Relative contribution of dietary protein level and aflatoxin B1 dose in generation of presumptive preneoplastic foci in rat liver

Male weanling F344 rats were orally gavaged with aflatoxin B1 (AFB1) in daily doses of 200, 235, 270, 300, and 350 micrograms/kg/day for a total of 10 doses over a 12-day period, and then 1 week after the last dose they were fed diets of varying protein (casein) content to compare the contribution of AFB1 dose and dietary protein level on the development of presumptive preneoplastic gamma-glutamyltransferase-positive (GGT+) foci in rat liver. All animals were fed the same 20% dietary casein level during the dosing period. One week after the end of the dosing period, one-half of the animals in each dose group were then continued on the 20% casein diet for the entire 12-week foci-development period; the remaining half in each dose group were fed lower levels of dietary casein during the foci-development period for the increasing AFB1 dose groups (20, 16, 12, 8, and 4% casein for the 235-, 250-, 270-, 300-, and 350-micrograms/kg/day groups, respectively). The AFB1 dose groups used were determined in a preliminary experiment. In this previous experiment, a clearly discernible threshold dose at about 100-150 micrograms AFB1/kg/day (below which no GGT+ foci were observed) and a steep slope between 150 and 400 micrograms/kg/day were produced. In the second experiment, while the expected positive slope of (AFB1) dose versus (GGT+ foci) response relationship was found for animals fed the 20% casein diet, the dose response for the animals fed the lower levels of casein was eliminated, providing evidence that nutrient intake during the postdosing foci development is more rate limiting toward the development of these preneoplastic lesions than is the carcinogen dose.     

Tumor promotion by the peroxisome proliferator nafenopin involving a specific subtype of altered foci in rat liver

The involvement of tumor promotion in the hepatocarcinogenic action of peroxisome proliferators has not been generally accepted. We studied the effect of nafenopin (NAF) as a model compound in a two-stage initiation-promotion protocol. Carcinogenesis was initiated by a single dose of aflatoxin B1 (AFB1) in female (AFB1, 5 mg/kg) and male (AFB1, 2 mg/kg) Wistar rats. After recovery NAF was fed via the diet, providing a daily dose of 100 mg/kg body weight. Phenobarbital (PB) (50 mg/kg body weight) was fed to female rats as a positive control. The following results were obtained. (a) At weeks 40, 55, 59, and 70, significantly more and larger liver tumors were present in AFB1-NAF-treated rats than in rats receiving either compound alone, and the effect of the combined treatment was clearly more than additive, in three independent experiments including both sexes. This suggests tumor promotion by NAF. Male rats responded more strongly than females. Similarly, PB enhanced the yield of liver tumors. Histologically, tumors were hepatocellular adenoma or carcinoma. In group AFB1-PB the majority consisted of eosinophilic and glycogenstoring cells. However, adenoma and carcinoma of groups AFB1-NAF and O-NAF consisted of weakly basophilic cells. (b) Phenotypically altered foci were evaluated in hematoxylin- and eosin-stained liver sections from the female rats. NAF treatment after AFB1 had little effect on number and size of eosinophilic-clear cell foci and decreased the number of trigroid foci. However, it led to a dramatic increase (20-fold after 70 weeks of NAF treatment) in number and size of foci of a special phenotype that was extremely rare after AFB1 alone and virtually absent in group AFB1-PB. Hepatocytes in these foci are characterized by weak diffuse basophilia and some eosinophilia, similar to the phenotype in adenoma and carcinoma, and by absence of gamma-glutamyltranspeptidase (GGT) expression. Based on these findings, we propose the hypothesis that NAF promotes the development of liver tumors via a mechanism involving amplification of a specific subtype of altered hepatic foci.     

Minimal dose and time protection by lindane (gamma-isomer of 1,2,3,4,5,6 hexachlorocyclohexane) against liver tumors induced by aflatoxin B1

This study was carried out in order to investigate the minimal exposure to lindane (LD, 99.72% gamma isomer of 1,2,3,4,5,6 hexachlorocyclohexane), a chlorinated hydrocarbon insecticide, required to protect against liver tumor induced by aflatoxin B1 (AFB1). Materials fed to Buffalo strain rats were as follows: LD 100 ppm; AFB1 1 ppm, LD 100 ppm plus AFB1 1 ppm; and control basal diet. The experimental animals were clinically observed and then serially killed at 1, 3, 5, 10, 15 and 82 weeks. Concurrent administration of LD with AFB1 to rats for more than 3 weeks totally inhibited the incidence of AFB1-induced hepatocellular carcinomas by week 82. Only 1 of 20 rats (5%) fed the same regimen for 1 week developed liver tumors. Animals given 1 ppm AFB1 singly for 15 weeks had a high liver tumor incidence (31.5%). No animals developed liver tumors in LD-treated and control groups. LD may inhibit AFB1-induced liver tumors by stimulating hepatic metabolism and excretion of AFB1 so that less carcinogen is available to liver tissue.     

The promotion effect of anorectic drugs on aflatoxin B(1)-induced hepatic preneoplastic foci.

The ability of three extensively used anorectic drugs, namely fenfluramine (FN), fluoxetine (FX) and amphetamine (AM), to alter the development of aflatoxin B(1) (AFB(1))-induced gamma-glutamyl-positive (GGT(+)) preneoplastic liver foci was investigated in 135 male weanling F344 rats. Following AFB(1) administration, 15 rats were killed, while the rest were divided into four groups and fed diets containing either FN, FX, AM or control diet, with half of the animals in each group subsequently being killed at 4 weeks and half at 10 weeks. All three anorectic drugs as expected suppressed initial food intake, growth rate, body weight gain and food efficiency. They also tended to suppress body fat mass and to decrease plasma levels of T(3) and T(4). FN significantly (P < 0.05) increased GGT(+) foci number/cm(2) and number/cm(3), while FX significantly increased GGT(+) foci number/cm(2) and the volume fraction of foci. Histopathological staining also revealed that FN- and FX-treated animals had more serious morphological alterations in their liver tissue. In contrast, foci development was, if anything, suppressed by AM feeding. These results indicate that serotoninergic drugs (FN and FX), as opposed to dopaminergic drugs (AM), may have tumor promoter activity, at least for liver tissue.     

Attenuation of preneoplastic lesion development by dietary protein intervention: apparent persistence and regression.

The effects of feeding high protein diets that promote the development of aflatoxin B1 (AFB1)-induced gamma-glutamyl transpeptidase positive (GGT+) preneoplastic lesions were examined in Fischer 344 (F344) rats. After administering AFB1 for 2 weeks (initiation), animals were fed diets over four successive 3-week periods (promotion). Either a low (5% casein) or high (20% casein) protein diet was fed for 3, 6, or 9 weeks before switching to the opposite diet to determine whether progressively longer periods of feeding the initial diet caused preneoplastic foci to become more refractory to the intervention effects of the second diet. The results from animals consuming the 20% casein diet for progressively longer periods suggest that longer exposure to the high protein diet progressively enhances the potential for future lesion growth. Results from animals consuming the 5% casein diet for progressively longer periods suggest that longer exposure to the inhibitory low protein diet progressively inhibits the potential for future lesion growth. These results suggest that a high protein diet is a potent promoter of preneoplastic growth and that progressively longer exposure to a particular promotive environment increasingly attenuates foci response to future dietary intervention.     

Inhibition by dietary organoselenium, p-methoxybenzene-selenol, of hepatocarcinogenesis induced by azoxymethane in rats

The effect of dietary p-methoxybenzeneselenol, a new organoselenium compound, on azoxymethane (AOM)-induced hepatocarcinogenesis was examined in female F344 rats. Semipurified diets containing 0 and 5 ppm p-methoxybenzeneselenol were fed to the rats, starting at 5 weeks of age until one week after the carcinogen treatment. At 7 weeks of age, all animals except the vehicle-treated controls were given weekly sc injections of AOM (15 mg/kg body weight, 3 times). At 34 weeks after the last AOM treatment, the liver neoplasm incidence and liver tumor multiplicity as well as the incidence of altered liver cell foci were significantly lower in AOM-treated rats fed the diet containing 50 ppm p-methoxybenzeneselenol (tumor incidence 19%, tumor multiplicity 0.45/rat, foci incidence 3.47/cm2) than in AOM-treated animals fed the diet without p-methoxybenzeneselenol (tumor incidence 66%, tumor multiplicity 2.24/rat, foci incidence 12.08/cm2). These results indicate that dietary p-methoxybenzeneselenol at a dose of 50 ppm inhibits AOM-induced hepatic tumorigenesis.     

Analysis of dietary fat, calories, body weight, and the development of mammary tumors in rats and mice: a review

We have extracted from the literature data from 100 animal experiments, involving 7838 rats and mice, which compared the effects of different levels of dietary fat and/or calorie intake on the development of mammary tumors. Both higher calorie intake (P less than 0.0001) and higher fat intake (P less than 0.0001) independently increased mammary tumor incidence in Sprague-Dawley rats and in mice, as judged from analyses combining ad libitum feeding experiments and restricted feeding experiments. The effect of fat was two thirds the magnitude of the calorie effect in both Sprague-Dawley rats and mice. In ad libitum feeding experiments, a modest but significant (P less than 0.0001) average increase in body weight was found in animals fed high fat diets. However, these differences in body weight did not correspond to differences in mammary tumor incidence. The effect of log body weight on the log odds of tumor incidence was not significant (P = 0.16), while dietary fat intake significantly increased tumor incidence (P less than 0.0001). The collection of animal experimental data supports the hypothesis that, in mammary tumor development, there is a specific enhancing effect of dietary fat, as well as a general enhancing effect of calories.     

Hepatic glutathione S-transferase activity and aflatoxin B1-induced enzyme altered foci in rats fed fractions of brussels sprouts

The aim of the present study was to determine whether the liver cytosol detoxication enzymes, glutathione S-transferases (GSTases) as well as gamma-glutamyl transpeptidase (GGT) foci induced by aflatoxin B1 (AFB) were changed by feeding weanling rats diets containing brussels sprouts, a glucosinolate fraction of brussels sprouts (extract), or a non-glucosinolate fraction (residue). All 3 of these diets induced high levels of hepatic GSTase specific activity as compared to purified-basal diet fed control rats. The brussels sprouts and the extract treatments, but not the residue dietary treatment, inhibited hepatic GGT foci induced by AFB. Thus, glucosinolates and non-glucosinolate fractions of brussels sprouts induce hepatic enzymes involved in detoxication mechanisms but the non-glucosinolate compound(s) apparently are not involved in all chemical carcinogen metabolic processes.     

Effect of dietary protein level on aflatoxin B1 actions in the liver of weanling rats.

The hepatocarcinogenic responses of rats to aflatoxin B1 (AFB1) are believed to depend on microsomal activation of the toxin, followed by macromolecular binding. Dietary protein insufficiency is reported to reduce the level of microsomal metabolism, and therefore would be expected to reduce the AFB1-induced carcinogenicity. Indeed, diminished hepatocarcinogenicity in low-protein diet fed weanling rats that had received AFB1 has been reported. In the present study, carcinogenicity and other toxic effects of AFB1 (0.5 p.p.m.) fed to weanling male Fischer F344 rats on a low-protein diet (5%) or normal-protein (20%) diet for up to 8 weeks were examined. In our study, in contrast with the previous report, all animals that had survived some initial toxicity were found to have developed hepatic tumors or hyperplastic gamma-glutamyltransferase-positive foci a year later. The low-protein diet also produced sub-acute toxicity after AFB1 exposure in the weanling rats, leading to severe histological changes, and the death of about half the animals after 3-4 weeks of exposure. Animals fed an AFB1-containing normal-protein diet also exhibited AFB1-induced hepatocarcinogenicity, but not the sub-acute toxicity. The levels of hepatic enzymes involved in AFB1 metabolism were examined in animals fed the low- or normal-protein diets in the absence of AFB1. The low-protein diet, fed to 3 week weanlings for the subsequent 5 weeks, decreased hepatic cytochrome P450 levels, as well as the in vitro capacity of microsomal fractions to form AFB1-8,9-dihydrodiol, an index of AFB1-8,9-epoxide formation. Rats on a normal-protein diet did not show these changes. This discrepancy between the observed increase in sub-acute toxicity and decrease in microsomal activities in the low-protein fed animals implies that the toxic effects observed in these rats were not directly related to metabolic activation of the toxin. In contrast to the diminished microsomal in vitro AFB1 activation, however, in vivo AFB1-DNA adduct formation ability in rats receiving the low-protein diet in the absence of AFB1 was found to become elevated more rapidly during the 5 week experimental feeding period, compared with animals receiving the normal-protein diet. This was accompanied by a more rapid fall in the levels of AFB1-glutathione S-transferase isozyme activity in the low-protein fed animals. The results of this study on weanling rats support the importance of AFB1-GSH in protecting against the carcinogenic responses to AFB1, and probably also the sub-acute toxicity of the latter.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of high and low dietary protein on the dosing and postdosing periods of aflatoxin B1-induced hepatic preneoplastic lesion development in the rat

Aflatoxin B1-induced liver lesion development is readily modified by dietary protein intake. Earlier work had shown that low-protein diets enhanced the acutely toxic lesion but depressed the carcinogenic lesion. This study examined the emergence of these lesions as a function of dietary protein intake, particularly with respect to whether the protein modification occurred during or after the aflatoxin B12 dosing period. High (20%) and low (5%) casein diets were fed to growing Fischer 344 rats during the dosing and postdosing periods of aflatoxin B2-induced hepatic preneoplastic lesion development. Focal areas of hepatocellular alteration were identified and quantitated by staining sections of liver for gamma-glutamyl transferase (GGT). Animals fed low casein diets during the dosing period displayed a characteristic spectrum of lesions including hepatomegaly, severe bile duct proliferation, cholangiofibrosis, and a tendency for developing large remodeling GGT-positive foci. These lesions were regarded as symptomatic of acute hepatoxicity. Animals fed high-protein diets during the dosing period had small, densely stained, GGT-positive foci, with only mild bile duct proliferation and no cholangiofibrosis, hepatomegaly, or large, remodeling GGT-positive foci. During the postdosing period, protein modulation markedly influenced the total number of foci. Animals fed high casein diets during this period exhibited an approximate 6-fold increase in the number of foci, regardless of the level of protein fed during the earlier dosing period. The marked increase in foci number (as well as area of liver occupied) in high casein diet animals during the postdosing period is regarded as an increased tendency for tumor development.     

The effects of alternating dietary restriction and ad libitum feeding of mice on the development of diethylnitrosamine-induced liver tumours and its correlation to insulinaemia.

The effects of alternating ad libitum feeding and 30% restriction of the dietary intake on the development of diethylnitrosamine (DEN)-induced hepatic neoplasia were investigated. Dietary restriction retarded the growth of glucose-6-phosphatase-deficient (G6Pd) preneoplastic foci and subsequently that of hepatocellular adenomas and adenocarcinomas. The number of foci in standardized liver sections increased from 4.44 foci/cm2 at 12 weeks to 9.65 foci/cm2 at 24 weeks in ad libitum fed animals but only from 2.35 foci/cm2 to 3.29 foci/cm2 in restricted animals. In animals fed first ad libitum for 12 weeks and then for 12 weeks on a restricted diet, the number of G6Pd foci dropped from 4.44 at 12 weeks to 3.54 at 24 weeks. This reduction appeared to be the result of a regression of the small sized G6Pd foci. Dietary restriction was most efficient in inhibiting the development of G6Pd foci when started early in life. Conversely, the growth of foci was stimulated when the mice first had restricted access to food and thereafter were fed ad libitum. The plasma insulin concentrations after a glucose challenge increased with age. Insulinaemia was much higher in ad libitum fed compared to the restricted mice. It was correlated to the number of G6Pd foci in the liver. This study suggests that insulin, which is a known mitogen for hepatocytes in vitro, may contribute to the promotion of DEN-induced liver tumours in mice.     

Interactive effects of methyl-deficiency and dietary restriction on liver cell proliferation and telomerase activity in Fischer 344 rats pretreated with aflatoxin B(1)

The effects of methyl-deficiency and dietary restriction (DR) on hepatic cell proliferation and telomerase activity was studied in male Fischer 344 rats pretreated with aflatoxin B(1) (AFB(1)). Five-week-old rats were gavaged 5 days per week for 3 weeks with AFB(1) (25 microg/rat per day) or solvent (100 microl 75% dimethylsulfoxide). Rats were then divided into four groups. Two groups were fed a methyl-sufficient (MS) diet either ab libitum (AL) or with DR. The other two groups were fed a methyl-deficient (MD) diet either AL or with DR. At 15, 20, and 32 weeks of age, hepatic cell proliferation, telomerase activity, and the number of glutathione S-transferase-P positive (GST-P(+)) foci were determined. DR reduced hepatic cell proliferation, while the MD diet and AFB(1) pretreatment increased cell proliferation. Telomerase activity was decreased by DR and increased by the MD diet and AFB(1) pretreatment. The same trend was observed with GST-P(+) foci: in AFB(1)-pretreated rats, methyl deficiency increased the number of foci, while DR decreased the number. These results are consistent with a role of telomerase in hepatocarcinogenesis.     

The influence of partial hepatectomy on the biphasic response of gamma glutamyl transpeptidase to aflatoxin B1

We previously observed a biphasic response in rat hepatic gamma glutamyl transpeptidase (GGT) activity to aflatoxin B1 (AFB1) feeding [9]. We have extended this observation to examine the effect of partial hepatectomy (PH) on the activity and distribution of GGT at different stages of the feeding regime. In control-fed animals GGT levels were elevated 3-7 days after PH with increased activity in periportal hepatocytes. In animals fed a sub-carcinogenic dose of AFB1 (up to 4 weeks) the effect of PH on GGT activity was similar to that in control animals, but increased activity was mainly due to biliary hyperplasia. There was no obvious difference between animals returned to control diet after PH and those returned to toxic diet. In animals fed 4-15 weeks the percentage increase in GGT activity 1 week after PH correlated with length of time on AFB1 diet before operation, with an increase in number and size of altered foci. These results further support the idea that there is a preliminary toxic response in GGT activity followed by a secondary response more closely related to the carcinogenic process.     

Promotion of aflatoxin B1 carcinogenesis by the natural tumor modulator indole-3-carbinol: influence of dose, duration, and intermittent exposure on indole-3-carbinol promotional potency

Indole-3-carbinol (13C), a secondary metabolite from cruciferous vegetables, inhibits aflatoxin B1 (AFB1) hepatocarcinogenesis in trout (Bailey et al., J. Natl. Cancer Inst., 78: 931-934, 1987) and rats (Selivonchick et al., unpublished results) when given prior to and with carcinogen but promotes carcinogenesis in both species when given continuously following AFB1 initiation. Since human 13C intake may not be continuous, and the promotional stimulation may be reversible, we have assessed 13C promotion using delayed and discontinuous exposure protocols. Following initiation with AFB1, 13C was fed to trout for varying periods of time, with varying lengths of delay after initiation and continuous or intermittent patterns of 13C treatment. Promotional enhancement of tumor incidence by 13C was found to be significant when 13C treatment was delayed for several weeks or months after the initial AFB1 challenge. Promotion also was found to increase with length of exposure to 13C treatment and to be decreased but still evident when 13C was given in alternating months or weeks, or twice per week only. These results do not support the idea that promotional stimulation in hepatocarcinogenesis is a reversible phenomenon. To quantify 13C promotional potency in terms of its dietary concentration, a series of AFB1 tumor dose-response curves was established, each with a different level of 13C fed continuously following AFB1 initiation. The resultant tumor dose-response curves, plotted as logit percentage of incidence versus log AFB1 dose, were displaced parallel toward lower AFB1 50% tumor take (TD50) values with increasing 13C concentration. The level of 13C that halves the AFB1 dose for 50% tumor incidence was calculated to be approximately 1000 ppm 13C, fed continuously, with no substantial threshold for promotion. By comparison, 13, when fed before and with AFB1, shows a 50% inhibitory value (13C concentration that doubles the dose of AFB1 for 50% tumor incidence) in trout of 1400 ppm 13C [Dashwood et al., Carcinogenesis (Lond.), 10: 175-181, 1989]. Thus the potential for 13C as a dietary additive to promote prior hepatic initiation events when fed continuously is approximately as great as its potential to inhibit concurrent AFB1 initiation.     

Resistance of aberrant crypt foci to apoptosis induced by azoxymethane in rats chronically fed cholic acid

We have previously shown that chronic feeding of cholic acidto carcinogen treated rats reduces the number of putativepreneoplasticlesions of colonic cancer, aberrant crypt foci(ACF), but enhancesthe growth of remaining ACF and the incidence of colonic tumors.The following study was conducted to further explore the effectsof cholic acid on ACF growth by determining if ACF in cholicacid-fed animals display resistance to apoptotic cell death.ACF were induced in male Sprague- Dawley rats with two injectionsof azoxy-methane (20 mg/kg body wt). Rats were divided intotwo groups and fed either the control AIN-76 diet or the AIN-76diet containing 0.2% cholic acid. After 18 weeks, colonic apoptoticcelldeath was induced with an acute low dose of azoxymethane(10 mg/kg body wt). The number of cells, apoptotic bodies andbromodeoxyuridine (BUdR)-labeled cells were determined in coloniccrypts comprising ACF and surrounding normal crypts in ratsfrom each diet group. The number of apoptotic bodies per 100cells was lower in ACF crypts than in normal-appearing crypts(P = 0.0034). Both normal and ACF crypts from rats fed the cholicacid diet had fewer apoptotic bodies per 100 cells than cryptsfrom rats fed the control diet (P =0.0102). These data suggestthat ACF harbor resistanceto induction of apoptosis. Chronicfeeding of a diet containing 0.2% cholic acid results in thedevelopment of increased resistance to apoptosis. The lowerrate of cell death in ACF may contribute to the enhanced growthof ACF and higher tumor incidence previously observed in cholicacid-fed animals.     

Progression of carcinogen-induced foci of y-glutamyltranspeptidase-positive hepatocytes to hepatomas in rats fed a choline-deficient diet

Following previous findings that feeding a choline-deficient (CD) diet to rats strongly promotes the evolution of liver cells, initiated by a chemical carcinogen, to foci of y-glutamyltranspeptidase (yGT)-positive hepatocytes, we investigated whether a CD diet could also promote the evolution of yGT-positive foci to hepatomas. yGT-positive foci were induced in male Sprague-Dawley rats by administration of a single dose of diethyInitrosamine followed by 2 weeks' feeding on a CD diet containing 0.02% acetylaminofluorene. Immediately thereafter, one group of rats was fed a plain CD diet and the other a choline-supplemented (CS) diet, and subgroups of animals were killed periodically for analysis of the number and size of yGT-positive foci and development of hepatomas. During the first 7 weeks after switching the diets, the number and size of the foci increased in both groups. At 12 and 16 weeks, the number and size of foci began to decline in rats fed the CS diet However, in the CD group, there was a progressive increase in the size with coalescence of the foci, as well as development of neoplastic nodules. These lesions were followed at 20 and 28 weeks by development of hepatomas at a high incidence rate. In rats fed the CS diet, a few scattered yGT-positive foci, and small neoplastic nodules remained in the liver at 20 and 28 weeks, but no hepatomas developed. These results show that the CD diet is a potent promoter of the evolution of foci of altered hepatocytes to hepatomas, and that most of the yGT-positive foci are reversible lesions.     

Initiation by bleomycin of hepatocellular foci in the rat.

The antitumor antibiotic, bleomycin, was tested for activity as an initiator of hepatocellular foci and neoplasms in rats. The compound was administered in a single dose via the portal vein 4 h after the proliferative stimulus of a two-thirds partial hepatectomy. Rats were subsequently fed diet containing phenobarbital for up to 41 weeks to promote the development of initiated hepatocytes. Bleomycin-treated livers displayed significantly increased frequencies of basophilic hepatocellular foci and hepatocellular foci which retain glycogen during fasting. Foci that express glutathione-S-transferase (placental form) were not initiated by bleomycin. Hepatocellular neoplasms were infrequently seen in bleomycin-treated livers (5% incidence). The results suggest that oxygen radical-mediated DNA damage may initiate, within populations of proliferating hepatocytes, new lineages of altered hepatocytes that form foci but have low probability of progressing to neoplasms during promotion with phenobarbital.     

Protection against aflatoxin B1-induced hepatocarcinogenesis in F344 rats by 5-(2-pyrazinyl)-4-methyl-1,2-dithiole-3-thione (oltipraz): predictive role for short-term molecular dosimetry.

Previous studies have demonstrated that dietary administration of the schistosomicidal drug 5-(2-pyrazinyl)-4-methyl-1,2-dithiole-3-thione (oltipraz) ameliorates the hepatotoxicity of aflatoxin B1 (AFB1). Notably, mortality, altered hepatic function, hepatic AFB1-DNA adduct levels, and expression of hepatic enzyme-altered foci were markedly reduced in the rat by concurrent feeding of oltipraz during exposures to AFB1. Collectively, these studies prompted us to evaluate the chemoprotective properties of oltipraz against AFB1-induced liver cancer. In addition, preliminary molecular dosimetry studies were undertaken to determine the utility of measurements of urinary aflatoxin-N7-guanine excretion as a marker of relative risk for hepatocarcinogenesis in AFB1-exposed rats. For the carcinogenesis studies, 5-wk-old male F344 rats were randomly divided into two groups. One group (55 rats) received the AIN-76A diet, and the other group (56 rats) received the AIN-76A diet supplemented with 0.075% oltipraz. The oltipraz-supplemented diet was fed for 4 wk. Beginning 1 wk after starting the experimental diets, all rats in both groups received 25 micrograms of AFB1/rat/day by gavage for 5 days per wk over the next 2 wk. One wk following cessation of dosing with AFB1, oltipraz was removed from the diet, and all rats were fed the AIN-76A diet for the remainder of the experiment. At 3 mo after dosing, livers of ten sentinel rats from each group were analyzed for the burden of gamma-glutamyltranspeptidase-positive foci. In accord with previous findings, rats fed the oltipraz-supplemented diet exhibited substantial reductions in the focal burden (97% reduction; P less than 0.05) of these AFB1-induced lesions. The remaining rats were maintained for the cancer study until they became moribund or the termination of the experiment at 23 mo. Gross liver lesions were identified at autopsy and confirmed by microscopic evaluation. An 11% incidence of hepatocellular carcinoma was observed in the AFB1-treated, control diet-fed rats. An additional 9% of this group had hepatocellular adenomas. Oltipraz afforded complete protection against both AFB1-induced hepatocellular neoplasms. Using Kaplan-Meier survival analyses, rats in the oltipraz group had a significantly (P less than 0.02) longer life span and an increased survival free of liver tumors (P less than 0.0002). Molecular dosimetry studies used rats fed either the oltipraz-supplemented or control diet for 1 wk and then challenged with a single dose of AFB1 to examine the initial rates of 8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 excreted in the urine.(ABSTRACT TRUNCATED AT 400 WORDS)     

Association between expression of transforming growth factor-alpha and progression of hepatocellular foci to neoplasms.

Hepatocarcinogenesis was initiated in rats with a single dose of either of two chemical mutagens--benzo[a]pyrene diolepoxide I and methyl(acetoxymethyl)nitrosamine--administered 15 h after partial hepatectomy. The development of hepatocellular foci and neoplasms was then promoted with dietary phenobarbital given for 45 or 62 weeks. Formalin-fixed tissue specimens that contained hepatic neoplasms and altered hepatocellular foci were screened for expression of the oncodevelopmental marker glutathione-S-transferase (placental form) (GSTP) and transforming growth factor-alpha (TGF-alpha) using immunohistochemistry. All (100%) hepatocellular carcinomas expressed both GSTP and TGF-alpha, as did most hepatocellular adenomas (greater than 80%). However, quantitative stereologic analysis of treated and control livers revealed that GSTP-positive foci were 10-30 times more frequent than TGF-alpha-positive foci. Foci with homogeneous expression of GSTP generally displayed heterogeneous expression of TGF-alpha with reaction product most prominent at their peripheries. Less frequently homogeneous TGF-alpha-positive foci were seen within GSTP-positive foci. The average volumes of those GSTP-positive foci that also expressed TGF-alpha were significantly greater than those of the entire sets of GSTP-positive foci. These results suggest that expression of TGF-alpha may distinguish a subset of GSTP-positive foci that have a growth advantage and increased probability of progression to neoplasia.