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Potentiation of aflatoxin B1-induced hepatocarcinogenesis in the rat by pretreatment with buthionine sulfoximine
CANCER ELSEVIER
Cancer Letters 113 (1997) 103-109
Potentiation of aflatoxin B1-induced hepatocarcinogenesis in the rat by pretreatment with buthionine sulfoximine Shingo Hiruma a*1,Masatomo Kimura a,l, Kiyoko Lehmanna, Prathima Gopalan-Kriczkya, Guo-Zhong Qin”, Hisashi Shinozukab, Kiyomi Sate”, Prabhakar D. LotlikaraT* “Fels Institute for Cancer Research and Molecular Biology and Department of Biochemistry, Temple University School of Medicine, Philadelphia, PA 19140, USA bDepartment of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA “Second Department of Biochemistry, Hirosaki University School of Medicine, Hirosaki 036, Japan
Received25 November1996;revisionreceived12December1996;accepted12December1996
Abstract A single i.p. doseof aflatoxin Bi (AFB,) (1.0 and 2.0 mg/kg body wt)-induced hepatocarcinogenesiswith phenobarbital as a promoter has heen examined in young male Fischer rats. Immunohistochemical method has been employed to detect AFBiinduced glutathione S-transferaseplacental form (GST-P)-positive hepatic foci observedfrom 3 week and 10 week to 40-48 week periods. With 2.0 mg AFBi dosing, the number, area and volume occupied by GST-P-positive hepatic foci increased significantly and progressively from 3 week, 10 week and 48 week periods. In long term studies (40-48 weeks), 1.0 mg and 2.0 mg AFBi doselevels yielded linear responsein areaand volume occupied by AFBi-induced hepatic foci. Pretreatmentof rats with L-buthionine sulfoximine (BSO), a GSH depleter, at a dose of 4 mmol/kg body wt 4 and 2 h before 1.0 or 2.0 mg AFB, treatmentenhancedthe number, area and volume of GST-P-positive hepatic foci, increasesbeing the largest at shorter time periods (3 and 10 weeks)comparedto longer time periods (40 and 48 weeks).This report appearsto be the first example of an enhancedchemical induced hepatocarcinogenesisin a long term study in any experimental animals speciesby a GSH depleting agent. 0 1997 Elsevier Science Ireland Ltd. Keyword,s:
Aflatoxin B,; Hepatocarcinogenesis;Rat; Buthionine sulfoximine; Enzyme altered foci
1. Introduction Epidemiological data suggest that aflatoxin Bl (AFBt), a mycotoxin produced by Aspergihs jhvus, contamination of food is one of the etiological factors responsible for human liver cancer [ 1,2]. Rat is the * Corresponding author.
’ Presentaddress:Departmentof Pathology,Kinki University Schoolof Medicine,Osaka589,Japan.
most susceptible laboratory animal species to AF’Bi hepatocarcinogenicity ([3,4] and references therein). Metabolic activation of AFBi to its 8,9-oxide, mediated via cytochrome P-450 enzyme system, is a prerequisite for its covalent interaction with DNA and initiation of carcinogenesis [5,6]. Several studies including our own have indicated that cytosolic glutat&one S-transferases (GSTs) play an important role in modulating AFBi-DNA binding and AI%, carcinogenicity in various susceptible and resistant species by
0304-3835/97/$17.00 0 1997ElsevierScienceIrelandLtd. All rightsreserved PII SO304-3835(97)04602-8
inactivating the reactive AFB,-epoxide by conjugating with GSH [4,7- 181. Induction of hepatic GST\ with antioxidants such as butylated hydroxyanisole. butylated hydroxytoluene. ethoxyquin and dithiolthiones in the rat has been largely responsible for the inhibition of AFB,-DNA binding and AFB, hepatocarcinogenesis [4,8,12-16.191. Among various enzyme markers. y-glutamyl transpeptidase (GGT) was widely used as a marker for detecting the production of enzyme altered foci in the liver during carcinogenesis by a variety of chemicals including AFB, [2O-22). It now appears that a new enzyme marker, (XT-placental form (GST-P) is more sensitive than GGT for detecting preneoplastic and neoplastic lesions during liver carcinogenesis 123-261. Studies with diethylnitrosamine (DEN) hcpatocarcinogenesis suggested that the early GST-P-positive populations could be the precursors for pre neoplastic foci and nodules 1271.Recent studies have confirmed a precursor role of GST-P-positive cell in the development of enzyme-altered foci expressing GST-P 128). A single dose of hepatocarcinogenh \uch as AFB, and DEN produces in a dose-dependent manner GST-P-positive single hepatocytes in rats 48 h after a carcinogen administration 129-3 11. Subsequently. GST-P-positive hepatic foci have been developed in rats in 3 weeks after a single dose of AFB, 132.331. Pretreatment of rats with buthionine sulfoximine (BSO). a potent inhibitor of y-glutamylcysteine synthetase. a key enzyme in GSH biosynthesis, reduces hepatic GSH levels without significantly affecting cytochrome P-450 [34-361. Our laboratory has chown that hepatic AFB,-DNA binding and GST-Ppositive hepatocytes and foci in short experiment> can he enhanced by pretreatment of rats with BSO 133.37.381. In the present study. we have exarnmed the effect OI BSO pretreatment of rats on the sequential formation of AFB I-induced and phenobarbital (PB)-promoted GST-P-positive hepatic foci and neoplastic nodules for an extended period of 40-48 weeks. The data presented in this report indicate that BSO pretreatment of rats does enhance AFB,-induced hepatocarcinogenesis in the rat. To our knowledge, this is the first example of an enhanced chemical carcinogenesis in a long term study in any animal species by a GSH depleting agent.
2. Materials and methods
AFB, was purchased from Moravek Biochemical>. Brea. CA. PB was obtained from Amend Drug and Chemical Co.. Irvin&ton, NJ. Dimethylsulfoxide (DMSO) and I.-BSO were purchased from Sigma Chemical Co.. St. Louis, MO. Vectastain AH<’ k~r li)r immunohistochemical staining ~‘a\ obtained from Vector Laboratories, Inc.. Burlingame. CA. PuI.ification of rat GST-P and preparation of rat GST-P antibody in rabbit were as described previously 123). AIN-76A diet 1391 was purchased from Dyets. Inc.. Bethlehem. PA. All other chemicals were of reagent grade.
Male Fischer ruts, h -7 weeks old (90- 100 g hod\ wt). purchased from Charles River Breeding Laboratories, Wilmington, MA were housed in plastic cage\ with hard wood chips for bedding in an air-conditioned room ;I( 23 f 2°C with 21 I? h light/dark cycle. They were maintained on a Purina rodent laboratory chow and given tap water for drinking for I week before use. Animals to be treated with BSO were injected suhcutaneously with I.-BSO (4 mmol/kg body wt) dissolved in isotonic saline 4 h and 2 h before the administration of AFB,. Other animals were injected with isotonic saline only. Where indicated, AFB, dissolved in DMSO was injected intraperitoneally at concentrations of 1.O and 2.0 mgikg body wt. Control animals were injected with DMSO only. Four days after DMSO or AFB, administration. all animals received AIN-76A diet and 0.1% PB in their drinking water until the); were sacriticed.
Animals were sacriticed by exsanguination either at .3weeks, 10 weeks, 40 weeks or 48 weeks after DMSO or AFB, administration. Livers were weighed and sections of the liver were fixed in 10% phosphate-buf.fered formalin. Liver tissues were processed ~OI histopathological examination using H and E and immunohistochemical stainings [40,41]. By taking
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S. Hiruma et al. /Cancer Letters 113 (1997) 103-109
two sectionsfrom each lobe, about 9-12 cm2of liver from each animal was examined for immunohistochemical staining. Histopathological criteria for the diagnosis of hepatic lesions were according to published procedures [42,43]. GST-P-positive foci and nodules were counted with the use of a light microscope.Diameters of these foci were determined with an eye piece micrometer. Number of foci per cm’ was converted to number of foci per cc with the conditional stereologic estimator of Enzmann et al. [44] as modified recently with the ‘Spheres’ software program developedby Bayer A.G. in Germany. The significance of the data was statistically evaluated using either Student’s t-test or x2 test. Values of P < 0.05 were considered statistically significant.
3. Results
On the basis of our previous studies [30,31,33,37], doselevel of L-BSO (4 mmol/kg body wt) and time of L-BSO administration 4 h and 2 h before AFBi administration were selected for examination of AFBiinduced hepatic foci in the rat. Data on the effect of BSO pretreatment on AFBi-induced GST-P-positive hepatic foci are presented in Table 1. In these and subsequent studies presented in this report, 4 days after a single dose of either DMSO or AFBi administration, all animals were given 0.1% PB in their drinking water until sacrifice. When animals were sacrificed at 40 weeks after a single dose of AFB i (1 mg/kg body wt), number of GST-P-positive hepatic
foci and area of these foci were 14-l&fold higher than those in animals without AFB, dose. Pretreatment of animals with L-BSO before AFBi dosing increasedthe number, area and volume of these foci by 40-50% above those without BSO pretreatment. A summary of data on AFBi-induced hepatic foci in rats pretreated with or without BSO before being injected i.p. with a single doseof AFBi (2 mg!kg body wt) and sacrificed at 3, 10 and 48 weeks after AFB, dosing is presentedin Table 2. In the absenceof AFB, dosing, there were no detectable foci with diameters of > 100 pm at 3 and 10 weeksof sacrifice; a few foci appearedat the 48 week period but the area occupied by thesefoci was small. The number, areaand volume occupied by GST-P-positive foci increased significantly and progressively after AFBi dosing from 3 week, 10 week and 48 week periods. Similarly, BSO pretreatment produced dramatic increases in these parameters at all time periods. The number, area and volume of GST-P-positive foci in animals with BSO pretreatment were significantly higher than those treated with AFBi alone at all time periods examined. In all of these studies, there was no significant differenceeither in body wt. or liver wt. of theseanimals among various groups at different time periods examined (data not shown). In short term studies with AFBi, (3 weeks and 10 weeks), H and E staining yielded both eosinophilic and basophilic foci, with the latter type predominating. However, in long term experiments (40-48 weeks), mostly eosinophilic and clear cell foci could
Table 1 Effect of BSO pretreatment on APB,-induced GST-P-positive hepatic foci in ratsa Pretreatment with
BSO
APB1 1w
+ f
GST-P-positive hepatic foci (>lOO pm) No./cm’
Area of foci (mm*/cm*)
No./cm3
No./liver
Volume of foci (mm3/hver)
2.4 f l.Ob 32.8 k 3.3d 44.7 f 8.3’
0.09 It 0.03 1.74 rfr 0.25d 2.65 f 0.64’
175 f 93 1611 * 2~54~ 2405 f 501’
3264 f 1787 32 190 f 5002d 46724 + 8674’
1.60 f 0.64 34.64 f 4.59d 52.51 AZ14.22’
“Each group of consisted of 6-7 animals. All animals were sacrificed 40 weeks after APB, dosing. Experimental details are described in Section 2 bMean + SD. C.dDatahighly significant with values of ‘P < 0.01 and dP < 0.001 when compared with respective data of animals without BSO pretreatment or without APB, dosing.
Table 2 Effect of BSO pretreatment on AFBI-induced GST-P-positive hepatic foci in rats at 3, IO and 48 weeks after AFBl dosing” BSO Pretreatment
AFB,
2w
Time of sacdice (week)
No./cm’ -. +
-If
i-
+ t
-1.
t +
3 3 3 10 IO 10 48 48 48
---.
GST-P-positive hepatic foci (>lOO pm)
-___-.--. 0 2.0 i I.? 8.0 L 2.0’ 0 4,s ?r I.2 <)i)I 3. 74 -. A,& 3.0 It 0.3 26.5 + 3. I’ 40.6 f 9.3’
Area of fixi (mm’lcm’) 0 0.08 + 0.07” 1.20 t 0.w 11 0.2x i 0.08’ 0.8 I -t 0.22’ 0.13 t: 0.04 3 37 i_ 0.77’ 5.81 + 1.03’ --_--l__
No./crrs3
No/liver
-._-_ 0 134F82’ 380 2 122” 0 382 i I W” 632 f II?” 359 1- 100 1471 + 1315“ 2087 i: 4iS‘ ---
0 1708 i. 3712 t 0 5903 + 9407 k 73301 31112 rt 42603 +
--_-. Volume of foci (n&/liver) 964’ 1456d 1492;‘ 1368” 1184 6093’ 5984”
0 0.90 i 0.77” I I .37 k 6.00’ 0 4.31? l.YY 12.21 + 3.20’ 2.55 k 1.27 7 1.28 5 14.99’ 120.15 Yk25.32”
“Each group sacrificed at either 3, 10 or 48 weeks after tither DMSO or I? mp .4FB, dosing consisted of 6--7 animals. Other experimental details are described in Section 2. bMean 5~SD. ‘d,eData highly significant with values of ’ P < 0.02. “P <.. 0.01 and ‘P -: 0.001 when compared with rcspecttve data of ammals without I350 pretreatment or without AFBl dosing.
be observed.No tigroid foci could be detectedeither in short or long term studies (data not shown). In animals sacrificed at 40 week periods, there was no incidence of either neoplastic nodule or HCC in any animals (O/7)given 1 mg AFB, only. Pretreatment with BSO before 1 mg AFBl yielded only If7 animals with neoplastic nodules without any BCC. In animals sacrificedat 48 weeksafter 2 mg AFB, dosing, 50% of the animals (3/6) developed neoplastic nodules without any HCC. Even though pretreatment with BSO before 2 mg AFB, dosing produced neoplastic nodulesin 100%of animals (7/7) with 30% of animals yielding HCCs, these data appear to be statistically not significant. 4. Discussion
Previous studiesin femalesusing GGT as a marker 1451and our preliminary data in maleswith MT-P as a marker enzyme [46] demonstratedthat PB can be a promoter in a single dose administration of AFB, hepatocarcinogenesisin the rat. Our present results indicate that a single doseinjection of AFBl administered to young male Fischer rats followed by promotion with PB producedGST-P-positive hepatic foci in theseanimals. The number, area and volume of these foci increasedover a time period from 3 week and 10
weeks to 40-48 weeks after an administration of AFB, . Treatment with BSO before AFB Ldosing produced enhancementof these various parameters of hepatic foci at all time periods examined. Induction of hepatic GSTs by various antioxidants has been shown to be responsible for inhibition of AFB,-DNA binding and AFB, hepatocarcinogenesis in the rat [4,7,8,12-16,191. Like GSTs, hepatic GSH levels can also be modulated.Studiesfrom our laboratory have indicated that pretreatmentwith BSO before AFBl dosing depletes hepatic GSH tevels and increases hepatic AFB,-DNA binding and AFB,induced GST-P-positive hepatocytes in rats 48 h after AFB, dosing [3OJ I ,37,38]. Thus, enhanced GST-P-positive hepatic foci observed in the present study by pretreatmentwith BSO before AFB I administration of rats can be explained by sequential increase in AFB,-DNA binding and GST-P-positive hepatocytesseenpreviously f30,31,37,38]. In long term studies (40-48 weeks) I .Oand 2.0 mg AFB, dose levels yielded linear responsein area and volume occupied by AFB{-induced hepatic foci (Tables 1 and 2). The growth of AFBI-induced foci increased by BSO pretreatment of rats, this growth being largest at shorter comparedto longer time periods. Wogan and Newberne 1471after administering a single oral dose of AFB, (5 mg/kg body wt) to male
S. Hiruma et al. /Cancer Letters 113 (1997) 103-109
Fischer rats observed no incidence of neoplastic nodules or HCC at 55 weeks. In their carcinogenicity studies, however, no promoter was employed. Even though males are more susceptible than female Fischer rats to AFBi hepatocarcinogenesis [3,47] recent studies by Kraupp-Gras1et al. [45] in female Fischer rats given a single oral dose of AFBi (5 mg!kg body wt) followed with PB as a promoter yielded, at 55 weeks, 44% of animals with neoplastic nodules and 22% with HCCs. In the present study, no neoplastic nodules or HCCs could be seenin animals (O/7) administered 1 mg AFBi and examined at the 40 week period. When animals were sacrificed at the 48 week period, 50% (3/6) animals given a 2 mg AFBi dose level developed neoplastic nodules. It is possible that the larger AFBi dose and longer time periods from 40 weeks to 48 weeks might have been responsible for some hepatic foci developing into neoplastic nodules. Pretreatment with BSO increased the incidence of neoplastic nodules in animals dosed with either 1 or 2 mg AFBi. Development of HCC was seen in 2/7 animals given BSO before 2 mg AFBi. It is tempting to speculate that these incidences of animals with neoplastic nodules and HCCs could have been statistically significant if larger number of animals were to be used in the present study. It hasbeenobservedin previous studiesthat PB asa promoter enhanced only GGT-positive foci which were also eosinophilic and clear cell foci in rats administered a single dose of AFBi [45]. These and other studieshave shown that tigroid foci are GGT-negative [45]. Our presentresults indicating absenceof tigroid foci in any of the animals administered AFBi with or without BSO pretreatmentare in agreementwith these earlier studies [45,48]. Like previous studies with DEN [27,28], our present and previous data [29-311 strongly suggestthat AFBi-induced GST-P-positive hepatocytes are precursors of AFBi-induced hepatic foci during AFBi hepatocarcinogenesis. In summary,BSO pretreatmentbefore AFBi dosing increased not only the incidence but the growth of GST-P-positive hepatic foci in male Fischer rats from early to late time periods. It appearsthat this report is the first example of an enhancedhepatocarcinogenesisin a long term study in any experimental animal speciesby a GSH depleting agent.
107
Acknowledgements The expert assistance of Israel Beauchamps in preparation of this manuscript is gratefully acknowledged. This research was supported in part by grant CA-31641 to PrabhakarD. Lotlikar and by grant CA-12227 from the National CancerInstitute to the Fels Institute.
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Cancer Letters 113 (1997) 103-109
Potentiation of aflatoxin B1-induced hepatocarcinogenesis in the rat by pretreatment with buthionine sulfoximine Shingo Hiruma a*1,Masatomo Kimura a,l, Kiyoko Lehmanna, Prathima Gopalan-Kriczkya, Guo-Zhong Qin”, Hisashi Shinozukab, Kiyomi Sate”, Prabhakar D. LotlikaraT* “Fels Institute for Cancer Research and Molecular Biology and Department of Biochemistry, Temple University School of Medicine, Philadelphia, PA 19140, USA bDepartment of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA “Second Department of Biochemistry, Hirosaki University School of Medicine, Hirosaki 036, Japan
Received25 November1996;revisionreceived12December1996;accepted12December1996
Abstract A single i.p. doseof aflatoxin Bi (AFB,) (1.0 and 2.0 mg/kg body wt)-induced hepatocarcinogenesiswith phenobarbital as a promoter has heen examined in young male Fischer rats. Immunohistochemical method has been employed to detect AFBiinduced glutathione S-transferaseplacental form (GST-P)-positive hepatic foci observedfrom 3 week and 10 week to 40-48 week periods. With 2.0 mg AFBi dosing, the number, area and volume occupied by GST-P-positive hepatic foci increased significantly and progressively from 3 week, 10 week and 48 week periods. In long term studies (40-48 weeks), 1.0 mg and 2.0 mg AFBi doselevels yielded linear responsein areaand volume occupied by AFBi-induced hepatic foci. Pretreatmentof rats with L-buthionine sulfoximine (BSO), a GSH depleter, at a dose of 4 mmol/kg body wt 4 and 2 h before 1.0 or 2.0 mg AFB, treatmentenhancedthe number, area and volume of GST-P-positive hepatic foci, increasesbeing the largest at shorter time periods (3 and 10 weeks)comparedto longer time periods (40 and 48 weeks).This report appearsto be the first example of an enhancedchemical induced hepatocarcinogenesisin a long term study in any experimental animals speciesby a GSH depleting agent. 0 1997 Elsevier Science Ireland Ltd. Keyword,s:
Aflatoxin B,; Hepatocarcinogenesis;Rat; Buthionine sulfoximine; Enzyme altered foci
1. Introduction Epidemiological data suggest that aflatoxin Bl (AFBt), a mycotoxin produced by Aspergihs jhvus, contamination of food is one of the etiological factors responsible for human liver cancer [ 1,2]. Rat is the * Corresponding author.
’ Presentaddress:Departmentof Pathology,Kinki University Schoolof Medicine,Osaka589,Japan.
most susceptible laboratory animal species to AF’Bi hepatocarcinogenicity ([3,4] and references therein). Metabolic activation of AFBi to its 8,9-oxide, mediated via cytochrome P-450 enzyme system, is a prerequisite for its covalent interaction with DNA and initiation of carcinogenesis [5,6]. Several studies including our own have indicated that cytosolic glutat&one S-transferases (GSTs) play an important role in modulating AFBi-DNA binding and AI%, carcinogenicity in various susceptible and resistant species by
0304-3835/97/$17.00 0 1997ElsevierScienceIrelandLtd. All rightsreserved PII SO304-3835(97)04602-8
inactivating the reactive AFB,-epoxide by conjugating with GSH [4,7- 181. Induction of hepatic GST\ with antioxidants such as butylated hydroxyanisole. butylated hydroxytoluene. ethoxyquin and dithiolthiones in the rat has been largely responsible for the inhibition of AFB,-DNA binding and AFB, hepatocarcinogenesis [4,8,12-16.191. Among various enzyme markers. y-glutamyl transpeptidase (GGT) was widely used as a marker for detecting the production of enzyme altered foci in the liver during carcinogenesis by a variety of chemicals including AFB, [2O-22). It now appears that a new enzyme marker, (XT-placental form (GST-P) is more sensitive than GGT for detecting preneoplastic and neoplastic lesions during liver carcinogenesis 123-261. Studies with diethylnitrosamine (DEN) hcpatocarcinogenesis suggested that the early GST-P-positive populations could be the precursors for pre neoplastic foci and nodules 1271.Recent studies have confirmed a precursor role of GST-P-positive cell in the development of enzyme-altered foci expressing GST-P 128). A single dose of hepatocarcinogenh \uch as AFB, and DEN produces in a dose-dependent manner GST-P-positive single hepatocytes in rats 48 h after a carcinogen administration 129-3 11. Subsequently. GST-P-positive hepatic foci have been developed in rats in 3 weeks after a single dose of AFB, 132.331. Pretreatment of rats with buthionine sulfoximine (BSO). a potent inhibitor of y-glutamylcysteine synthetase. a key enzyme in GSH biosynthesis, reduces hepatic GSH levels without significantly affecting cytochrome P-450 [34-361. Our laboratory has chown that hepatic AFB,-DNA binding and GST-Ppositive hepatocytes and foci in short experiment> can he enhanced by pretreatment of rats with BSO 133.37.381. In the present study. we have exarnmed the effect OI BSO pretreatment of rats on the sequential formation of AFB I-induced and phenobarbital (PB)-promoted GST-P-positive hepatic foci and neoplastic nodules for an extended period of 40-48 weeks. The data presented in this report indicate that BSO pretreatment of rats does enhance AFB,-induced hepatocarcinogenesis in the rat. To our knowledge, this is the first example of an enhanced chemical carcinogenesis in a long term study in any animal species by a GSH depleting agent.
2. Materials and methods
AFB, was purchased from Moravek Biochemical>. Brea. CA. PB was obtained from Amend Drug and Chemical Co.. Irvin&ton, NJ. Dimethylsulfoxide (DMSO) and I.-BSO were purchased from Sigma Chemical Co.. St. Louis, MO. Vectastain AH<’ k~r li)r immunohistochemical staining ~‘a\ obtained from Vector Laboratories, Inc.. Burlingame. CA. PuI.ification of rat GST-P and preparation of rat GST-P antibody in rabbit were as described previously 123). AIN-76A diet 1391 was purchased from Dyets. Inc.. Bethlehem. PA. All other chemicals were of reagent grade.
Male Fischer ruts, h -7 weeks old (90- 100 g hod\ wt). purchased from Charles River Breeding Laboratories, Wilmington, MA were housed in plastic cage\ with hard wood chips for bedding in an air-conditioned room ;I( 23 f 2°C with 21 I? h light/dark cycle. They were maintained on a Purina rodent laboratory chow and given tap water for drinking for I week before use. Animals to be treated with BSO were injected suhcutaneously with I.-BSO (4 mmol/kg body wt) dissolved in isotonic saline 4 h and 2 h before the administration of AFB,. Other animals were injected with isotonic saline only. Where indicated, AFB, dissolved in DMSO was injected intraperitoneally at concentrations of 1.O and 2.0 mgikg body wt. Control animals were injected with DMSO only. Four days after DMSO or AFB, administration. all animals received AIN-76A diet and 0.1% PB in their drinking water until the); were sacriticed.
Animals were sacriticed by exsanguination either at .3weeks, 10 weeks, 40 weeks or 48 weeks after DMSO or AFB, administration. Livers were weighed and sections of the liver were fixed in 10% phosphate-buf.fered formalin. Liver tissues were processed ~OI histopathological examination using H and E and immunohistochemical stainings [40,41]. By taking
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S. Hiruma et al. /Cancer Letters 113 (1997) 103-109
two sectionsfrom each lobe, about 9-12 cm2of liver from each animal was examined for immunohistochemical staining. Histopathological criteria for the diagnosis of hepatic lesions were according to published procedures [42,43]. GST-P-positive foci and nodules were counted with the use of a light microscope.Diameters of these foci were determined with an eye piece micrometer. Number of foci per cm’ was converted to number of foci per cc with the conditional stereologic estimator of Enzmann et al. [44] as modified recently with the ‘Spheres’ software program developedby Bayer A.G. in Germany. The significance of the data was statistically evaluated using either Student’s t-test or x2 test. Values of P < 0.05 were considered statistically significant.
3. Results
On the basis of our previous studies [30,31,33,37], doselevel of L-BSO (4 mmol/kg body wt) and time of L-BSO administration 4 h and 2 h before AFBi administration were selected for examination of AFBiinduced hepatic foci in the rat. Data on the effect of BSO pretreatment on AFBi-induced GST-P-positive hepatic foci are presented in Table 1. In these and subsequent studies presented in this report, 4 days after a single dose of either DMSO or AFBi administration, all animals were given 0.1% PB in their drinking water until sacrifice. When animals were sacrificed at 40 weeks after a single dose of AFB i (1 mg/kg body wt), number of GST-P-positive hepatic
foci and area of these foci were 14-l&fold higher than those in animals without AFB, dose. Pretreatment of animals with L-BSO before AFBi dosing increasedthe number, area and volume of these foci by 40-50% above those without BSO pretreatment. A summary of data on AFBi-induced hepatic foci in rats pretreated with or without BSO before being injected i.p. with a single doseof AFBi (2 mg!kg body wt) and sacrificed at 3, 10 and 48 weeks after AFB, dosing is presentedin Table 2. In the absenceof AFB, dosing, there were no detectable foci with diameters of > 100 pm at 3 and 10 weeksof sacrifice; a few foci appearedat the 48 week period but the area occupied by thesefoci was small. The number, areaand volume occupied by GST-P-positive foci increased significantly and progressively after AFBi dosing from 3 week, 10 week and 48 week periods. Similarly, BSO pretreatment produced dramatic increases in these parameters at all time periods. The number, area and volume of GST-P-positive foci in animals with BSO pretreatment were significantly higher than those treated with AFBi alone at all time periods examined. In all of these studies, there was no significant differenceeither in body wt. or liver wt. of theseanimals among various groups at different time periods examined (data not shown). In short term studies with AFBi, (3 weeks and 10 weeks), H and E staining yielded both eosinophilic and basophilic foci, with the latter type predominating. However, in long term experiments (40-48 weeks), mostly eosinophilic and clear cell foci could
Table 1 Effect of BSO pretreatment on APB,-induced GST-P-positive hepatic foci in ratsa Pretreatment with
BSO
APB1 1w
+ f
GST-P-positive hepatic foci (>lOO pm) No./cm’
Area of foci (mm*/cm*)
No./cm3
No./liver
Volume of foci (mm3/hver)
2.4 f l.Ob 32.8 k 3.3d 44.7 f 8.3’
0.09 It 0.03 1.74 rfr 0.25d 2.65 f 0.64’
175 f 93 1611 * 2~54~ 2405 f 501’
3264 f 1787 32 190 f 5002d 46724 + 8674’
1.60 f 0.64 34.64 f 4.59d 52.51 AZ14.22’
“Each group of consisted of 6-7 animals. All animals were sacrificed 40 weeks after APB, dosing. Experimental details are described in Section 2 bMean + SD. C.dDatahighly significant with values of ‘P < 0.01 and dP < 0.001 when compared with respective data of animals without BSO pretreatment or without APB, dosing.
Table 2 Effect of BSO pretreatment on AFBI-induced GST-P-positive hepatic foci in rats at 3, IO and 48 weeks after AFBl dosing” BSO Pretreatment
AFB,
2w
Time of sacdice (week)
No./cm’ -. +
-If
i-
+ t
-1.
t +
3 3 3 10 IO 10 48 48 48
---.
GST-P-positive hepatic foci (>lOO pm)
-___-.--. 0 2.0 i I.? 8.0 L 2.0’ 0 4,s ?r I.2 <)i)I 3. 74 -. A,& 3.0 It 0.3 26.5 + 3. I’ 40.6 f 9.3’
Area of fixi (mm’lcm’) 0 0.08 + 0.07” 1.20 t 0.w 11 0.2x i 0.08’ 0.8 I -t 0.22’ 0.13 t: 0.04 3 37 i_ 0.77’ 5.81 + 1.03’ --_--l__
No./crrs3
No/liver
-._-_ 0 134F82’ 380 2 122” 0 382 i I W” 632 f II?” 359 1- 100 1471 + 1315“ 2087 i: 4iS‘ ---
0 1708 i. 3712 t 0 5903 + 9407 k 73301 31112 rt 42603 +
--_-. Volume of foci (n&/liver) 964’ 1456d 1492;‘ 1368” 1184 6093’ 5984”
0 0.90 i 0.77” I I .37 k 6.00’ 0 4.31? l.YY 12.21 + 3.20’ 2.55 k 1.27 7 1.28 5 14.99’ 120.15 Yk25.32”
“Each group sacrificed at either 3, 10 or 48 weeks after tither DMSO or I? mp .4FB, dosing consisted of 6--7 animals. Other experimental details are described in Section 2. bMean 5~SD. ‘d,eData highly significant with values of ’ P < 0.02. “P <.. 0.01 and ‘P -: 0.001 when compared with rcspecttve data of ammals without I350 pretreatment or without AFBl dosing.
be observed.No tigroid foci could be detectedeither in short or long term studies (data not shown). In animals sacrificed at 40 week periods, there was no incidence of either neoplastic nodule or HCC in any animals (O/7)given 1 mg AFB, only. Pretreatment with BSO before 1 mg AFBl yielded only If7 animals with neoplastic nodules without any BCC. In animals sacrificedat 48 weeksafter 2 mg AFB, dosing, 50% of the animals (3/6) developed neoplastic nodules without any HCC. Even though pretreatment with BSO before 2 mg AFB, dosing produced neoplastic nodulesin 100%of animals (7/7) with 30% of animals yielding HCCs, these data appear to be statistically not significant. 4. Discussion
Previous studiesin femalesusing GGT as a marker 1451and our preliminary data in maleswith MT-P as a marker enzyme [46] demonstratedthat PB can be a promoter in a single dose administration of AFB, hepatocarcinogenesisin the rat. Our present results indicate that a single doseinjection of AFBl administered to young male Fischer rats followed by promotion with PB producedGST-P-positive hepatic foci in theseanimals. The number, area and volume of these foci increasedover a time period from 3 week and 10
weeks to 40-48 weeks after an administration of AFB, . Treatment with BSO before AFB Ldosing produced enhancementof these various parameters of hepatic foci at all time periods examined. Induction of hepatic GSTs by various antioxidants has been shown to be responsible for inhibition of AFB,-DNA binding and AFB, hepatocarcinogenesis in the rat [4,7,8,12-16,191. Like GSTs, hepatic GSH levels can also be modulated.Studiesfrom our laboratory have indicated that pretreatmentwith BSO before AFBl dosing depletes hepatic GSH tevels and increases hepatic AFB,-DNA binding and AFB,induced GST-P-positive hepatocytes in rats 48 h after AFB, dosing [3OJ I ,37,38]. Thus, enhanced GST-P-positive hepatic foci observed in the present study by pretreatmentwith BSO before AFB I administration of rats can be explained by sequential increase in AFB,-DNA binding and GST-P-positive hepatocytesseenpreviously f30,31,37,38]. In long term studies (40-48 weeks) I .Oand 2.0 mg AFB, dose levels yielded linear responsein area and volume occupied by AFB{-induced hepatic foci (Tables 1 and 2). The growth of AFBI-induced foci increased by BSO pretreatment of rats, this growth being largest at shorter comparedto longer time periods. Wogan and Newberne 1471after administering a single oral dose of AFB, (5 mg/kg body wt) to male
S. Hiruma et al. /Cancer Letters 113 (1997) 103-109
Fischer rats observed no incidence of neoplastic nodules or HCC at 55 weeks. In their carcinogenicity studies, however, no promoter was employed. Even though males are more susceptible than female Fischer rats to AFBi hepatocarcinogenesis [3,47] recent studies by Kraupp-Gras1et al. [45] in female Fischer rats given a single oral dose of AFBi (5 mg!kg body wt) followed with PB as a promoter yielded, at 55 weeks, 44% of animals with neoplastic nodules and 22% with HCCs. In the present study, no neoplastic nodules or HCCs could be seenin animals (O/7) administered 1 mg AFBi and examined at the 40 week period. When animals were sacrificed at the 48 week period, 50% (3/6) animals given a 2 mg AFBi dose level developed neoplastic nodules. It is possible that the larger AFBi dose and longer time periods from 40 weeks to 48 weeks might have been responsible for some hepatic foci developing into neoplastic nodules. Pretreatment with BSO increased the incidence of neoplastic nodules in animals dosed with either 1 or 2 mg AFBi. Development of HCC was seen in 2/7 animals given BSO before 2 mg AFBi. It is tempting to speculate that these incidences of animals with neoplastic nodules and HCCs could have been statistically significant if larger number of animals were to be used in the present study. It hasbeenobservedin previous studiesthat PB asa promoter enhanced only GGT-positive foci which were also eosinophilic and clear cell foci in rats administered a single dose of AFBi [45]. These and other studieshave shown that tigroid foci are GGT-negative [45]. Our presentresults indicating absenceof tigroid foci in any of the animals administered AFBi with or without BSO pretreatmentare in agreementwith these earlier studies [45,48]. Like previous studies with DEN [27,28], our present and previous data [29-311 strongly suggestthat AFBi-induced GST-P-positive hepatocytes are precursors of AFBi-induced hepatic foci during AFBi hepatocarcinogenesis. In summary,BSO pretreatmentbefore AFBi dosing increased not only the incidence but the growth of GST-P-positive hepatic foci in male Fischer rats from early to late time periods. It appearsthat this report is the first example of an enhancedhepatocarcinogenesisin a long term study in any experimental animal speciesby a GSH depleting agent.
107
Acknowledgements The expert assistance of Israel Beauchamps in preparation of this manuscript is gratefully acknowledged. This research was supported in part by grant CA-31641 to PrabhakarD. Lotlikar and by grant CA-12227 from the National CancerInstitute to the Fels Institute.
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