevation in patients with liver cirrhosis or hepatitis indicate a "primary reaction". These patients may eventually develop HCC under the influence of some unknown carcinogenic factors
300
N. R. Gopal Naidu, B. K. Aikat, R. J. Dash and S. Sehgal Table 1. Showing the serum AFP levels, time of appearance OfHCC and histological grading in various groups of animals in azodye carcinogenesis
Group
A. B. C. D. E.
AFP response Biphasic or monophasic
Biphasic Biphasic Monophasic Monophasic None
Maximum AFP levels (#g/ml)
Time of appearance of HCC (months)
Histological grading
250-1110 7-15 7-15 2-4 < 0.1
8-10 16-24 16-24 ---
Grades III and IV Grades I and II Grades I and II ---
like naturally occurring aflatoxin or nitrosamine. In the present study it is possible to divide the tumor-bearing animals into two distinct groups- " q u i c k " and "slow" responders on the basis of their capacity to synthesize AFP. In "quick responders" the tumors developed by 8-10 months with high A F P values ranging from 250 to 1110 #g/ml (Group A). Majority of the tumors in this group were poorly differentiated H C C . T h e slow responders on the other hand developed H C C after 16 months and they were better differentiated and the serum AFP levels were significantly lower (15 #g/ml) than the other group. Relatively poorly differentiated (grade I I I ) are able to synthesize A F P in larger-quantity. Highly a n a p l a s t i c H C C behaving as quick responders, yet could not attain very high levels of AFP. (Table 1 ). These
observations are consistent with those of Takahashi [18] who reported similar findings in h u m a n H C C . There were no tumors in groups D and E, though some of the animals did show transient initial rise of AFP. There is an apparent discrepancy regarding AFP levels in aflatoxin induced hepatoma. Kroes and his coworkers [7] for first time demonstrated the rise of serum AFP in aflatoxin induced H C C in young Fischer strain rats and the reported levels r a n g e d from" as low as 1 ttg to as high as 100 ttg [19]. In our study 1/10 rats developed H C C only at the end of 26 months and the serum A F P levels did not exceed 2 ttg/ml. This difference could possibly be attributed to the strain used, lower dose of aflatoxin B1, higher age of the animal in our study as well as tempo of carcinogenesis during aflatoxin B1 therapy.
REFERENCES 1. G.I. ABELEV,S. D. PEROVA,N. I. KHRAMKOVA,Z. A. POSTNIKOVAand I. S. IRLIN, Production of embryonal alpha-fetoprotein by transplantable mouse hepatomas. Transplantation 1, 174 (1963). 2. V.S. TATARINOV, Detection of embryospecific alpha-globulin in the blood of • patients with primary liver tumor. Vop. reed. Khim. 10, 90 (1964). 3. G . I . ABELEV,Antigenic structure of chemically induced hepatomas. Progr. exp. Tumor Res. 7, 104 (1965). 4. H. WATABE,Early appearance of embryonic alpha-globulin in rat serum during carcinogenesis with 4-dimethyl-aminoazobenzene. Cancer Res. 31, 1192 (1971). 5. M. STANISLAWSKI-BIRENCWAJG,J. URIEL and P. GRABAR, Association of embryonic antigen with experimentally induced hepatic lesions in the rat. Cancer Res. 97, 1990 (1967). 6. R. MASSEYEFF,J GILLI,B. KREBS,A. CALLUAUDand C. BONET, Evolution of alpha-fetoprotein serum levels throughout life in humans and rats and during pregnancy in rats. Ann. N. Y. Acad. Sci. 259, 17 (1975). 7. R. KROES,J. M. SONTAOand J. H. WEISBURGER,Alpha fetoprotein in rats with hepatomas induced in aflatoxin B1. Nature (Lond.) 240, 240 (1972). 8.- N. NISHI and H. HIRAI, Radioimmunoassay of alpha-fetoprotein in hepatomas, other liver diseases and pregnancy. Gann Monogr. 14, 79 (1973). 9. F.C. GREENWOOD,W. M. HUNTER and J. S. GLOVER, The preparation of 131i, labelled human growth hormone of high specific radioactivity. Biochem. 07. 89, 114 (1963). 10. V. PETTS and I. M. ROITT, Peroxidase conjugates for demonstration of tissue antibody. Evaluation of technique. Clin. exp. Immunol. 9, 407 (1971).
Serum Alpha-fetvprotein 11. 12. 13. 14.
15. 16.
17. 18. 19.
SAINTE-MARIE, A paraffin embedding immunofluorescence. J. Histochem. Cytochem. 10~ 250 (1962). H.A. EDMONOSONand P. E. STEIN, Primary carcinoma of the liver. Cancer (Philad.) 7, 462 (1954). H. HmAI, S. NISHI, H. WATABE and Y. TSUKADA,Some chemical experimental and clinical investigation of alpha-fetoprotein. Gann Monogr. l t , 19 (1973). T. ONOE, M. MARIA, A. KANEKO,K. DEMPOand K. GWAWA,Significance-of the appearance ofalpha-fetoprotein in the early stage ofazodye hepatocarcinogenesis. Tumour Res. 8, 75 (1975). T. ONOE, A. KANEKO,K. DEMPO, K. OGAWAand T. MINASE,Alpha-fetoprotein and early histological changes of hepatic tissue in DAB hepatocarcinogenesis. Ann. N.Y. Acad. Sci. 259, 168 (1975). S. SELL, M. NICHOLAS,F. F. BEC~ERand H. L. LEFFERT,Hepatocyte proliferation and alpha-fetoprotein in pregnant, neonatal and partial hepatectomized rats. Cancer Res. 3'1, 865 (1974). P.A. DATE, S. V. SOTHOSKARand S. V. BHIDA, Effect of partial Hepatectomy on tumor incidence and metabolism of mice fed thioacetamide. J. nat. Cancer Inst. 56, 493 (1976). M. TAKAHASHI,A. KAWAGUCHI,K. KAWAMATA and T. YASUMA,Alphafetoprotein by the immunofluorescent anti-body technique in primary liver carcinoma and its histopathological basis. Gann Monogr. 14~ 219 (1973). R. KROES,J. M. SONTAG,S. SELL, G. M. WILLIAMSand J. H. WEISBURGER, Elevated concentration of Serum alpha-fetoproteins in rats with chemically induced liver tumours. Cancer Res. 35, 1214 (1975).
301
Serum Alpha-fetoprotein in Experimental Hepatocellular Carcinomas m Rats* N. R. GOPAL NAIDU,t B. K. AIKAT,t R. J. DASH~ and S. SEHGAL~ ~Department of Immunopathology, +Department of Endocrinology, Postgraduate Institute of Medical Education and Research, Chandigarh, India
Abstract-- The present study has beencarriedout mainly to study the sequentialchangesof serum alpha-fetoprotein (AFP) levels with the evolution o.1"experimental hepatocellular carcinoma (HCC) and ~ocorrelate with the histology of the tumors. Ratsfed with 0.06% of azodyes developedHCC by 8-24 months and on the basis of their capacity to synthesizeAFP, the animals can be divided into two distinct major group~ " qukk responders" and "slow responders". In thejqrsl group the animals developed tumors by 8-10 months and mostly of poorly differentiated type with high serum AFP levels. The slow respondersdevelopedtumors only after 16 months and they weremostly of well differentiated with comparatively low AFP values. Both the groups showed biphasic response of AFP during the evolution of HCC. The animals in aJlatoxin B1 group did not show AFP levels more than 2#g/ml.
The present investigation was undertaken to study the serum AFP levels using R I A during the evolution of liver cancer using different types of chemical carcinogens and to correlate the morphological type of lesions with AFP synthesis.
INTRODUCTION ALPHA-FETOPROTEIN is a feto specific serum globulin synthesised by fetal liver and secreted into the serum during embryonal life of mammalian species. Abelev et al. [1], for the first time reported the presence of AFP in blood of mice bearing transplantable hepatomas. Reappearance of fetal antigens is now recognised as common phenomenon during oncogenesis. Elevated levels of serum AFP during hepatic carcinogenesis has subsequently been reported in different species of animals including man [2, 3]. Besides its appearance in rats with chemically-induced H C C its early elevation has also been demonsLrated within weeks after the administration of" chemical carcinogens [4]. However, certain chemical carcinogens like aflatoxin induced '.HCC which are not associated with significant rise of AFP [5]. The techniqtle of agar gel immunodiffusion has been used fi)r the estimation of serum AFP, but the sensildvity is relatively low. Among the more sophisticated methods for the early detection of HCC, radioimmunoassay (RIA) happens to be most sensitive one. In fact the use of R I A has revealed that even normal individuals and animals have a minimum quantity of AFP in circulation [6].
MATERIAL A N D M E T H O D S Wistar strain rats of both sexes weighing 90-120 g were kept in groups of four in wire mesh cages and maintained on laboratory diet. The carcinogens used were as follows: Batch 1 : ( 5 0 animals) was fed with 0.06% of 4-dimethyl-aminoazobenzene (DAB). Batch 2 (52 animals) with same dose of 3-methyl-4dimethylaminoazobenzene (3-Me-DAB) and batch 3 (10 animals) was administered aflaxtoxin B1 (gift from E. K. Weisburger, National Cancer Institute, Bethesda, Maryland, U.S.A.) orally 25 ~ug in normal saline for 5 consecutive days for 8 weeks [7]. Control animals were maintained for each group. The azodye containing diet was fed to the rats for 6 months and then they were kept on normal diet. Due to lack of azodyes during the experimental period for some time, feeding with the experimental diet was discontinued after 10 weeks in 15 animals in each group and they were observed for 24 months for the development of HCC. The animals were sacrificed after the end of the experiment. The animals were either sacrificed or biopsied at monthly intervals and tissues processed after
Accepted 16 September 1977. *This study was supported in part by Indian Council of Agricultural Research, New Delhi, India. Reprint requests to B. K. Aikat. 295
296
N. R. Gopal Naidu, B. K. Aikat, R. J. Dash and S. Sehgal
fixing in t)esh Carnoy's fluid for 24 hr. Sections were cut at 4/~m, stained by H & E and Toluidine blue. Blood was collected at monthly interval by puncturing the occular sinus, serum was separated and stored at - 2 0 ° C until further use. Radioimmunoassay. Quantitative estimation o f serum AFP was done by RIA using double antibody technique [8]. Pure rat AFP was kindly supplied by International Agency for Research on Cancer, Lyon, France. Monospecific antibody raised in sheep was kindly supplied by Dr. Helena Phiko.* Antisheep gamma-globulin was raised in rabbit in our laboratory. Purified rat AFP was iodinated by chloramine-T method [9]. The radioimmunoassay was carried out as shown below:--
PBS, 0.05M, p.H. 7.0 (0.1% gel) Serum or standard Labeled AFP (10,000 counts per 100 sec) Sheep antirat AFP (1 in 4000 dilution) Normal sheep serum (1 in 100 dilution) Mixed, incubated at 4°C for 24 hr Rabbit antisheep gamma-globulin (i in 3 dilution) Mixed, incubated at 4°C for 24 hr 2000 rev/min for 30 min Counted the precipitate
0.4 ml 0.1 ml 0.1 ml 0.1 ml 0.1 ml
0.1 ml
The antibody dilution binding at least 20% of the precipitable counts was selected for the inhibition test. Standard inhibition curve was obtained by diluting pure AFP by two fold dilution ranging from 1000 to 1 ng and standard curve was plotted. Serum samples were tested in duplicate and the values were read against the standard curve (Fig. 1). IO0[
Standardcurve of RIA Of AFP
\ o 40
113 k~
0
I
L
i
I0
I00
I000
Standard AFP in n9
Fig. 1.
Standard curve of radioimmunoassay of rat AFP.
*Department of Serology and Bacteriology, University of Helsinki, Finland.
From the standard curve obtained under the condition of the assay used, quantitation of purified rat AFP above 1 ng/0.t ml could be reproducibly demonstrated. Hence a quantity of 10 ng of AFP per ml of serum sample could be detected with confidence. A set of dilution of known serum sample containing AFP and a set of standard AFP were used for calibration. The curves plotted on the same graph showed parallelism indicating specificity. The assay precision as determined by the coefficient of variation of inter and intra assay of reference sample did not exceed 10°/o. Synthesis of AFP in the tumor tissue was demonstrated by immunoperoxidase technique [10] and the tissues were processed according to the Sainte-Marie's method [1 !]" RESULTS
The serum AFP levels of normal rats were 10-80 ng/ml, the average being 49 ng/ml. Males showed higher values (57+9.4 ng/ml) than females (41+14.1 ng/ml) and also the incidence of HCC in males was significantly higher. A biphasic reaction of serum AFP levels was observed in most of the animals during the evolution of HCC with azodye carcinogens. The rats that were fed with diet containing 0.06% ofazodyes for 6 months developed HCC during 8-24 months of observation. The serum AFP levels began to rise in the first month and increased to attain a peak between second and third month with the mean of 4/~g/ml. By the third month a clear suppression of AFP synthesis could be appreciated. From the fourth month onwards a majority of the animals showed subsequent progressive rise in AFP with the appearance of HCC. The sequential changes in AFP levels are depicted in Fig. 2. Animals that showed the maximum AFP levels during the first 3 months were the earliest to develop HCC by 8-10 months (Group A). The subsequent serum AFP levels of these animals ranged from 250 to 1100 #g/ml. Two animals, which showed the serum AFP levels 7/~g/ml. during the first peak period, developed HCC as early as 8th month, showing a maximu'm AFP levels of 1100 /~g/ml. Animals which showed gradual rise of AFP levels from fifth month onwards developed HCC by 16-24 months and the leVels were less than 15 #g/ml (Group B). Three animals (Group C) which failed to show any rise of serum AFP during the first peak period, yet developed HCC by 16-24 months and the AFP levels were low as
Serum Alpha-fetoprotein
Fig. 3. Fig. 4. Fig. 5.
Hepatocellular carcinoma of grade H in DAB carcinogenesis. H
297
and E x 110.
Hepatocellular carcinoma of grade IV (anaplastic type) showing spindle shaped cells with numerous giant cells.
H and E x 110.
(a) Tumor showing lack of AFP in highly ill differentiated cells (Top right). The adjoining poorly differentiated cells show intense reaction. Immunoperoxidase x 440. (b) Random distribution of AFP synthesis by tumor cells lying singly or in clusters with intermixing AFP negative cells. Immunoperoxidase x 440.
299
Serum Alpha-fetoprotein IO00
•
f , , ~
incidence of AFP levels were observed in grade III where the values were 250-1110#g/ml. Grade II HCC showed levels between 7 and 15 #g/ml. The synthesis of AFP was comparatively less marked in grades I and IV. Two tumors were recorded in aflatoxin B1 group: one is HCC of grade III with serum AFP levels less than 2 #g/ml and the other is a cholangiocarcinoma with no rise of AFP levels. The findings were correlated using immunoperoxidase staining for AFP synthesis in these tumor tissues (Fig. 5). The poorly differentiated HCC showed intensive staining reaction indicating that all the cells synthesize AFP. Whereas in the tumors of grades ! and II the AFP ceils were distributed either singly or in clusters with intermixing AFP negative cells.
A
100
.E [3-
i ';'2.:5::.-. IO
~
7"--
i-- - --~,~x--x--~-E
5
I0
15
20
Months
DISCUSSION Fig. 2. Serum AFP levels in 3-Me-DAB carcinogenesis. Group A quick responders: groups B and C slow responders. Group D showed only primary reaction a~d group E no rise of serum AFP levels. No tumors d,;veloped i n groups D and E.
compared to animals which showed biphasic response. Four animals (Group D) which showed initial vise of AFP during the first three months did not develop HCC and there was no subsequent rise of AFP. Four animals (Group E) did not show any rise of AFP during the whole period of experiments. Since the animals were being sacrificed at regular intervals to study sequential changes, it was not possible to document either the percentage of animals that developed HCC nor the precise percentage of animals showing absence of a second peak of AFP synthesis.. However, the results of AFP levels of some of the animals have been depicted in the graph. Fig. 2. Twelve animals (7 in 3-Me-DAB and 5 in 4-DAB group), which were fed with experimental diet for 2½ months only, also developed HCC after 20 months. None of the animals in aflatoxin B1 group showed rise of AFP levels more than 12 #g/ml and 2 animals developed tumors after 26 months. Histology Tumors were classified into four grades. Grade I, well differentiated; grade II (Fig. 3), moderately differentiated; grade III, poorly differentiated and grade IV, undifferentiated (Fig. 4) according to Edmonson [12]. Animals which developed HCC by 8-10 months were mostly of grade III and some of grade IV. High
Early appearance of serum AFP in experimental rats has been designated as "Primary reaction" [13]. This is characterized by proliferation of the controversial "oval cells" and small hepatocytes in the liver [14]. The sequential changes observed in the rat liver during oncogenesis are as follows: injury, regeneration, proliferation of neoplastic nodule and ultimately to frank HCC [15]. In this study the synthesis of AFP started from first month onwards ranging 1-4/~g/ml (Fig. 2) presumably associated with the onset of regeneration. Sell et al. [16] observed a marked rise of AFP synthesis after partial hepatectomy in rats during the period of regeneration and serum AFP levels were six times the normal values. The observed rise in AFP during the "primary reaction" is most likely due to regeneration. In the present study 12/30 animals were fed the carcinogenic diet for 10 weeks only yet developed HCC. These observations are in accordance with those made by Hirai et al. [13], in which the DAB containing diet was replaced by a normal diet before the 7th week: none of the animals developed HCC; however, when DAB feed was replaced on the 12th week, HCC developed in most of the animals. It possibly indicated that the carcinogen has to act on the regenerating cells to cause mafignant changes. Mice given 0.15% thioacetamide (TAA) after partial hepatectomy developed HCC earlier than those with TAA diet alone [17]. It may thus be speculated that the transitory AFP elevation in patients with liver cirrhosis or hepatitis indicate a "primary reaction". These patients may eventually develop HCC under the influence of some unknown carcinogenic factors
300
N. R. Gopal Naidu, B. K. Aikat, R. J. Dash and S. Sehgal Table 1. Showing the serum AFP levels, time of appearance OfHCC and histological grading in various groups of animals in azodye carcinogenesis
Group
A. B. C. D. E.
AFP response Biphasic or monophasic
Biphasic Biphasic Monophasic Monophasic None
Maximum AFP levels (#g/ml)
Time of appearance of HCC (months)
Histological grading
250-1110 7-15 7-15 2-4 < 0.1
8-10 16-24 16-24 ---
Grades III and IV Grades I and II Grades I and II ---
like naturally occurring aflatoxin or nitrosamine. In the present study it is possible to divide the tumor-bearing animals into two distinct groups- " q u i c k " and "slow" responders on the basis of their capacity to synthesize AFP. In "quick responders" the tumors developed by 8-10 months with high A F P values ranging from 250 to 1110 #g/ml (Group A). Majority of the tumors in this group were poorly differentiated H C C . T h e slow responders on the other hand developed H C C after 16 months and they were better differentiated and the serum AFP levels were significantly lower (15 #g/ml) than the other group. Relatively poorly differentiated (grade I I I ) are able to synthesize A F P in larger-quantity. Highly a n a p l a s t i c H C C behaving as quick responders, yet could not attain very high levels of AFP. (Table 1 ). These
observations are consistent with those of Takahashi [18] who reported similar findings in h u m a n H C C . There were no tumors in groups D and E, though some of the animals did show transient initial rise of AFP. There is an apparent discrepancy regarding AFP levels in aflatoxin induced hepatoma. Kroes and his coworkers [7] for first time demonstrated the rise of serum AFP in aflatoxin induced H C C in young Fischer strain rats and the reported levels r a n g e d from" as low as 1 ttg to as high as 100 ttg [19]. In our study 1/10 rats developed H C C only at the end of 26 months and the serum A F P levels did not exceed 2 ttg/ml. This difference could possibly be attributed to the strain used, lower dose of aflatoxin B1, higher age of the animal in our study as well as tempo of carcinogenesis during aflatoxin B1 therapy.
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