Int. J. Cancer: 41, 727-731 (1988) 0 1988 Alan R. Liss, Inc.

Publication of the International Union Against Cancer Publication de I'Union lnternationale Contre le Cancer

ALBUMIN-NEGATIVE HEPATOCYTES IN SPRAGUE-DAWLEY X ANALBUMINEMIC F 1 RATS TREATED WITH HEPATIC CARCINOGENS Katsuhiro OGAWA~, Kin-ichi YOKOTA', Tomoko SONODA~, Zhe Si PIAO', Michio Mom' and Sumi NAGASE~ 'Department of Pathology, Sapporo Medical College, S. I , W 17, Chuo-ku, Sapporo 060; and 2Department of Chemistry, Sasaki Institute, Surugadai 2-2, Kanda, Chiyoda-ku, Tokyo 101, Japan.

Analbuminemic rats (NAR) are a mutant breed with an inherent inability to synthesize albumin. However, heterozy- gous rats born of a pair of NAR and Sprague-Dawley (SD) rats can synthesize albumin. lmmunohistochemical staining for albumin shows that, although the majority of hepatocytes of SD x NAR F, (female SD x male NAR) rats are positive for albumin, a small number of hepatocytes are negative. These albumin-negative hepatocytes are frequently found in the form of clusters which appear cytologically normal. When the rats were given a dietary regimen of 2-acetyl-aminofluorene (2-AAF), there was a significant increase in the number of albumin-negative hepatocytes. O n the other hand, diethylni- trosamine (DEN) or 5-azacytidine did not increase the number of albumin-negative hepatocytes. 2-AAF and DEN also in- duced enzyme-altered hepatocytes but the albumin-negative hepatocytes were of a completely different class from the enzyme-altered hepatocytes. The results of this study indicate that some kind(s) of carcinogens induce mutated hepatocytes which are probably not involved in carcinogenesis.

Nagase's analbuminemic rats (NAR) represent a mutant breed isolated from Sprague-Dawley (SD) rats (Nagase et al., 1979). The genetic abnormality in these rats is the deletion of 7 base pairs in the 9th intron of the albumin gene, which is considered to result in a defect in the processing of albumin- mRNA (Esumi et al., 1983). However, when the livers of NAR are immunohistochemically stained with anti-rat albumin antibodies, a small number of hepatocytes are positive for albumin. Recently, Makino et al. (1986) have reported that the number of albumin-positive hepatocytes increased with age or upon administration of certain kinds of hepatic carcinogens. These albumin-positive hepatocytes are different from hepa- tocytic nodule cells (presumably pre-neoplastic populations) and seem to be unrelated to the carcinogenic process.

On the other hand, as the mutant gene of NAR is recessive, the hepatocytes of heterozygous NAR have the ability to pro- duce albumin (Nagase et al., 1980). After finding that a small number of hepatocytes are albumin-negative in SD X NAR F1 rats, we investigated whether the numbers of such albumin- negative hepatocytes are increased by treatment with chemical carcinogens or a demethylating agent for DNA (5-azacyti- dine), either with or without partial hepatectomy. In this re- port, we show that the numbers of albumin-negative hepatocytes are increased by administration of 2-acetylamino- fluorene (2-AAF), but not by either diethylnitrosamine (DEN) or 5-azacytidine. By examining albumin and glutathione s- transferase-placental form (GST-P), we show that these hepa- tocytes were different populations from the enzyme-altered hepatocytes.

MATERIAL AND METHODS

Animals and treatment NAR were bred in the Sasaki Institute, and SD rats were

purchased from Clea (Tokyo, Japan). Male NAR and female SD were mated at the age of 8-10 weeks. Offspring (SD X NAR F1) were weaned 3 weeks after birth, given a chow diet (Oriental Yeast, Tokyo) and water ad libitum in a room with a 12-hr light/12-hr dark cycle. Male SD X NAR F1 rats, weigh- ing 215 * 20 g, were treated with chemicals as follows (Fig.

1). 2-AAF was admixed into the diet at a concentration of 0.02% and given for 6 weeks from the age of 6 weeks. DEN or 5-azacytidine was dissolved with physiological saline, and a dose of 10 mglkg was injected into the peritoneal cavity twice a week from the age of 6 weeks. Control rats were not treated with the carcinogens. Each group of animals was di- vided into 2 subgroups, ,and two-thirds partial hepatectomies were performed according to the method of Higgins and An- derson (1931) in one subgroup 4 weeks after the beginning of treatment with the chemicals. The rats of both the control and experimental groups were killed at the end of the 6th week. All were given bromodeoxyuridine (BUdR) dissolved in di- methylsulfoxide (1 mg/kg body weight) 1 hr before killing to label the nuclei of proliferating hepatocytes. Immunohistochemical staining

Livers were perfusion-fixed by a paraformaldehyde-lysine- periodate solution (McLean and Nakane, 1974) for 5 min, and immersed in the same fixative for an additional 6-hr period. Serial sections, 4 pm thick, were cut from paraffin-embedded blocks and immunohistochemically stained for rat albumin and GST-P (Satoh et al . , 1985). Antibodies to rat albumin were purchased from Cooper Biochemical (Malvern, PA), and an- tibodies to GST-P were a gift from Dr. K. Satoh. Immuno- staining was carried out by the ABC method (Hsu et al . , 1981). The sections were sequentially treated with 1% H20? in methanol, primary antibodies, biotin-conjugated anti-rabbit immunoglobulin and avidin-biotinylated peroxidase complex. As formaldehyde blocked endogenous hepatic avidin-binding activity, no specific treatment was needed to inhibit the endog- enous activity. The localization of the antigens was visualized by incubation in the diaminobenzidine/H202 solution, and nuclei were counter-stained with methyl green. The number of albumin-negative or GST-P-positive cell clusters was counted so as to separate groups depending on the nuclear number. At least 5 sections, each containing different slices taken from each liver lobe, were used; the total area examined was 16-25 cm2 per animal. To compare the expression of albumin and GST-P, the stained sections were photographed, and 278 clusters consisting of more than 10 cells were marked on the photos, The albumin-negative hepatocytes were ana- lyzed for expression of GST-P. Measurement of proliferating hepatocytes

Pairs of deparaffinized sections were treated with 0.1N HCl, washed with phosphate-buffered saline (PBS) and reacted with anti-BUdR monoclonal antibodies (Becton Dickinson, Moun- tain View, CA) (Gratzner, 1982). Localization of anti-BUdR antibodies within the tissues was demonstrated by the ABC method. One of the sections was further immunostained for albumin in the same way as described above, and the other for GST-P. Nuclei were counter-stained with hematoxylin. For determination of the labelling index of the albumin-positive hepatocytes, the number of labelled and unlabelled nuclei was counted in GST-P-negative areas of BUdRIGST-P stained sec-

Received: July 2 1 , 1987 and in revised form November 11, 1987.

728 OGAWA ET AL.

kill I control I- 1

PH

2 - A A F D U I . I v v v v v v v v v v v v

DEN I I I

5-azacytidine I

PH V V . ? V V V V V V V V

PH

Ow Iw 2 w 3w 4 w 5 w 6 w

FIGURE 1 - Schematic representation of the treatment of rats with chemicals. (V) Intraperitoneal injection of DEN (10 mg/kg body weight); (V) intraperitoneal injection of 5-azacytidine (10 mglkg body weight); PH, two-thirds partial hepatectomy.

tions by means of an ocular grid, and the proportion of labelled nuclei against total nuclei was calculated for each rat. As the number of albumin-negative cells was smaller than that of albumin-positive cells, the values were assumed to substan- tially represent the labelling index of albumin-positive cells. For the labelling index of albumin-negative hepatocytes and that of GST-P positive cells, BUdR/albumin and BUdR/GST- P double-stained sections were used, respectively. For each rat, the labelling index was determined by examining more than l@ cells for albumin-positive hepatocytes, and lo3 cells for albumin-negative or GST-P-positive cells, respectively. Measurement of serum albumin level

Serum albumin level was measured by a modification of the method of Demetriou et al. (1986). Briefly, serum taken from normal SD and SD X NAR F1 rats was diluted 1:lOO with distilled water and applied to SDS-PAGE using the Phast System (Pharmacia, Uppsala, Sweden). Purified rat albumin was used as the standard. The gels of SDS-PAGE were densi- tometrically scanned and the area of the peak corresponding to albumin was measured by means of an image analyzer. The albumin concentrations were calculated from the standard values. Statistical analysis

difference between the values. Student’s t-test was applied to statistical analysis of the

RESULTS

Albumin immunohistochemistry On immunohistochemical staining for albumin, 100% of the

hepatocytes of SD rats were positive (Horikawa et al . , 1976; Lebouton and Masse, 1980; Yokota and Fahimi, 1981). The hepatocytes showed granular staining predominantly localized near the bile canalicules and a fine reticular staining through- out the cytoplasm. On the other hand, a small number of albumin-negative hepatocytes were seen in the livers of SD X NAR F1 rats, whereas the majority were positively stained (Fig. 2a-c). The albumin-negative hepatocytes formed small clusters ranging from 1 to 40 cells (Fig. 2a-c). The shape of the clusters was usually irregular and they did not show any compressive figures against the surrounding hepatic tissue.

FIGURE 2 - Immunostaining of the liver of a control SD X NAR F, rat using antibodies against rat albumin antibodies. (a) A single albumin- negative hepatocyte (arrow); (b) group of albumin-negative hepatocytes; (c) a larger cluster of albumin-negative hepatocytes. Bar scale 50 pm (a,b), 100 pm (4.

ALBUMIN-NEGATIVE HEPATOCYTES IN SD X NAR F1 RATS 729

TABLE I - INCIDENCE OF ALBUMIN-NEGATIVE AND GST-P-POSITIVE FOCI (NUMBER OF F O C I ~ C M ~ OF SECTION)

Albumin ( - j GST-P( +)

I 2-9 10-50 < 502 1 2-9 10-50 < 50’ Treatment PH’ Number of rats

Control - 5 9.1 +4.73 3.9k 1.7 2.5k2.0 0 0 0 0 0 + 5 7.2k2.1 3.3k2.2 2.0k1.7 0 0 0 0 0

+ 8 29.2+10.15 15.5+6.95 5.3k3.8 0.5k0.3 2.5rtl.l 2.4k1.5 1.1ki.o 0

+ 5 8.5L2.8 4.1k3.1 1.0i0.9 0 12.8k4.8 I9.4rt9.9 7.3+5.1 0 5-azacytidine - 5 10.5k6.6 4.0k1.0 1.0*0.5 0 0 0 0 0 + 6 7.6k4.6 3.4+1.2 2.3ki.8 0 0 0 0 0

0 0.7*0.6

7 10.2k6.2 4.4k2.3 2.912.3 0 5.2_+0.5 3.5k1.8 1.2k1.0 0.2k0.2

2-AAF - 6 14.Ok7.O4 6.0k3.8 1.5+ 1.4 0 3.8k2.1 9.0k3.0

DEN -

‘Two-thirds partial hepatectomy.-*Number of cells in the ~ lus t e r . -~Mean ? standard de~idtion.-~Significantly different from control, p < 0.05.-5Significantly different from control, piO.01.

TABLE I1 - BUdR NUCLEAR LABELING INDEX (%)OF AVERAGE, ALBUMIN-NEGATIVE AND GST-P-POSITIVE HEPATOCYTES

Treatment PH’ Albumin (+) Albumin ( - j GST-P( +) Number of rats

Control - 5 0.8 k 0.72 0 .o + 0.02 - + 5 0.8k0.8 o.o+o.o - 2-AAF - 6 2.5k1.7 4.8k2.6 6.5f2.5* + 8 8.7k2.0 10.4k5.5 21.6k5.9

7 8.0k3.9 12.7k4.6 57.7 + 24.0 DEN + 5 34.7+_ 15.2’ 6 1.2 k 24.9’ 73.2 k 42.9

+ 6 4 .9 i4 .3 6.7k9.6

-

5-azacytidine - 5 5.3k4.9 4.7+4.8 - -

‘Two-thirds partial hepatectomy.-’Mean j~ sn.-’The difference is significant between the values for the average and albumin (-)cells. p<O.O5.

TABLE 111 - SERUM ALBUMIN LEVEL

Strain Age (months) Number of rats Albumin (g/dl)

SD 6 8 2.4k0.4 SD X NAR F1 8 11 0.9k0.4

Partial hepatectomy did not influence the incidence of albu- min-negative hepatocytes in control SD X NAR F1 rats.

Upon administration of 2-AAF, the incidence of albumin- negative hepatocytes increased significantly (Table I). Cou- pling a partial hepatectomy with 2-AAF increased not only the number but also the size of the clusters of albumin-negative cells; the largest clusters consisted of more than 100 cells (Fig. 3a). Administration of DEN or 5-azacytidine resulted in no significant increase in the number of albumin-negative cells as compared to controls (Table I).

Irnrnunohistochemistry of enzyme markers GST-P-positive foci were seen in the groups treated with 2-

AAF or DEN, although they were never observed in the livers of control or 5-azacytidine-treated rats. These cells formed clusters of various sizes, ranging from a single cell to over 100 cells (Figs. 3b and 4). GST-P was essentially localized in the cytoplasm. The incidence of GST-P-positive cells was highest in the group which had received DEN plus partial hepatectomy (Table I). Serial sections of the livers which had been treated with 2-AAF or DEN were examined to compare the expression of albumin and GST-P. Of 278 albumin-nega- tive foci consisting of more than 10 cells examined in the

present study, none showed positive staining for GST-P as a whole (Fig. 3a,b). On the other hand, singlets or doublets of albumin-negative hepatocytes were observed within large GST- P-positive nodular areas on rare occasions. Incidence of proliferating cells

The incidence of nuclear labelling with BUdR varied in each experimental group. When compared to the control, the groups treated with DEN, 2-AAF or 5-azacytidine showed a higher proliferating activity of hepatocytes (Table 11). The incidence of proliferating cells was further increased in the 2-AAF- or DEN treated groups by performance of a partial hepatectomy in the 4th week. Although the labelling index (LI) of GST-P- positive cells was significantly higher than that of the sur- rounding hepatocytes, the LI of albumin-negative hepatocytes was almost the same as, or at most 1.9 times higher than, the latter (Table 11).

The serum albumin level of SD X NAR FI rats was less than half of that seen in SD rats (Table 111).

DISCUSSION

Our results show that a small number of hepatocytes in SD X NAR F1 are negative for albumin, and that they were increased in number by administration of 2-AAF. Such albu- min-negative hepatocytes are never seen in normal or carcin- ogen-treated SD rats. The observation of the presence of single albumin-negative cells is suggestive of their clonal origin, as has been argued from observations on enzyme-altered hepato- cytes (Scherer and Hoffmann, 1971; Rabes et al., 1982; Wil- liams et al., 1983). It is thought that, as SD X NAR F1 cells have a single normal albumin gene, dysfunction of the mater- nal normal albumin gene by somatic mutation may lead to a loss of albumin production.

FIGURE 4 - Higher magnification of GST-P-positive hepatocytes in- duced by DEN. Bar scale 100 pm.

Although 2-AAF increased the number of albumin-negative hepatocytes, DEN and 5-azacytidine did not. Since most al- bumin-negative cells were in the form of singlets or doublets, it is suspected that the increase was mainly due to conversion of albumin-positive cells to albumin-negative cells rather than colonization of pre-existing albumin-negative cells. On the other hand, the number of GST-P-positive cells was increased more by DEN than by 2-AAF. It seems that the mechanism of occurrence of albumin-negative cells and GST-P-positive cells is dependent on the kind of chemicals which may produce different genetic alterations. There was no increase in the number of albumin-negative hepatocytes following administra- tion of 5-azacytidine, suggesting that hypomethylation of DNA has no connection with the appearance of albumin-negative hepatocytes .

The appearance of albumin-negative hepatocytes in SD X NAR F1 rats resembles that of the small number of hepatocytes that are albumin-positive in NAR rats (Makino et al., 1986). That is, (1) the numbers of such cells are increased by the administration of 2-AAF or 3’-methyl-4-dimethylaminoazo- benzene but not by DEN. (2) They form clusters ranging from only 1 cell to over 80 cells. (3) They are different from enzyme-altered cells. However, Makino et al. (1986) have shown that the incidence of albumin-positive hepatocytes was 12/106 cells after the administration of 0.03% 2-AAF for 14 weeks (10 times higher than in untreated controls of the same age). On the other hand, when the incidence of albumin- negative hepatocytes was expressed in the same terms, it was 0.6/106 cells in SD X NAR F1 treated with 2-AAF for 6 weeks, indicating that the incidence is much lower than that of albumin-positive cells in NAR.

It has been shown that early populations of carcinogen- altered hepatocytes induced by chemical carcinogens exhibit various biochemical markers, although they are heterogeneous (Moore and Kitagawa, 1986). It became clear, however, upon examination of biochemical markers, that albumin-negative hepatocytes are different cell populations from enzyme-altered hepatocytes. This suggests that, besides classically known en- zyme-altered hepatocytes, carcinogens may also induce hepa- tocytes with genetic abnormalities but without pre-neoplastic potential.

The Proliferation rate of albumin-negative hepatocytes was almost the Same or, at most, twice as high as that in the surrounding hepatic tissue. It is not clear why such differences arise. The albumin-negative cells may be less sensitive to the

FIGURE 3 - Serial sections of the liver of a SD X NAR F, rat treated with 2-AAF. Immunostaining using anti-rat albumin antibodies (a). The areas encircled by a dotted line are GST-P-positive in (b). None of the albumin-negative clusters coincide with GST-€‘-positive areas. Bar scale, 500 pm.

ALBUMIN-NEGATIVE HEPATOCYTES IN SD X NAR F1 RATS 73 1

cytotoxicity of carcinogens due to different metabolic activities of the carcinogens, as argued from observations of enzyme- altered hepatocytes (Farber, 1984). On the other hand, the proliferation rate of GST-P-positive cells was 2 to 7 times higher than that in the surrounding hepatic tissue. This indi- cates that, although both albumin-negative and GST-P-positive cell populations form clusters when exposed to carcinogens, only the latter are able to form nodules compressed against the surrounding tissue.

The number of albumin-negative clusters was lower in the DEN-treated group than in the 2-AAF-treated group. Initially this appeared inconsistent with the finding that the labelling index of albumin-negative cells was higher in the DEN-treated than in the 2-AFF-treated group. However, when carcinogenic treatment is prolonged, inhibition of cell proliferation occurs concomitantly with stimuli for restorative hyperplasia which

results from cell loss (Craddock, 1976). Therefore, this is a complex situation involving simultaneous inhibition and stim- ulation, which sometimes results in the occurrence of a wave of cell proliferation at a certain time after the beginning of the treatment. It is thus possible that the time at which we killed our animals might have coincided precisely with the phase of high cell proliferation in the DEN-treated group. The size of the clusters might be expected to become larger at later times only.

ACKNOWLEDGEMENTS

This work was supported by grants from the Ministry of Education, Culture and Science of Japan and from the Hok- kaido Society for the Promotion of Gerontology.

REFERENCES

CRADDOCK, V.M., Cell proliferation and experimental liver cancer. In: H.M. Cameron, D.A. Linsell and G.P. Warwick (eds.), Liver cell cancer, pp. 153-201, Elsevier, Amsterdam (1976). DEMETRIOU, A.A., LAVENSON, S.M., NOVIKOFF, P.N., NOVIKOFF, A.B., CHOWDHURY, N.R.. WHITING, J., REISNER, A., and CHOWDHURY, J.R..

the liver of analbuminemic rats and their increase on treatment with hepatocarcinogenesis. Gann, 77, 153-159 (1986). MCLEAN, I.W., and NAKANE, P.K., Periodate-lysine-paraformaldehyde fixation: a new fixative for immunoelectron microscopy. J. Hisrochem. Cvtochem., 22, 1077-1083 (1974).

Survival, organization, and function of microcarrier-attached hepatocytes transplanted in rats. Proc. nat. Acad. Sci. (Wash.), 83, 7475-7479 (1986). ESUMI, H., TAKAHASHI, Y., SATO, S., NAGASE, S., and SUGIMURA, T., A seven-base-pair deletion in an intron of the albumin gene of analbumi- nemic rats. Proc. nut Acad. Sci. (Wash.), 80,95-99 (1983). FARBER, E., Cellular biochemistry of the stepwise development of cancer with chemicals. Cancer Res., 44, 5463-5474 (1984). GRATZNER, H.G., Monoclonal antibody to bromo- and 5-io-dodeoxyuri- dine: a new reagent for detection of DNA replication. Science, 218, 474- 475 (1982). HIGGINS, G.M., and ANDERSON, R.M., Experimental pathology of the liver. I. Restoration of the liver of the white rat following partial surgical removal. Arch. Path., 12, 186-202 (1931). HORIKAWA, M., CHISAKA, N., YOKOYAMA, S., and ONOE, T., Effect of stirring during fixation uoon immunofluorescence results with distribution of alb;min-p;oducing cells in the liver. J. Hisrochem. Cytochern., 24, 926-932 (1976). Hsu, S.M., RAINE, L., and FANGER, H., Use of avidin-biotin-peroxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures. J. Histochem. Cytochem., 29,577-580 (I98 1). LEBOUTON, A.V., and MASSE, P.J., A random arrangement of albumin- containing hepatocytes seen with histo-immunologic methods. I. Verifi- cation of the artifact. Anut. Rec., 197, 183-194 (1980). MAKINO, R., SATO, S., ESUMI, H., NEGISHI, C., TAKANO, M., SUGIMURA, T., NAGASE, S., and TANAKA, H., Presence of albumin-positive cells in

MOORE, M., and KITAGAWA, T., Hepatocarcinogenesis in the rat: the effect of promoters and carcinogens in vivo and in vitro. Inr. Rev. Cyrol., 101,

NAGASE, S., SHIMAMUNE, K., and SHUMIYA, S., Albumin-deficient rat mutant. Science, 205, 590-591 (1979). NAGASE, S. , SHIMAMUNE, K., and SHUMIYA, S., Albumin-deficient rat mutant: an animal model for analbuminemia. Enp. Anim., 29, 33-38 ( 1980). RABES, H.M., BUECHER, TH., HARTMANN, A,, LINKE, I., and DUENE- WALD, M., Clonal growth of carcinogen-induced enzyme-deficient preneo- plastic cell populations in mouse liver. Cancer Res., 42, 3220-3227 (1982). SATOH, K., KITAHARA, A., SOMA, Y., HATAYAMA, I., and SATO, K., Purification, induction and distribution of placental glutathione transfer- ase: a new marker enzyme for preneoplastic cells in the rat chemical carcinogenesis. Proc. nat. Acad. Sci. (Wash.), 82, 3964-3968 (1985). SCHERER, E., and HOFFMANN, H., Probable clonal genesis of cellular islands induced in rat liver by diethylnitrosamine. Europ. J. Cancer, 7,

WILLIAMS, E.D., WARENHAM, K.A., and HOWELL, S., Direct evidence for the single-cell origin of mouse liver tumors. Brit. J. Cancer, 47, 723- 726 (1983). YOKOTA, S., and FAHIMI, H.D., Immunocytochemical localization of albumin in the secretory apparatus of rat liver parenchymal cells. Proc. nut. Acad. Sci. (Wash.), 78,4970-4974 (1981).

125-173 (1986).

369-371 (1971).