Journal of the American College of Nutrition
ISSN: 0731-5724 (Print) 1541-1087 (Online) Journal homepage: http://www.tandfonline.com/loi/uacn20
Genetics and diet: synergism in hepatocarcinogenesis in rats. M F Melhem, M E Kazanecki, K N Rao, H W Kunz & T J Gill 3rd To cite this article: M F Melhem, M E Kazanecki, K N Rao, H W Kunz & T J Gill 3rd (1990) Genetics and diet: synergism in hepatocarcinogenesis in rats., Journal of the American College of Nutrition, 9:2, 168-173, DOI: 10.1080/07315724.1990.10720367 To link to this article: http://dx.doi.org/10.1080/07315724.1990.10720367
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Date: 20 June 2016, At: 15:06
Genetics and Diet: Synergism in Hepatocarcinogenesis in Rats Mona F. Melhem, Mary E. Kazanecki, Kalipatnapu N. Rao, Heinz W. Kunz, and Thomas J. Gill HI Veterans Administration Medical Center, Pittsburgh (M.F.M.) and Department of Pathology, University of Pittsburgh, School of Medicine, Pittsburgh (M.F.M., M.E.K., K.N.R., H.W.K., and T.J.G.)
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Key words: genetics, growth and reproduction complex, diet, choline deficiency, hepatocarcinogenesis In previous studies we have shown that male rats carrying genes controlling growth and reproduction (gre) linked to the major histocompatibility complex (MHC) to be more susceptible to N-2-acetylaminofluorene than rats without gre genes. In the present studies we show that by manipulation of the diet of gre. rats, hepatocarcinogenesis induced by another carcinogen can be altered. Male rats with the gre gene (R16) and wild type (ACP) were initiated with diethylnitrosamine (DEN) (200 mg/kg body weight). Some were fed laboratory chow (LC) for 9 months; others were fed a choline-supplemented (CS) or a choline-deficient (CD) diet. The rats were killed at various time periods and the liver sections were stained with H&E and for γ-glutamyltranspeptidase (GGT). After 9 months on LC, the livers of R16 showed greater size and number of GGT-positive foci, bile duct proliferation, cellular atypia, cirrhosis, and nodular hyperplasia than the ACP. While the first hepatocellular carcinoma in R16 fed either a CS or LC was seen at 9-10 months, one R16 rat fed a CD diet had liver cancer at 4 months. On a CS diet the R16 showed greater GGT-positive foci at 2 months than the ACP. On a CD diet the R16 showed even greater size and number of GGT-positive foci. At 12 months, 15 of 22 (68%) of the Rl 6 rats on a CD diet had liver cancer and seven of 24 (29%) of the R16 on a CS diet. Of the ACP, none of 15 (0%) on CS and one of 18 (6%) on CD diet had liver tumors. The results show that the gre genes confer high susceptibility to liver cancer, which is enhanced by a CD diet, suggesting synergism between genetics and diet.
INTRODUCTION The clinical literature indicates the existence of dietary and hereditary influences on many types of can cer. Differences in the incidence of various cancers oc curring in human populations are often correlated with differences in the diet [1]. In addition, in humans there are known to be genotypes that predispose to cancer development, cancer family syndromes, and epidemiologie relationships between certain neoplasms and specific HLA types [2]. The rates of colon and rectal cancer in European immigrants to the United States more closely resemble those of the United States than those of their countries of origin [3]. There is a higher risk of bowel cancer [4] and a lower risk of stomach cancer [5] in Japanese immigrants to Hawaii who eat a Western diet. The question then arises: Does diet alter the progression of cancer in susceptible individuals? Be cause of the highly complex nature of neoplastic dis eases and variations in dietary habits in humans, it is best
to examine this question by employing experimental animal model systems. The rat is the preferred animal in studies dealing with carcinogenesis, and we have one of the world's major resources of inbred strains, congenic lines, and recombinants. The discovery of the growth and reproduction com plex (gre) in our laboratory was the first demonstration of genes affecting growth and development linked to the major histocompatibility complex (MHC) since the dis covery of the i-haplotypes of the mouse [6]. The in creased susceptibility of gm rats to the induction of hepatic cancer by N-2-acetylaminofluorene is one of the most interesting and potentially significant observations we have made in recent years [7]. The availability of congenic gre, strains and their wild type counterparts and the extensive work in our department on the promotion of hepatic cancer by a choline-deficient diet (CD) [8] gave us the opportunity to study the interaction of dietary influences and genetic predisposition to the development of hepatic cancer.
Addressreprintrequeststo Thomas J. Gill III, M.D., Chairman, Department of Pathology, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15261.
Journal of the American College of Nutrition, Vol. 9, No. 2, 168-173 (1990) © 1990 John Wiley & Sons, Inc.
CCC 0731-5724/90/020168-06$04.00
Genetics, Diet, and Hepatocarcinogenesis Table 1. Histopathology of Livers of Rats Initiated with DEN and Fed Laboratory Chow for 9 Months
Strain
Number of foci/cm2
Foci area/ section area
ACP
2.4 + 0.5
0.16 ±0.01
R16
49.3 ± 22.3*
2.95 ± 1.3*
Cellular atypia
Cirrhosis
Nodular hyperplasia
0-+
0-+
0
0
++
+++
+
+
Bile duct proliferation
*p < 0.05 considered significant when compared with the ACP. Values are mean ± SE of six animals. ACP = wild type rat strain without gre. R16 = rat strains with gre. DEN = diethylnitrosamine. The degree of bile duct proliferation, cellular atypia, cirrhosis, and nodular hyperplasia were scored blindly on a scale of 0 to +++.
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Table 2. Histopathology of Livers of Rats Initiated with DEN and Fed Either a CS or a CD Diet for 2 Months Strain
Diet
Group
Number of foci/cm2
Foci area/ section area
ACP ACP R16 R16
CS CD CS CD
a b c d
1.1 ±0.3 4.8 ± 0.4a* 5.1 ±1.1» 12.9 ± 5 . 5 "
0.024 + 0.01 0.152 + 0.04" 0.226 ± 0.1 a 0.545 ±0.17 a *
*p < 0.05 considered significant when compared with the group indicated with a superscript. Values are mean ± SE of four animals. CS - choline-supplemented diet. CD = choline-deficient diet. ACP = wild type rat strain without gre. R16 = rat strain with gre. DEN = diethylnitrosamine.
Our study is based on the evidence that cell prolifera tion is a prerequisite for the development of cancer [8,9], and rats with the grc. show increased rates of cell proliferation in the liver [7]. In models of hepatocar cinogenesis, promotion typically involves stimulation of hepatocyte proliferation, for example, by partial hepatectomy [10] or by a CD diet [8]. In this study we have tested the hypothesis that the effects of genetics and diet on cell proliferation are additive, and when they are combined they enhance promotion of diethylnitrosamine (DEN)-induced hepatocellular carcinoma to a greater ex tent than either factor alone.
but does not have grc. genes [11]. AU rats, 4-18 rats per group, were 6-8 weeks of age at the beginning of the experiment. The ACP males weighed about 250 g, and the R16 males of the same age weighed 150 g, the smaller size of the R16 reflecting their growth and reproduction complex [6]. In addition fo laboratory chow (LC), two semisynthetic semipurified diets, a cholinesupplemented (CS) and a choline-deficient (CD) diet, were used (Dyets Ine, Bethlehem, PA). The composition and preparation of the CS and CD diets were described elsewhere [12]. Experimental Protocol
MATERIALS AND METHODS Animals and Diets The response of the liver to DEN-induced hepatocar cinogenesis was studied in two strains of male rats ob tained from our colony at the University of Pittsburgh School of Medicine. The susceptible R16 strain is a recombinant (A° B" Da E" gre) between RIO (An B1 D1 E~ grc) and ACP (Aa Ba D" E~ grc+). ACP, the normal counterpart of the R16 strain, has identical MHC genes
The experimental protocol is designed to understand the development of DEN-induced hepatocarcinogenesis due to grc genes alone, CD diet alone, and the combined effects of grc. genes and CD diet. Both ACP and R16 male rats were injected intraperitoneally with a single dose of DEN (200 mg/kg of body weight) (Sigma Chemical Company, St. Louis, MO). The animals were divided into three groups and placed in wire-bottomed steel cages, two per cage in an air-conditioned room with temperature and humidity controls and with a 12-hr light (7 a.m. to 7 p.m.) and dark (7 p.m. to 7 a.m.) cycle. All
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Genetics, Diet, and Hepatocarcinogenesis
B Fig. 1. Histopalhological changes in livers of R16 and ACP rats.
rats were allowed to eat LC for the next 2 weeks; then the first group of rats had free access to LC. the second group a CS diet, and the third group a CD diet. The first group was sacrificed after 9 months, and the second and third groups were sacrificed at different time intervals. Complete autopsies were performed and the livers and tumors, if any, were processed for histological examina tion.
Other Procedures
Histology
The results of the histological examination of liver of R16 and ACP rats initiated with a single necrogenic dose of DEN and fed LC for 9 months are presented in Table 1. The number as well as the size of GGT-positive foci were greater in RI6 strain when compared with the ACP strain. The architecture of the livers of the R16 was dis rupted with fibrosis, cirrhosis, and hyperplastic nodules. These livers showed severe cellular atypia with large nuclei and prominent nucleoli. There were many binucleated cells, increased mitotic figures (2-3/high power field), and bile duct and oval cell proliferation. The above changes were rare or absent in the ACP rat livers.
Liver and tumor sections were stained (H&E) for his tological examination. In addition, liver sections were stained for γ-glutamyltranspeptidase (GGT) (7]. Liver sections were evaluated for cirrhosis, nodules, fatty in filtrate, and tumors. Sections were also scored for GGTpositive foci, bile duct proliferation, disruption of hepatic architecture, and degree of cellular atypia using a scale of 0 to +++ [13]. The GGT-positive foci were measured and quantitated |13] and expressed as foci area/section area and number of foci/cm2 of liver sec tions. respectively.
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Statistical analysis of the data was performed using analysis of variance, and the difference between means was considered significant if p < 0.05 [ 14|.
RESULTS
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Genetics, Diet, and Hepatocarcinogenesis
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Fig. 2. GGT staining of livers of R16 (A) and ACP (B) rats on a CD diet for 4 months after initiation with DEN showing a large GGT-positive focus in the R16 liver. H&E-stained sections of R16 (C) and ACP (D) livers showing increased fatty changes, cellular atypia, and bile duct proliferation in R16 (x200).
DEN-induced hepatocarcinogenesis in R16 and ACP with promotion (CD diet) and without promotion (CS diet) was examined (the results are presented in Table 2). After 2 months of feeding either a CS or CD diet, the livers of R16 rats showed significantly greater number and size of GGT-positive foci than those of ACP. When promoted with a CD diet, both ACP and R16 showed greater number and size of GGT-positive foci compared to the same rats fed a CS diet. Therefore, R16 on a CD diet showed much greater enzymatically altered foci than either R16 on a CS diet or ACP on a CD diet. At 4 months the histological differences between R16 and ACP rats fed CD, i.e., fatty changes, cellular atypia, and bile-duct and oval cell proliferation, became more prominent (Fig. 1). A greater degree of cirrhosis, fatty change, and nodularity, and one hepatocellular car cinoma were seen among R16 rats (Fig. 2). As early as 12 months, 68% of the R16 rats on CD and 29% on CS diets developed hepatocellular carcinoma, while only
one ACP rat (6%) on CD diet had a similar tumor and none on the CS diet. The lab chow diet gave similar results to the CS diet, with 30% R16 and 0% ACP developing liver malignancies (Table 3).
DISCUSSION Cell proliferation is a prerequisite for cancer develop ment [8,9]. Rats homozygous for the gre genes have a higher rate of cell proliferation and are more susceptible to the development of chemically induced hepatocellular carcinoma [7]. CD diet acts as a promoter by increasing liver cell proliferation [8]. The effects of grc genes and CD diet on cell proliferation should be synergistic in the development of DEN-induced hepatocarcinomas. DEN is a hepatocarcinogen which, when administered to rats intraperitoneally in a single necrogenic dose, produces hepatocellular carcinoma in 25% of albino strain of rats
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Genetics, Diet, and Hepatocarcinogenesis Table 3. The Difference in Hepatocellular Carcinoma Incidence in In bred Strains of Rat and the Effect of Different Diets on Tumor Development 12 Months after DEN
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Hepatocellular carcinoma Strain
Diet
n
n
%
R16
LC CS CD
9 24 22
3 7 15
30 29 68
ACP
LC CS CD
11 15 18
0 0 1
0 0 6
DEN = diethylnitrosamine; LC = lab chow; CD = choline-deficient; CS = choline-supplemented; R16 = grc-bearing rats; ACP = wild counterpart.
fed a lab chow diet after 85-115 weeks [15]. Its latent period with CS feeding is not known. For the R16 strain of rats this latent period appears to be 10 months for both the CS and the lab chow diet. Within 9 months of initiation with a single necrogenic dose of DEN and feeding LC, rats homozygous for gre (RI6) developed greater number and size of GGT-positive foci, bile duct and oval cell proliferation, and disrup tion of the lobular architecture than control rats (ACP) fed LC (Table 1). Similarly, the R16 rats on a control CS diet developed these changes in the liver to a greater extent than ACP rats on CS diet. These preneoplastic and hyperplastic changes are known to be associated with the eventual development of cancer. Therefore, the enhanced susceptibility of R16 rats without dietary promotion can be attributed to the gre genes. Both R16 and ACP rats showed greater preneoplastic changes when fed CD diet than when fed CS diet (Table 2, Fig. 1). Therefore, CD diet acts as a promoter independently of genetic strain. Rats carrying the gre and fed a CD diet, i.e., having both genetic susceptibility and dietary promotion, showed greater preneoplastic changes than rats having either genetic susceptibility alone (R16 rats fed CS) or dietary promotion alone (ACP rats fed CD). Also, the incidence of hepatocellular carcinoma at 12 months was more than doubled among R16 rats fed CD, when com pared to those fed either a CS diet or lab chow. There fore, the effects of the gre genes and CD diet are synergistic in DEN-induced hepatocarcinogenesis. The mechanism by which gre genes confer enhanced susceptibility to cancer and the synergism between gre genes and CD diet in hepatocarcinogenesis is not com pletely understood, but may be related to increased cell proliferation.
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CONCLUSION The gre genes confer unusually high susceptibility to the carcinogen DEN, resulting in liver tumors. This sus ceptibility is enhanced by a choline-deficient diet. The combination of the effects on cell proliferation of the gre and a choline-deficient diet provide a unique model for the study of the synergism between genetic predisposi tion and dietary manipulation in carcinogenesis.
ACKNOWLEDGMENTS This work was supported by grants from the National Institute of Health (CA 18659 and HD 08662). The Anafred N. Halpem young investigator award to Mona F. Melhem and Mary E. Kazanecki by the American Col lege of Nutrition is gratefully appreciated.
REFERENCES 1. National Research Council: "Diet, Nutrition, and Cancer." Washington, DC: National Academy Press, 1982. 2. Chaganti RSK, German JL: "Genetics in Clinical Oncol ogy." Oxford: Oxford University Press, 1985. 3. Haenszel W: Cancer mortality among the foreign born in the United States. J Nati Cancer Inst 26:37-132, 1961. 4. Haenszel W, Berg JW, Segi M, Kurihara M, Locke FB: Large-bowel cancer in Hawaiian Japanese. J Nati Cancer Inst 51:1765-1779, 1973. 5. Haenszel W, Kurihara M, Segi M, Lee RKC: Stomach cancer among Japanese in Hawaii. J Nati Cancer Inst 49:969-988, 1972.
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Genetics, Diet, and Hepatocarcinogenesis 6. Gill TJ III, Kunz HW: Gene complex controlling growth and fertility linked to the major histocompatibility com plex in the rat. Am J Pathol 96:185-206, 1979. 7. Rao KN, Shinozuka H, Kunz HW, Gill TJ III: Enhanced susceptibility to a chemical carcinogen in rats carrying MHC-linked genes influencing development (gre). Int J Cancer 34:113-120, 1984. 8. Giambaressi LI, Katyal SL, Lombardi B: Promotion of liver carcinogenesis in the rat by a choline-devoid diet: role of liver cell necrosis and regeneration. Br J Cancer 46:825-829, 1982. 9. Färber E: Chemical carcinogenesis. Am J Pathol 106:271— 296, 1982. 10. MacDonald RA, Rogers AE: Control of regeneration of the liver: lack of effect of plasma from partially hepatectomized, cirrhotic and normal rats upon deoxynribonucleic acid synthesis and mitosis in rat liver. Gastroenterology 41:33-38, 1961. 11. Gill TJ III: Immunogenetic control of pregnancy and development. In Talwar GP (ed): "Symposium on Con traception Research for Today and the Nineties." New York: Springer-Verlag, pp 161-169, 1988.
12. Young RJ, Lucas CC, Patterson JM, Best CH: Lipotropic dose-response studies in rat: comparisons of choline, betaine and methionine. Can J Biochem Physiol 34:713720, 1956. 13. Melhem MF, Rao KN, Kunz HW, Gill TJ III: Suscep tibility of grc-bearing Rats to DEN and its relationship to the HMP-pathway. In Feo F, Pani P, Columbano A, Garcea R (eds): "Chemical Carcinogenesis: Models and Mechanisms." New York: Plenum, pp 485-493, 1988. 14. Steel RGD, Torrie JH: "Principles and Procedures of Statistics: A Biomedicai Approach," 2nd ed. New York: McGraw-Hill, 1980. 15. Craddock VM: Effect of a single treatment with the alkylating carcinogens dimethylnitrosamine, diethylnitrosamine, and methyl methane sulphonate on liver regeneration after partial hepatectomy. Chem Biol Interac tions 10:313-321, 1975.
Received September 1988; revision accepted October 1989.
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