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Spontaneous Altered Hepatocellular Foci (AHF) in Captive Aged Male Cotton Rats (Sigmodon hispidus)
J.
Camp. Path. 1993 Vol. 109,439-445
Spontaneous Altered Hepatocellular Foci (AHF) in Captive Aged Male Cotton Rats (Sig'l'nodon hispidus) M. G. Paranjpe, C. W. Qualls Jr, A. M. S. Chandra and R. L. Lochmiller* Department of VeterinalY Pathology, College of Veterinary Medicine and *Department oj Zoology, Oklahoma Slate University, Stillwater, OK 74078, U.S.A.
Su:m:mary
Spontaneous altered hepatocellular loci (AHF) of basophilic type were observed in adult male cotton rats. The histological features of these foci were similar to those observed in Fischer 344 rats. However, an immunohistochemical technique with antibody to glutathione S-transferase placental form (CST-P) fltiled to stain these foci. Also normal bile ducts were not immunoreactive for CST-Po The presence of a gall bladder ancl of non-CST-P immunoreactive liver foci and bile ducts suggests that these cotton rats are phylogenetically closer to mice than rats.
Introduction AHF occur spontaneously and increase in number as a function of age in laboratory rat strains such as Fischer 344 (F344), Wistar, and Sprague-Dawley (Ogawa et al., 1981; Hendrich et al., 1988). AHF are virtually non-existent in F344 rats less than 3 months old, are commonly seen by 6 to 12 months of age, and occur in virtually 100 per cent ofrats by 15 to 24· months of age (Harada et al., 1989; Eustis et al., 1990). Up to weaning, the incidence of AHF is similar in male and female rats, but thereafter it increases significantly in male rats (Mitaka and Tsukada, 1987; Pitot et al., 1989). AHF are considered by some authors to be pre-neoplastic and have been used as an index ofhepatocarcinogenicity (Williams, 1980; Bannasch et al., 1986). AHF appear earlier and increase in number in rats exposed to chemical hepatocarcinogens (Williams, 1980; Bannasch et al., 1986). AHF have been classified by their tinctorial qualities with haematoxylin and eosin (HE) stain into five types-basophilic, eosinophilic, clear, vacuolated and mixed (Harada et al., 1989; Eustis et al., 1990). Quantification of these foci after HE staining, by means of a computerassisted image analyzer in conjunction with a camera lucida (Harada et al., 1989) is possible, though difficult. End point markers such as garnma-glutamyl transpeptidase (GGT), adenosine triphosphatase (ATPase), glucose-6-phosphate dehydrogenase (G6PD) ancl eSTop have been tried as a basis for the identification of AHF by Correspondence to: C. W. Qualls, Jr. 0021-9975(93(080439 + 07 $08.00(0
©
1993 Academic Press Limited
440
M. G. Paranjpe et al.
immunohistochemical staining techniques. The GGT, ATPase, and G6PD markers have several disadvantages such as high background staining and elaborate methodology (Imaida et al., 1983; Tamano et al., 1983; Thamarit et al., 1985). GST-P, introduced as a novel endpoint marker enzyme by Sato et al. (1984), appears to be very sensitive and a more consistent marker for all types of AHF than other markers. The AHF demonstrated by this technique arc clearly distinguishable, there are minimal alterations in tinctorial qualities, there is a high contrast, they are uniformly stained, and they permit the recognition of all of the cellular stages in hepatocarcinogenesis (Tatematsu et al., 1985, 1987, 1988). Even single GST-P positive cells have been considered as potentially initiated cells (Moore et al., 1987). After immunohistochemical staining, the quantitation of AHF is facilitated by computerized semiautomated morphometric analysis equipment (Ito et al., 1989a). The cotton rat has an abundant distribution in the United States, a lifeexpectancy ofless than 6 months in the wild, and a confined area of movement of less than 1 hectare. We have used this species to evaluate the effects of environmental contamination on genotoxicity, metabolism (cytochrome P-450 induction), immunotoxicity, and population dynamics (Elangbam et al., 1989, 1991). The purpose of this report is to document the spontaneous occurrence of basophilic AHF in aged male cotton rats raised in a breeding colony. Materials and Methods
A small breeding colony of cotton rats originated from wild cotton rats captured in the vicinity ofStiHwater, OK, U.S.A. To maintain genetic diversity, wild cotton rats were occasionally introduced into the breeding colony. Six male cotton rats born in the breeding colony were killed by carbon dioxide euthanasia when 15 months old and weighing between 210 and 300 g. Throughout their life these rats were housed singly in polycarbonated wire top cages and maintained on a 12 h light cycle. All rats were fed Purina Rat Chow and given tap water ad libitum. At necropsy, the following tissues were collected in 10 per cent neutral buffered formalin: adrenal glands, brain, caecum, colon, duodenum, epididymis, heart, ileum, jejunum, kidneys, liver, lung, lymph node (popliteal), oesophagus, pancreas, pituitary, seminal vesicles, skeletal muscle, spleen, stomach, testes, thyroids and parathyroids, trachea and urinary bladder. All tissues were routinely processed, sectioned at 5-6 11m, and stained with HE. The avidin-biotin-peroxidase complex (ABC) method introduced by Hsu et al. (1981) was used to determine the location of GST-P binding in the liver. Affinity-purified biotin-labelled goat anti-rabbit IgG and ABC were obtained from Vector Laboratories, Burlingame, CA, U.S.A. Paraffin wax sections were routinely passed through petroleum benzene and a graded alcohol series and then treated sequentially with appropriate dilutions of normal goat serum, rabbit anti-GST-P, biotin-labelled goat anti-rabbit IgG and ABC. The sites of peroxidase binding were demonstrated by diaminobenzidine. Sections were counterstained by haematoxylin for microscopical examination. As a negative control for specificity of anti-GST-P antibody binding, pre-immune rabbit serum was used instead of antiserum. As a positive control, normal livers and AHF from F344 rats were stained.
Results and Discussion At necropsy, the livers of all six rats were mildly to moderately pale and only slightly mottled. Individual livers weighed between 9 and 15 g. None of the
Altered Hepatocellular Foci in Cotton Rats
Fig. I.
441
An altered hepatocellular focus in a cotton rat is outlined by arrowheads. Negative staining with ,tnti-GST-P antibody, haematoxylin counterstain. x 100.
rats had any other macroscopical lesions. Microscopical examination revealed basophilic AHF in each of the liver sections examined. These foci were randomly scattered, clearly delineated and characterized by increased localized cytoplasmic basophilia and hyperchromasia of nuclei. The basophilic cells were slightly smaller than the normal hepatocytes. In addition, all livers had mild to moderate fatty change. The immunohistochemical technique with antibody to GST-P failed to stain these foci (Fig. l). Even the biliary epithelium was nonimmunoreactive. The biliary epithelium used as a positive control from normal F344 rat livers gave positive staining, as also did the AHF in F344 rat livers (Fig. 2). The histological features of basophilic foci observed in cotton rats were similar to those described in F344 rats (Harada et al., 1989; Eustis et al., 1990), but the eosinophilic, clear, mixed, or vacuolated foci seen in F344 rats aged ~ 6 months were not observed. In our earlier studies (Elangbam et al., 1989, 1991), no AHF of any type were observed in liver sections from more than 300 male and female wild cotton rats weighing between 80 and 120 g (approximately 23 months of age). Similarly, no AHF were observed in an earlier study of the livers of 13 captive female cotton rats aged 12 to 18 months (unpublished). GST-P has been found to be the most reliable endpoint marker for chemically induced foci. Ito et al. (1988, 1989a,b) usecl it as a marker in their medium term bioassays ofhepatocarcinogenicity and tested the hepa tocarcinogenic potential of more than 140 chemicals. Although aST-p is the most suitable marker, several others such as GGT, ATPase, and G6PD have also been tried. Zerban et al. (1989) emphasize that, to da te, there is no single universal marker for hepatic foci. When multiple markers are used at least
442
Fig. 2.
M. G. Paranjpe et ai.
An altered hepatocellular (ocus in an F344- rat. Positive staining with anti-GST-P antibody, haematoxylin counterstain. x 100.
10 per cent more AHF may be identified (Hendrich et al., 1987). None of the foci in this study stained positively for GST-P, although appropriate control slides did so. It is unknown whether these foci would have stained positively with other markers such as GGT, ATPase, or G6PD. As the observation of spontaneous basophilic foci in the cotton rats was purely incidental, liver tissues were not processed in a manner suitable for other markers such as GGT (Imaida et ai., 1983; Tamano et ai., 1983; Thamarit et al., 1985). Ward and Henneman (1990) recently reported that most of the spontaneously occurring basophilic foci in female F344 rats were GST-P negative. The spontaneous occurrence of AHF in mice is also well documented (Ward et al., 1983, 1986). A complete lack of staining of foci and even of biliary epithelium, as seen in the present study, has also been noted in B6C3Fl mouse livers with GST-P U. M. Ward, personal communication). As stated elsewhere (Niethammer, 1990), the cotton rat is classified under the "New World mice", subfamily Sigmodontinae. Phy1ogenetically, the cotton rat is far removed from the Old World Muridae, which includes commonly used laboratory rats and mice such as F344 and B6C3Fl, respectively. Cotton rats, unlike laboratory rats, have a gall bladder. B6 and C3H mice have very few spontaneous AHF, yet their spontaneous liver tumour rates are high; on the other hand, F344 rats have a high incidence of spontaneous AHF but their spontaneous liver tumour rate is low (Frith and Wiley, 1982; Moore, 1989). Neither spontaneous AHF nor hepatic turnoUTS have been documented in cotton rats. We have observed very few aged cotton rats (12-18 months) and none with liver tumours. It would be interesting to
Altered Hepatocellular Foci in Cotton Rats
443
examine more such animals (aged ~ 24 months) for liver tumours. Information pertaining to chemically induced hepatic neoplasms in cotton rats is scant. Miller and Miller (1955) stated that cotton rats were resistant to the development of hepatic neoplasms when exposed to 2-acetylaminofluorcne. In summary, this paper reports the spontaneous occurrence of basophilic AHF in captive aged male cotton rats as an incidental finding at necropsy. These foci were not immunoreactive for GST-P. Acknowledgments
This study was funded by grants from the Oklahoma Center for the Advancement of Science and Technology (HR9-017), the University Center for "Vater Research, Oklahoma State University, and the U.S. Air Force Office of Scientific Research, Air Force Systems Command, USAF (AFBSR91-0316). The authors acknowledge Dr J. M. Ward for GST-P staining and for reviewing the manuscript, and SherI Holesko for assistance in preparation of the manuscript. References
Bannasch, P., Griesemer, R. A., Anders, F., Becker, R., Cabral,]. R., Della Porta, G., Feron, V. J., Henschler, D., Ito, N., Kroes, R., :Magee, P. N., wlontesano, R., Napalkov, N. P., Neshow, S., Rao, G. N. and Wilbourn, J. (1986). Early preneoplastic lesions. In: Long- Term and Short- Term Assa.YSJor Carcinogens: A Critical AfJPraisal. R. Montesano, H. Bartsch, H. Vainaio,]. Wilbourn and H. Yamasaki, Eds, International Agency for Research on Cancer/International Programme Oil Chemical Safety, Commission of the European Communities, Lyon. Publication No. 83, pp. 85-101. EIangbam, C. S., Qualls, C. W. and LochmiIIer, R. 1. (1991). O-dealkylation of resorufin ethers as an indication ofhepatic cytochrome P-450 isoenzyme induction in the cotton rat (Sigmodon hispidus): a method for monitoring environmental contamination. Bulletin oJ Environmental Contamination and Toxicology, 47, 23-28. EIangbam, C. S., Qualls, C. W., Lochmiller, R. L. and Novak,]. (1989). Development of the cotton rat (Sigmodon Izispidus) as a biomonitor of environmental contamination with emphasis on hepatic cytochrome P-450 induction and population dynamics. Bulletin ql Environmental Contamination and 'Toxicology, 42, 482-488. Eustis, S. L., Boorman, G. A., Harada, T. and Popp,]. A. (1990). Liver. In: Patholo,t{Y (!/ the Fischer Rat. G. A. Boorman, S. 1. Eustis, M. R. Elwell and G. A. Montgomery, Eds, Academic Press, San Diego, pp. 71-94. Frith, C. H, and Wiley, L. (1982). Spontaneous hepatocellular neoplasms and hepatic haemangiosarcomas in several strains of mice. LaboratOl} Animal Science, 32, 157-162. Harada, T, Maronpot, R. R., Morris, R. W., Stitzel, K. A. and Boorman, G. A. (1989). NIorphological and stereological characterization of hepatic !(wi uf cellular alteration in control Fischer 344 rats. Toxicologic Pathology, 17, 579-593. Hendrich, S., Campbell, H. and Pitot, H. C. (1987). Quantitative stereological evaluation of four histochemical markers of altered foci in multistage hepatocareinogenesis in the rat. Carcinogenesis, 8, 1245-1250. Hendrich, S., Glauert, H. P. and Pitot, H. C. (!988). Dietary effects of initiation and promotion ofhepatocarcinogenesis in the rat. Journal f!/Cancer Re.l'ftlI'ch and Clinical Oncology, 114, 14-9-157. Hsu, S. M., Raine, L. and Fanger, H. (1981). Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison bet"veen ABC and unlabelled antibody (PAP) procedures. Journal of Histochemist~v lind G)tochemistl~Y, 29, 577-580.
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Imaida, K., Fukushima, S., Shirai, T., Ohtani, M., Nakanishi, K. and Ito, N. (1983). Promoting activities of butylated hydroxyanisole and butylated hydroxytoluel1e on 2-stage urinary bladder carcinogenesis and inhibition of y-glutamyl transpeptidase-positive foci development in the liver of rats. Carcinogenesis, 4, 895-899. Ito, N., Imaida, K., Hasegawa, R. and Tsuda, H. (1989a). Rapid bioassay methods for carcinogens and modifiers of hepatocarcinogenesis. Critical Reviews qf Toxicology, 19,385-415. Ito, N., Tatematsu, M., Hasegawa, R. and Tsuda, H. (1989b). Medium-term bioassay system for detection of carcinogens and modifiers of hepatocarcinogenesis utilizing the GST-P positive liver cell focus as an endpoint marker. Toxicologic Pathology, 17, 630-641. Ito, N., Tsuda, H., Tatematsu, M., Inoue, T., Tagawa, Y., Aoki, T., Uwagawa, S., Kagawa, M., Ogiso, T., Masue, T., Imaida, K., Fukushima, S. and Asamoto, M. (1988). Enhancing effects of various hepatocarcinogens on induction of preneoplastic glutathione S-transferase placental form positive foci in rats-An approach for new medium-term bioassay system. Carcinogenesis, 9, 387-394. Miller, E. C. and Miller, J. A. (1955). Biochemical investigations on hepatic carcinogenesis. Journal of the National Cancer Institute, 15, 1571-1990. Mitaka, T. and Tsukada, H. (1987). Sexual difference in the histochemical characteristics of "altered cell foci" in the liver of aged Fischer 344 rats. JajJanese Journal qfCancer Research (Gann), 78, 785-790. Moore, J. A. (1989). Discussion relative to regularity issues. To:r:icologic Pathology, 17, 729-735. Moore, M. A., Nakagawa, K., Satoh, K., Ishikawa, T. and Sato, K. (1987). Single GST-P positive liver cells-putative initiated hepatocytes. Carcinogenesis, 8, 4·83-4·86. Niethammer, J. (1990). New world mice (subfamily Sigmodontinae). In: Grzimek's Encyclopedia of Mammals, Vol. 3. McGraw-Hill Publishing Co., NY, p. 215. Ogawa, K., Onoe, T. and Takeuchi, M. (1981). Spontaneous occurrence of glutamyl transpeptidase-positive hepatocyte foci in 105-week-old Wistar and 72-week-old Fischer 344 male rats. Journal of the National Cancer Institute, 67, 4·07-412. Pitot, H. C., Campbell, H. A., Maronpot, R., Bawa, N., Rizvi, T. A., Xu, Y. H., Sargent, L., Dragan, Y. and Pyron, M. (1989). Critical parameters in the quantitation of the stages of initiation, promotion and progression in one model of hepatocarcinogenesis in the rat. Toxicologic Pathology, 17, 594-612. Sato, K., Kitahara, A., Satoh, K., Ishikawa, T., Tatematsu, M. and Ito, N. (1984). The placental form of glutathione S-transferase as a new marker protein for preneoplasia in rat chemical hepatocarcinogenesis. Japanese Journal of Cancer Research (Gann), 75, 199-202. Tamano, S., Tsuda, H., Fukushima, S., Masui, T., Hosoda, K. and Ito, N. (1983). Dose and sex dependent effects of 2-acety1aminofluorene, 3'-methyl-4-dimethylaminoazobenzene and DL-ethionine in induction of y-glutamyl-transpeptidase-positive liver cell foci in rats. Cancer Letters, 20, 313-321. Tatematsu, M., Mera, Y., Inoue, T., Satoh, K., Sato, K. and Ito, N. (1988). Stable phenotypic expression ofy-glutathione S-transferase placental type and unstable phenotypic expression of y-glutamyl transferase in rat liver preneoplastic and neoplastic lesions. Carcinogenesis, 9, 215-220. Tatematsu, M., Mera, Y., Ito, N., Satoh, K. and Sato, K. (1985). Relative merits of immunohistochemical demonstrations of placental, A, Band C forms of glutathione s-transferase as markers of altered foci during liver carcinogenesis in rats. Carcinogenesis 6, 1621-1626. Tatematsu, M., Tsuda, H., Shirai, '1'., Masui, T. and Ito, N. (1987). Placental glutathione S-transferase (GST-P) as a new marker for hepatocarcinogenesis: in vivo short-term screening for hepatocarcinogens. Toxicologic Pathology, 15, 60-68.
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Thamarit, W., Tatematsu, M" Ogiso, T., Mera, Y., Tsuda, H. and Ito, N. (1985). Dose-dependent effects of butylated hydroxyaniso1e, butylated hydroxytoluene and ethoxyquin in induction offoci of rat liver cells containing the placental form of glutathione S-transferase. Cancer Letters, 27, 295-303. Ward, J. M., Diwan, B. A., Ohshima, M., Hu, H., Schuller, H. M. and Rice, J. M. (1986). Tumor-initiating and promoting activities of di (2-ethylhexy1) phthalate in vivo and in vitro. Environmental Health Perspective, 65, 279-291. Ward, ]. M. and Henneman, J. R. (1990). Naturally-occurring age dependent glutathione s-transferase 1t immunoreactive hepatocytes in ageing female F344 rat liver as potential promotable targets for nongenotoxic carcinogens. Cancer Letters, 52, 187-195. Ward, J. M., Rice, J. M., Creasia, D., Lynch, P. and Riggs, C. (1983). Dissimilar patterns of promotion by di(2-ethylhexyl)phthalate and phenobarbital of hepatocellular neoplasia initiated by diethylnitrosamine in B6C2Fl mice. Carcinogenesis, 4, 1021-1029. Williams, G. M. (1980). The pathogenesis of rat liver cancer caused by chemical carcinogens. Biochimica et Biopll)lsica Acta, 605, 167-189. Zerban, H" Preussmann, R. and Bannasch, P. (1989). Quantitative morphometric comparison between the expression of two different marker enzymes in preneoplastic liver lesions induced in rats with low doses of N-nitrosodiethanolamine. Cancer Letters, 43, 99-104·.
Received,]une4·th,1993 ] [ AccejJted, September 6th, 1993
Camp. Path. 1993 Vol. 109,439-445
Spontaneous Altered Hepatocellular Foci (AHF) in Captive Aged Male Cotton Rats (Sig'l'nodon hispidus) M. G. Paranjpe, C. W. Qualls Jr, A. M. S. Chandra and R. L. Lochmiller* Department of VeterinalY Pathology, College of Veterinary Medicine and *Department oj Zoology, Oklahoma Slate University, Stillwater, OK 74078, U.S.A.
Su:m:mary
Spontaneous altered hepatocellular loci (AHF) of basophilic type were observed in adult male cotton rats. The histological features of these foci were similar to those observed in Fischer 344 rats. However, an immunohistochemical technique with antibody to glutathione S-transferase placental form (CST-P) fltiled to stain these foci. Also normal bile ducts were not immunoreactive for CST-Po The presence of a gall bladder ancl of non-CST-P immunoreactive liver foci and bile ducts suggests that these cotton rats are phylogenetically closer to mice than rats.
Introduction AHF occur spontaneously and increase in number as a function of age in laboratory rat strains such as Fischer 344 (F344), Wistar, and Sprague-Dawley (Ogawa et al., 1981; Hendrich et al., 1988). AHF are virtually non-existent in F344 rats less than 3 months old, are commonly seen by 6 to 12 months of age, and occur in virtually 100 per cent ofrats by 15 to 24· months of age (Harada et al., 1989; Eustis et al., 1990). Up to weaning, the incidence of AHF is similar in male and female rats, but thereafter it increases significantly in male rats (Mitaka and Tsukada, 1987; Pitot et al., 1989). AHF are considered by some authors to be pre-neoplastic and have been used as an index ofhepatocarcinogenicity (Williams, 1980; Bannasch et al., 1986). AHF appear earlier and increase in number in rats exposed to chemical hepatocarcinogens (Williams, 1980; Bannasch et al., 1986). AHF have been classified by their tinctorial qualities with haematoxylin and eosin (HE) stain into five types-basophilic, eosinophilic, clear, vacuolated and mixed (Harada et al., 1989; Eustis et al., 1990). Quantification of these foci after HE staining, by means of a computerassisted image analyzer in conjunction with a camera lucida (Harada et al., 1989) is possible, though difficult. End point markers such as garnma-glutamyl transpeptidase (GGT), adenosine triphosphatase (ATPase), glucose-6-phosphate dehydrogenase (G6PD) ancl eSTop have been tried as a basis for the identification of AHF by Correspondence to: C. W. Qualls, Jr. 0021-9975(93(080439 + 07 $08.00(0
©
1993 Academic Press Limited
440
M. G. Paranjpe et al.
immunohistochemical staining techniques. The GGT, ATPase, and G6PD markers have several disadvantages such as high background staining and elaborate methodology (Imaida et al., 1983; Tamano et al., 1983; Thamarit et al., 1985). GST-P, introduced as a novel endpoint marker enzyme by Sato et al. (1984), appears to be very sensitive and a more consistent marker for all types of AHF than other markers. The AHF demonstrated by this technique arc clearly distinguishable, there are minimal alterations in tinctorial qualities, there is a high contrast, they are uniformly stained, and they permit the recognition of all of the cellular stages in hepatocarcinogenesis (Tatematsu et al., 1985, 1987, 1988). Even single GST-P positive cells have been considered as potentially initiated cells (Moore et al., 1987). After immunohistochemical staining, the quantitation of AHF is facilitated by computerized semiautomated morphometric analysis equipment (Ito et al., 1989a). The cotton rat has an abundant distribution in the United States, a lifeexpectancy ofless than 6 months in the wild, and a confined area of movement of less than 1 hectare. We have used this species to evaluate the effects of environmental contamination on genotoxicity, metabolism (cytochrome P-450 induction), immunotoxicity, and population dynamics (Elangbam et al., 1989, 1991). The purpose of this report is to document the spontaneous occurrence of basophilic AHF in aged male cotton rats raised in a breeding colony. Materials and Methods
A small breeding colony of cotton rats originated from wild cotton rats captured in the vicinity ofStiHwater, OK, U.S.A. To maintain genetic diversity, wild cotton rats were occasionally introduced into the breeding colony. Six male cotton rats born in the breeding colony were killed by carbon dioxide euthanasia when 15 months old and weighing between 210 and 300 g. Throughout their life these rats were housed singly in polycarbonated wire top cages and maintained on a 12 h light cycle. All rats were fed Purina Rat Chow and given tap water ad libitum. At necropsy, the following tissues were collected in 10 per cent neutral buffered formalin: adrenal glands, brain, caecum, colon, duodenum, epididymis, heart, ileum, jejunum, kidneys, liver, lung, lymph node (popliteal), oesophagus, pancreas, pituitary, seminal vesicles, skeletal muscle, spleen, stomach, testes, thyroids and parathyroids, trachea and urinary bladder. All tissues were routinely processed, sectioned at 5-6 11m, and stained with HE. The avidin-biotin-peroxidase complex (ABC) method introduced by Hsu et al. (1981) was used to determine the location of GST-P binding in the liver. Affinity-purified biotin-labelled goat anti-rabbit IgG and ABC were obtained from Vector Laboratories, Burlingame, CA, U.S.A. Paraffin wax sections were routinely passed through petroleum benzene and a graded alcohol series and then treated sequentially with appropriate dilutions of normal goat serum, rabbit anti-GST-P, biotin-labelled goat anti-rabbit IgG and ABC. The sites of peroxidase binding were demonstrated by diaminobenzidine. Sections were counterstained by haematoxylin for microscopical examination. As a negative control for specificity of anti-GST-P antibody binding, pre-immune rabbit serum was used instead of antiserum. As a positive control, normal livers and AHF from F344 rats were stained.
Results and Discussion At necropsy, the livers of all six rats were mildly to moderately pale and only slightly mottled. Individual livers weighed between 9 and 15 g. None of the
Altered Hepatocellular Foci in Cotton Rats
Fig. I.
441
An altered hepatocellular focus in a cotton rat is outlined by arrowheads. Negative staining with ,tnti-GST-P antibody, haematoxylin counterstain. x 100.
rats had any other macroscopical lesions. Microscopical examination revealed basophilic AHF in each of the liver sections examined. These foci were randomly scattered, clearly delineated and characterized by increased localized cytoplasmic basophilia and hyperchromasia of nuclei. The basophilic cells were slightly smaller than the normal hepatocytes. In addition, all livers had mild to moderate fatty change. The immunohistochemical technique with antibody to GST-P failed to stain these foci (Fig. l). Even the biliary epithelium was nonimmunoreactive. The biliary epithelium used as a positive control from normal F344 rat livers gave positive staining, as also did the AHF in F344 rat livers (Fig. 2). The histological features of basophilic foci observed in cotton rats were similar to those described in F344 rats (Harada et al., 1989; Eustis et al., 1990), but the eosinophilic, clear, mixed, or vacuolated foci seen in F344 rats aged ~ 6 months were not observed. In our earlier studies (Elangbam et al., 1989, 1991), no AHF of any type were observed in liver sections from more than 300 male and female wild cotton rats weighing between 80 and 120 g (approximately 23 months of age). Similarly, no AHF were observed in an earlier study of the livers of 13 captive female cotton rats aged 12 to 18 months (unpublished). GST-P has been found to be the most reliable endpoint marker for chemically induced foci. Ito et al. (1988, 1989a,b) usecl it as a marker in their medium term bioassays ofhepatocarcinogenicity and tested the hepa tocarcinogenic potential of more than 140 chemicals. Although aST-p is the most suitable marker, several others such as GGT, ATPase, and G6PD have also been tried. Zerban et al. (1989) emphasize that, to da te, there is no single universal marker for hepatic foci. When multiple markers are used at least
442
Fig. 2.
M. G. Paranjpe et ai.
An altered hepatocellular (ocus in an F344- rat. Positive staining with anti-GST-P antibody, haematoxylin counterstain. x 100.
10 per cent more AHF may be identified (Hendrich et al., 1987). None of the foci in this study stained positively for GST-P, although appropriate control slides did so. It is unknown whether these foci would have stained positively with other markers such as GGT, ATPase, or G6PD. As the observation of spontaneous basophilic foci in the cotton rats was purely incidental, liver tissues were not processed in a manner suitable for other markers such as GGT (Imaida et ai., 1983; Tamano et ai., 1983; Thamarit et al., 1985). Ward and Henneman (1990) recently reported that most of the spontaneously occurring basophilic foci in female F344 rats were GST-P negative. The spontaneous occurrence of AHF in mice is also well documented (Ward et al., 1983, 1986). A complete lack of staining of foci and even of biliary epithelium, as seen in the present study, has also been noted in B6C3Fl mouse livers with GST-P U. M. Ward, personal communication). As stated elsewhere (Niethammer, 1990), the cotton rat is classified under the "New World mice", subfamily Sigmodontinae. Phy1ogenetically, the cotton rat is far removed from the Old World Muridae, which includes commonly used laboratory rats and mice such as F344 and B6C3Fl, respectively. Cotton rats, unlike laboratory rats, have a gall bladder. B6 and C3H mice have very few spontaneous AHF, yet their spontaneous liver tumour rates are high; on the other hand, F344 rats have a high incidence of spontaneous AHF but their spontaneous liver tumour rate is low (Frith and Wiley, 1982; Moore, 1989). Neither spontaneous AHF nor hepatic turnoUTS have been documented in cotton rats. We have observed very few aged cotton rats (12-18 months) and none with liver tumours. It would be interesting to
Altered Hepatocellular Foci in Cotton Rats
443
examine more such animals (aged ~ 24 months) for liver tumours. Information pertaining to chemically induced hepatic neoplasms in cotton rats is scant. Miller and Miller (1955) stated that cotton rats were resistant to the development of hepatic neoplasms when exposed to 2-acetylaminofluorcne. In summary, this paper reports the spontaneous occurrence of basophilic AHF in captive aged male cotton rats as an incidental finding at necropsy. These foci were not immunoreactive for GST-P. Acknowledgments
This study was funded by grants from the Oklahoma Center for the Advancement of Science and Technology (HR9-017), the University Center for "Vater Research, Oklahoma State University, and the U.S. Air Force Office of Scientific Research, Air Force Systems Command, USAF (AFBSR91-0316). The authors acknowledge Dr J. M. Ward for GST-P staining and for reviewing the manuscript, and SherI Holesko for assistance in preparation of the manuscript. References
Bannasch, P., Griesemer, R. A., Anders, F., Becker, R., Cabral,]. R., Della Porta, G., Feron, V. J., Henschler, D., Ito, N., Kroes, R., :Magee, P. N., wlontesano, R., Napalkov, N. P., Neshow, S., Rao, G. N. and Wilbourn, J. (1986). Early preneoplastic lesions. In: Long- Term and Short- Term Assa.YSJor Carcinogens: A Critical AfJPraisal. R. Montesano, H. Bartsch, H. Vainaio,]. Wilbourn and H. Yamasaki, Eds, International Agency for Research on Cancer/International Programme Oil Chemical Safety, Commission of the European Communities, Lyon. Publication No. 83, pp. 85-101. EIangbam, C. S., Qualls, C. W. and LochmiIIer, R. 1. (1991). O-dealkylation of resorufin ethers as an indication ofhepatic cytochrome P-450 isoenzyme induction in the cotton rat (Sigmodon hispidus): a method for monitoring environmental contamination. Bulletin oJ Environmental Contamination and Toxicology, 47, 23-28. EIangbam, C. S., Qualls, C. W., Lochmiller, R. L. and Novak,]. (1989). Development of the cotton rat (Sigmodon Izispidus) as a biomonitor of environmental contamination with emphasis on hepatic cytochrome P-450 induction and population dynamics. Bulletin ql Environmental Contamination and 'Toxicology, 42, 482-488. Eustis, S. L., Boorman, G. A., Harada, T. and Popp,]. A. (1990). Liver. In: Patholo,t{Y (!/ the Fischer Rat. G. A. Boorman, S. 1. Eustis, M. R. Elwell and G. A. Montgomery, Eds, Academic Press, San Diego, pp. 71-94. Frith, C. H, and Wiley, L. (1982). Spontaneous hepatocellular neoplasms and hepatic haemangiosarcomas in several strains of mice. LaboratOl} Animal Science, 32, 157-162. Harada, T, Maronpot, R. R., Morris, R. W., Stitzel, K. A. and Boorman, G. A. (1989). NIorphological and stereological characterization of hepatic !(wi uf cellular alteration in control Fischer 344 rats. Toxicologic Pathology, 17, 579-593. Hendrich, S., Campbell, H. and Pitot, H. C. (1987). Quantitative stereological evaluation of four histochemical markers of altered foci in multistage hepatocareinogenesis in the rat. Carcinogenesis, 8, 1245-1250. Hendrich, S., Glauert, H. P. and Pitot, H. C. (!988). Dietary effects of initiation and promotion ofhepatocarcinogenesis in the rat. Journal f!/Cancer Re.l'ftlI'ch and Clinical Oncology, 114, 14-9-157. Hsu, S. M., Raine, L. and Fanger, H. (1981). Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison bet"veen ABC and unlabelled antibody (PAP) procedures. Journal of Histochemist~v lind G)tochemistl~Y, 29, 577-580.
444
M. G. Paranjpe et al.
Imaida, K., Fukushima, S., Shirai, T., Ohtani, M., Nakanishi, K. and Ito, N. (1983). Promoting activities of butylated hydroxyanisole and butylated hydroxytoluel1e on 2-stage urinary bladder carcinogenesis and inhibition of y-glutamyl transpeptidase-positive foci development in the liver of rats. Carcinogenesis, 4, 895-899. Ito, N., Imaida, K., Hasegawa, R. and Tsuda, H. (1989a). Rapid bioassay methods for carcinogens and modifiers of hepatocarcinogenesis. Critical Reviews qf Toxicology, 19,385-415. Ito, N., Tatematsu, M., Hasegawa, R. and Tsuda, H. (1989b). Medium-term bioassay system for detection of carcinogens and modifiers of hepatocarcinogenesis utilizing the GST-P positive liver cell focus as an endpoint marker. Toxicologic Pathology, 17, 630-641. Ito, N., Tsuda, H., Tatematsu, M., Inoue, T., Tagawa, Y., Aoki, T., Uwagawa, S., Kagawa, M., Ogiso, T., Masue, T., Imaida, K., Fukushima, S. and Asamoto, M. (1988). Enhancing effects of various hepatocarcinogens on induction of preneoplastic glutathione S-transferase placental form positive foci in rats-An approach for new medium-term bioassay system. Carcinogenesis, 9, 387-394. Miller, E. C. and Miller, J. A. (1955). Biochemical investigations on hepatic carcinogenesis. Journal of the National Cancer Institute, 15, 1571-1990. Mitaka, T. and Tsukada, H. (1987). Sexual difference in the histochemical characteristics of "altered cell foci" in the liver of aged Fischer 344 rats. JajJanese Journal qfCancer Research (Gann), 78, 785-790. Moore, J. A. (1989). Discussion relative to regularity issues. To:r:icologic Pathology, 17, 729-735. Moore, M. A., Nakagawa, K., Satoh, K., Ishikawa, T. and Sato, K. (1987). Single GST-P positive liver cells-putative initiated hepatocytes. Carcinogenesis, 8, 4·83-4·86. Niethammer, J. (1990). New world mice (subfamily Sigmodontinae). In: Grzimek's Encyclopedia of Mammals, Vol. 3. McGraw-Hill Publishing Co., NY, p. 215. Ogawa, K., Onoe, T. and Takeuchi, M. (1981). Spontaneous occurrence of glutamyl transpeptidase-positive hepatocyte foci in 105-week-old Wistar and 72-week-old Fischer 344 male rats. Journal of the National Cancer Institute, 67, 4·07-412. Pitot, H. C., Campbell, H. A., Maronpot, R., Bawa, N., Rizvi, T. A., Xu, Y. H., Sargent, L., Dragan, Y. and Pyron, M. (1989). Critical parameters in the quantitation of the stages of initiation, promotion and progression in one model of hepatocarcinogenesis in the rat. Toxicologic Pathology, 17, 594-612. Sato, K., Kitahara, A., Satoh, K., Ishikawa, T., Tatematsu, M. and Ito, N. (1984). The placental form of glutathione S-transferase as a new marker protein for preneoplasia in rat chemical hepatocarcinogenesis. Japanese Journal of Cancer Research (Gann), 75, 199-202. Tamano, S., Tsuda, H., Fukushima, S., Masui, T., Hosoda, K. and Ito, N. (1983). Dose and sex dependent effects of 2-acety1aminofluorene, 3'-methyl-4-dimethylaminoazobenzene and DL-ethionine in induction of y-glutamyl-transpeptidase-positive liver cell foci in rats. Cancer Letters, 20, 313-321. Tatematsu, M., Mera, Y., Inoue, T., Satoh, K., Sato, K. and Ito, N. (1988). Stable phenotypic expression ofy-glutathione S-transferase placental type and unstable phenotypic expression of y-glutamyl transferase in rat liver preneoplastic and neoplastic lesions. Carcinogenesis, 9, 215-220. Tatematsu, M., Mera, Y., Ito, N., Satoh, K. and Sato, K. (1985). Relative merits of immunohistochemical demonstrations of placental, A, Band C forms of glutathione s-transferase as markers of altered foci during liver carcinogenesis in rats. Carcinogenesis 6, 1621-1626. Tatematsu, M., Tsuda, H., Shirai, '1'., Masui, T. and Ito, N. (1987). Placental glutathione S-transferase (GST-P) as a new marker for hepatocarcinogenesis: in vivo short-term screening for hepatocarcinogens. Toxicologic Pathology, 15, 60-68.
Altered Hepatocellular Foci in Cotton Rats
445
Thamarit, W., Tatematsu, M" Ogiso, T., Mera, Y., Tsuda, H. and Ito, N. (1985). Dose-dependent effects of butylated hydroxyaniso1e, butylated hydroxytoluene and ethoxyquin in induction offoci of rat liver cells containing the placental form of glutathione S-transferase. Cancer Letters, 27, 295-303. Ward, J. M., Diwan, B. A., Ohshima, M., Hu, H., Schuller, H. M. and Rice, J. M. (1986). Tumor-initiating and promoting activities of di (2-ethylhexy1) phthalate in vivo and in vitro. Environmental Health Perspective, 65, 279-291. Ward, ]. M. and Henneman, J. R. (1990). Naturally-occurring age dependent glutathione s-transferase 1t immunoreactive hepatocytes in ageing female F344 rat liver as potential promotable targets for nongenotoxic carcinogens. Cancer Letters, 52, 187-195. Ward, J. M., Rice, J. M., Creasia, D., Lynch, P. and Riggs, C. (1983). Dissimilar patterns of promotion by di(2-ethylhexyl)phthalate and phenobarbital of hepatocellular neoplasia initiated by diethylnitrosamine in B6C2Fl mice. Carcinogenesis, 4, 1021-1029. Williams, G. M. (1980). The pathogenesis of rat liver cancer caused by chemical carcinogens. Biochimica et Biopll)lsica Acta, 605, 167-189. Zerban, H" Preussmann, R. and Bannasch, P. (1989). Quantitative morphometric comparison between the expression of two different marker enzymes in preneoplastic liver lesions induced in rats with low doses of N-nitrosodiethanolamine. Cancer Letters, 43, 99-104·.
Received,]une4·th,1993 ] [ AccejJted, September 6th, 1993