Elevated concentrations of serum alpha-fetoprotein in rats with chemically induced liver tumors.

The study was undertaken to determine whether aflatoxin B1 (AFB1)-induced liver tumors in rats produced alpha1-fetoprotein (AFP) and whether the age of the animals would influence such as appearance, a finding suggested by data seen in man. Other liver carcinogens (N-hydroxy-N-2-fluorenylacetamide, N-2-fluorenylacetamide, and diethylnitrosamine) were tested for their ability to induce liver tumors producing AFP. The presence of AFP. The presence of AFP in the serum was determined by double diffusion in agarose and by comparison also by quantitative radioimmunoassay. Using double diffusion, AFP was detected in the majority of tumor-bearing rats that had received either N-2-fluorenylacetamide or N-hydroxy-N-2-fluorenylacetamide. Sera of diethylinitrosamine-treated rats with liver tumors were all positive, whereas sera of rats bearing AFB1-induced tumors were positive in only a few cases. However, all sera of tumor-bearing rats examined had elevated AFP levels by radioimmunoassay. Nonetheless, the average level of AFP in the sera of rats bearing AFB1-induced tumors was considerably lower, compared to the sera of rats with tumors caused by diethylnitrosamine, N-2-fluorenylacetamide, or N-hydroxy-N2-fluorenylacetamide. Rats started on AFB1 when 6 weeks old had more mixed liver tumors with neoplastic hepatocytes and bile ducts and higher AFP levels than did rats started at 26 weeks of age. However, the histological grade of differentiation of inducted tumors did not seem to influence the AFP level.     

In situ hybridization studies on expression of albumin and alpha-fetoprotein during the early stage of neoplastic transformation in rat liver

The expressions of albumin and alpha-fetoprotein (AFP) genes were studied in early preneoplastic liver lesions produced by the Solt-Farber protocol using "in situ" hybridization with single stranded RNA probes. In normal rat liver, albumin was expressed at a lower level in the centrilobular than in the periportal areas of the liver acinus, whereas the bile duct epithelium did not show any expression. Five weeks after initiation with diethylnitrosamine, islands of hepatocytes were present which showed heterogeneous expression of albumin and were surrounded by cells comprised of albumin negative hepatocytes and oval cells. gamma-Glutamyltranspeptidase positive foci of enzyme altered cells were located in albumin positive areas. Albumin expression gradually decreased in permanent nodules but increased in the hepatocytes outside the nodules during the first five months after initiation with diethylnitrosamine. Remodeling nodules, which were partly gamma-glutamyltranspeptidase and albumin positive, were also present. However, no consistent correlation was found between gamma-glutamyltranspeptidase positive and albumin negative areas during the first 5 months after initiation. Occasionally, cells showing an elevated expression of albumin were found in permanent nodules. These cells were located in the vicinity of oval type cells, which also showed a weak expression of albumin. AFP was expressed at high level in oval cells 5 weeks after the initiation. However, oval cells observed at later time points, either around the neoplastic nodules or inside the nodules showed only low expression of AFP. Hepatocytes in the enzyme-altered foci and in neoplastic nodules were always negative for AFP. The presence of strongly albumin positive cells inside the neoplastic nodules in close proximity to oval type cells suggests that these cells may be derived from primitive "stem-cell"-like oval cells.     

Reliability of a short-term test for hepatocarcinogenesis induced by aflatoxin B1.

The reliability of a short-term test for hepatocarcinogenesis induced by aflatoxin B1 (AFB1) was tested by comparing the early appearance of gamma-glutamyl transpeptidase (GGT)-positive foci with the occurrence of primary liver cancer at a later stage. All rats received a basic short-term treatment with AFB1 intraperitoneally, during which three experimental groups received Chinese green tea or 2000 or 5000 ppm butylated hydroxyanisole in the diet and a control group received basic diet. Some of the rats in each group were sacrificed at the end of the short-term procedure, and the remainder were observed up to 92 weeks. The livers of all animals were examined for GGT-positive foci or primary liver tumours. The GGT-positive foci were most numerous and largest and the incidence of liver tumours was highest in the control group. These findings suggest that GGT-positive foci are a valuable preneoplastic marker for AFB1-induced hepatocarcinogenesis, that the short-term model is fairly reliable, and that both Chinese green tea and butylated hydroxyanisole inhibit AFB1-induced hepatocarcinogenesis.     

Inhibition of hepatocarcinogenesis by adrenocorticotropin in aflatoxin B1-treated rats.

We examined whether hormones would modify the carcinogenic action of aflatoxin B1 (AFB1). Four groups of inbred Fischer rats received AFB1, 125 mug per animal, weekly per os. In three of the groups, certain hormones were administered simultaneously: One group received 1 U growth hormone (GH) sc weekly, another was given 4 U adrenocorticotropin (ACTH) weekly, and a third received 0.5 U insulin weekly sc. AFB1, ACTH, and insulin were given for 20 weeks; GH was given for only 10 weeks. The control group did not receive hormone adjuvant. In each group, 4 animals were killed at 7, 14, 21, 28, and 35 weeks; the remaining rats were killed at 77 weeks. Their livers were carefully examined and samples prepared for light and electron microscopy. Animals receiving AFB1 and ACTH failed to exhibit hepatocellular carcinoma. On the other hand, malignant lymphoma appeared at 56 weeks in 3 of the 6 surviving males on this regime. AFB1, alone or when given with insulin or GH, caused hepatocellular carcinoma in all animals; in these, lymphoma was not observed. Lymphoma comprised two cell types, each with similar neclear characteristics but differing in their nucleocytoplasmic ratios and in the amount and distribution of cytoplasmic organelles. Alterations leading to hepatocellular carcinoma were examined at various stages of development. "Basophilic hyperplasia" reflected an increase in free ribosomes. "Hyperplastic nodules" were composed of hepatocyte aggregates with characteristics similar to those encountered in the earlier stage. Both the "neoplastic nodules" and hepatocellular carcinomas were formed by cells containing large, "smooth fingerprints" and free ribosomal aggregates. These features supported the concept that AFB1 impairs ribosomal binding to endoplasmic reticulum membranes. The failure of ACTH-treated animals to develop hepatocellular carcinoma was ascribed to the effect of adrenal cortical stimulation upon membrane-polysome binding.     

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.     

A single dose-response effect of aflatoxin B1 on rapid liver cancer induction in two strains of rats

To investigate rapid liver cancer induction in rats by aflatoxin B1 (AFB1), different single oral doses of AFB1 were given to 3 groups of 1-year-old Buffalo and Wistar rats. The animals were treated once and all survivors were killed 6 weeks later. Control animals received an equal volume of solvent (DMSO), and both groups of animals were maintained under identical conditions throughout the period of experiment. The survival rates were 40% with low and medium doses in AFB1-treated Buffalo and Wistar rats, and 0% in the high-dose Buffalo rats. Slight ante-mortem elevations in serum concentrations of glutamic pyruvic transaminase (SGOT) and glutamic oxaloacetic transaminase (SGPT) were indicative of the persistent damage effect of AFB1 at week 6. Total protein and albumin concentrations were not altered. The percent incidence of altered cell foci (areas) and neoplastic nodules was higher in Wistar than in Buffalo rats given a similar low dose. Various stages of well differentiated hepatocellular carcinomas (0.1-0.2 cm in diameter) developed in 3 of 8 Wistar rats. It thus appears that Wistar rats are more susceptible to hepatocarcinogenesis following a single oral dose of AFB1 than Buffalo rats.     

Cholangiocellular carcinomas induced in Syrian golden hamsters administered aflatoxin B1 in large doses

The hepatocarcinogenicity of aflatoxin B1 (AFB1) was compared in male Syrian golden hamsters and inbred F344 rats. AFB1 was administered by gavage 5 days/week for 6 weeks at doses of 1 and 2 mg/kg body weight/day to rats and hamsters, respectively; rate did not survive beyond 3 weeks with doses of 2 mg/kg/day. After 6 weeks the animals either received no further treatment or were given 0.1% phenobarbital sodium in the drinking water. ATPase-deficient foci of hepatic parenchymal cells, neoplastic nodules, and hepatocellular carcinomas were observed in liver sections from AFB1-treated rats killed at 6, 14, or 23 weeks; they were not seen in sections from AFB1-treated hamsters killed at 6, 14, or 46 weeks. Each of the 50 AFB1-treated rats developed hepatocellular carcinomas by 46 weeks; many also had cholangiocarcinomas and mixed hepatocellular-cholangiocellular carcinomas. Hepatocellular carcinomas were found in only 2 of 49 AFB1-treated hamsters by 78 weeks. At this time cholangiocarcinomas were found in 15 hamsters, and microscopic cholangiomas were seen in all of the livers. Compared to the rat, the hamster was resistant to the hepatotoxic and hepatocellular carcinogenic effects of AFB1.     

Proliferation of preneoplastic lesions after discontinuation of chronic DEN feeding in the development of hepatomas in rat.

Diethylnitrosamine (DEN, 10 mg/kg/day) was fed to rats for 2, 4 and 6 weeks. At different times after feeding with DEN was stopped, growth of preneoplastic lesions has been correlated with pathological evolution (preneoplastic foci, neoplastic nodules and hepatomas). The proliferating fraction in the foci, the cell content, and relative volume of foci increase as a function of the duration of the treatment. The proliferating fraction increases evenly throughout the liver, but, in all experimental modalities, preneoplastic cells show a proliferative advantage over the phenotypically normal tissue. In each experimental group, the proliferative rate correlates with the pathological evolution. After 2 weeks of DEN feeding the growth activity of foci remains very low, and neoplastic nodules are not detectable until the median time of death (14 months). After 4 and 6 weeks, a critical size of the foci is reached, corresponding to the neoplastic transformation, and an increased labelling index is triggered in the lesions and in the phenotypically normal tissue. It is speculated that the "growth pressure" induced by the first carcinogen treatment, associated with the subsequent disturbance of the mitotic control regulation, may be implicated in the process of malignant transformation of preneoplastic lesions.     

Sequential analysis of chemically induced hepatoma development in rats by two dimensional electrophoresis

Using the Solt-Farber hepatocarcinogenesis model, a large population of preneoplastic and neoplastic nodules were induced in male Fischer 344 rats. Total cellular polypeptides from normal liver and individual preneoplastic and neoplastic nodules were analyzed for both qualitative and quantitative changes using computer assisted high resolution two-dimensional electrophoresis. Approximately 800-1000 cytosolic and 1200-1400 membrane associated polypeptides were readily separated and detected using an ultrasensitive silver stain. The polypeptide patterns were remarkably similar for each tissue and only four qualitative polypeptide differences were noted. One cytosolic polypeptide, 6.8/57 (designated pl/Mr X 10(-3), and three membrane associated polypeptides, 6.25/41, 6.75/24, and 6.05/21, were expressed in both preneoplastic and neoplastic nodules but not in normal liver. No qualitative polypeptide differences were detected among the individual preneoplastic or individual neoplastic nodules or between preneoplastic and neoplastic nodules. Numerous quantitative changes in both known markers for hepatocarcinogenesis and in as yet unidentified polypeptides were noted. In particular, the Ya subunit of glutathione S-transferase B, the Yb subunit of glutathione S-transferase A, as well as the three isoelectric point variants of the Yp subunit of glutathione S-transferase P were increased 2-, 4-, and 7-fold, respectively, in preneoplastic and neoplastic nodules. Whereas DT-diaphorase was increased 2-3-fold in hyperplastic nodules as compared to normal liver, no differences in the expression of albumin were noted. Although no differences were observed in the expression of aldehyde dehydrogenase in preneoplastic and neoplastic nodules, polypeptide b (6.9/54) was shifted slightly toward the basic region in normal liver. alpha-Fetoprotein was not detected in either preneoplastic or neoplastic nodules. In addition to these changes in known markers, comparison of 500-800 cytosolic and 750-1000 membrane associated polypeptides showed that roughly 4-10% of the polypeptides were undergoing quantitative changes of at least 4-fold during these stages of hepatocarcinogenesis. Thirty (10 cytosolic and 20 membrane) polypeptides were significantly down-regulated while 22 (7 cytosolic and 15 membrane) polypeptides were up-regulated in both preneoplastic and neoplastic nodules. In all cases the direction and magnitude of change were the same in both preneoplastic and neoplastic nodules with the exception of three polypeptides.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Chemoprevention of aflatoxin B1 hepatocarcinogenesis by coumarin, a natural benzopyrone that is a potent inducer of aflatoxin B1-aldehyde reductase, the glutathione S-transferase A5 and P1 subunits, and NAD(P)H:quinone oxidoreductase in rat liver

Structurally diverse compounds can confer resistance to aflatoxin B1 (AFB1) hepatocarcinogenesis in the rat. Treatment with either phytochemicals [benzyl isothiocyanate, coumarin (CMRN), or indole-3-carbinol] or synthetic antioxidants and other drugs (butylated hydroxyanisole, diethyl maleate, ethoxyquin, beta-naphthoflavone, oltipraz, phenobarbital, or trans-stilbene oxide) has been found to increase hepatic aldo-keto reductase activity toward AFB1-dialdehyde and glutathione S-transferase (GST) activity toward AFB1-8,9-epoxide in both male and female rats. Under the conditions used, the natural benzopyrone CMRN was a major inducer of the AFB1 aldehyde reductase (AFAR) and the aflatoxin-conjugating class-alpha GST A5 subunit in rat liver, causing elevations of between 25- and 35-fold in hepatic levels of these proteins. Induction was not limited to AFAR and GSTA5: treatment with CMRN caused similar increases in the amount of the class-pi GST P1 subunit and NAD(P)H: quinone oxidoreductase in rat liver. Immunohistochemistry demonstrated that the overexpression of AFAR, GSTA5, GSTP1, and NAD(P)H:quinone oxidoreductase affected by CMRN is restricted to the centrilobular (periacinar) zone of the lobule, sometimes extending almost as far as the portal tract. This pattern of induction was also observed with ethoxyquin, oltipraz, and trans-stilbene oxide. By contrast, induction of these proteins by beta-naphthoflavone and diethyl maleate was predominantly periportal. Northern blotting showed that induction of these phase II drug-metabolizing enzymes by CMRN was accompanied by similar increases in the levels of their mRNAs. To assess the biological significance of enzyme induction by dietary CMRN, two intervention studies were performed in which the ability of the benzopyrone to inhibit either AFB1-initiated preneoplastic nodules (at 13 weeks) or AFB1-initiated liver tumors (at 50 weeks) was investigated. Animals pretreated with CMRN for 2 weeks prior to administration of AFB1, and with continued treatment during exposure to the carcinogen for a further 11 weeks, were protected completely from development of hepatic preneoplastic lesions by 13 weeks. In the longer-term dietary intervention, treatment with CMRN before and during exposure to AFB1 for a total of 24 weeks was found to significantly inhibit the number and size of tumors that subsequently developed by 50 weeks. These data suggest that consumption of a CMRN-containing diet provides substantial protection against the initiation of AFB1 hepatocarcinogenesis in the rat.     

Sequential histologic study of rat liver during peroxisome proliferator [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio]-acetic acid (Wy-14,643)-induced carcinogenesis

The livers of male inbred F344 rats fed Wy-14,643 [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio] acetic acid (CAS: 50892-23-4) in the diet at a concentration of 0.1% (wt/wt) were examined sequentially at 5, 10, 17, 21, 26, 30, 35, 40, 52, 60, and 70 weeks. At 5 weeks the livers were markedly enlarged and histologically showed markedly enlarged hepatocytes with prominent nucleoli. At 21 weeks altered acidophilic areas were seen in 2 of 3 animals that were killed. Between 26 and 52 weeks neoplastic nodules were noted of 1-8 mm containing cells with morphologic features similar to those observed in altered areas. Hepatocellular carcinomas (HCC) were observed in 1 animal killed at 30 weeks and in all the animals sacrificed at 60 weeks and later. [3H]thymidine nuclear labeling studies showed marked proliferative activity of cells in altered areas, neoplastic nodules, and HCC. Altered areas, neoplastic nodules, and HCC were consistently negative for gamma-glutamyltransferase and showed decreased ATPase activity. Glucose-6-phosphatase (Glc-6-Pase) activity was decreased in altered areas and neoplastic nodules. However, some of the HCC showed a strong positive reaction for Glc-6-Pase.     

Localization of alpha1-fetoprotein and DNA-synthesis in liver cell populations during experimental hepatocarcinogenesis in rats.

Six-, 12- and 20-week-old rats were fed N-nitrosomorpholine (NNM) at low concentrations (6 mg/kg/day) or high concentrations (20 mg/kg/day) for 6 or 12 weeks. Irrespective of the age of the rats, both NNM schedules resulted in development of hepatomas and during the early stages of hepatoma induction, liver histotoxic patterns depended only on the dose of carcinogen employed. Necrosis of hepatocytes and proliferation of small, oval-shaped cells occurred when high doses of NNM were applied. Parallel to the proliferation of oval-shaped cells, resurgence of alpha₁-fetoprotein (AFP) in rat sera was observed and production of this protein was confined to the oval-shaped cells as shown by immunoperoxidase staining. During proliferation of bile duct epithelium, induced by galactosamine injections, those cells could also stain for AFP, and proliferation of oval-shaped cells concomitant with intracellular AFP staining resulted from restitution of heavily damaged liver. In pulse-chase labelling experiments with ³H-thymidine, oval-shaped cells were seen in continuous development towards hepatocytes, and distinct areas of hyperplastic appearance occurred. Normal hepatocytes and hyperplastic areas did not stain for AFP. Upon low-dose feeding of NNM, no cellular AFP and no serum AFP were detected unless hepatoma cells had developed. At the stage of malignant conversion, distinct AFP-staining nodules were localized which consisted of neoplastic hepatocytes. AFP-staining and non-AFP-staining nodules were seen concomitantly in the same animal. AFP was localized only in neoplastic hepatocytes. Pulse-labelling experiments with [³H]thymidine showed the proliferative character of hepatoma cells from which the AFP-staining population in particular was involved. No correlation was found between the presence of AFP and histological grading of hepatomas. The wide range of both serum AFP levels and their rising rates in individual rats indicated the highly heterogeneous character of the induced hepatomas with respect to AFP production.     

Concanavalin A-induced agglutinability of normal, preneoplastic, and neoplastic mouse mammary cells.

The concanavalin A (Con A)-induced agglutinability of normal, preneoplastic, and neoplastic mouse mammary epithelial cells was examined. Cells freshly dissociated from normal mammary glands, hyperplastic alveolar nodules, or primary mammary adenocarcinomas by collagenase digestion in the presence of bovine serum albumin were strongly agglutinated by low concentrations of Con A. After short-term culture in vitro, however, cells from all three types of tissue were only weakly agglutinated by Con A, as measured by both suspension and hemadsorption assays. By comparison, cells of three established mammary tumor culture lines agglutinated strongly in the presence of the lectin. Treatment of the normal, preneoplastic, and neoplastic mammary cells in primary cultures with either trypsin or collagenase had little or no effect on their agglutinability, whereas hyaluronidase significantly increased their reactivity. Studies with fluorescein-tagged Con A indicated that all three cell types were capable of binding the lectin. The results were consistent with previous evidence suggesting that neoplastic transformation of mouse mammary epithelial cells is not manifested in vitro by several of the alterations in growth patterns, intercellular interactions, and surface properties that usually accompany transformation of fibroblastic cells.     

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.     

Bone marrow-derived cells fuse with hepatic oval cells but are not involved in hepatic tumorigenesis in the choline-deficient ethionine-supplemented diet rat model

Bone marrow cells (BMCs) have been reported to behave as tissue-specific stem cells in some organs and to participate in tumorigenesis. However, the roles of BMCs in hepatic regeneration and carcinogenesis are still unknown. A choline-deficient, ethionine-supplemented (CDE) diet leads to the appearance of oval cells, a type of hepatic progenitor cell, and activates their replication. Furthermore, this type of diet induces preneoplastic nodules and hepatocellular carcinomas (HCCs) derived from oval cell progenitors. The aims of this study were to determine whether oval cells are derived from BMCs and whether preneoplastic nodules or HCCs originate from BMCs in the CDE diet rat model. To clarify the origin of constituent cells in the liver, we transplanted BMCs from green fluorescent protein (GFP) transgenic female rats into male Lewis rats, which were then exposed to a CDE diet to induce hepatocarcinogenesis. Some oval cells showed both donor-derived GFP expression and the recipient-specific Y chromosome, indicating that donor BMCs fused with recipient oval cells. Several preneoplastic nodules (precancerous lesions) identified by their glutathione S-transferase placental (GSTp) positivity were induced by CDE treatment. However, these preneoplastic GSTp-positive nodules were not GFP positive. In conclusion, this study has produced two major findings. First, BMCs fuse with some oval cells. Second, BMC-fused oval cells and BMCs might not have malignant potential in the CDE-treated rat model.     

Reducing effects of rodent malaria on hepatic carcinogenesis induced by dietary aflatoxin B1

The present study was undertaken to evaluate the effects of Plasmodium berghei infection on the development of liver tumors induced in male Buffalo rats by aflatoxin B1 (AFB1). Intraperitoneal (i.p.) injection of 10(6) parasitized red blood cells (pRBC) into the rat 12 days prior to administration of 2 ppm dietary AFB1 for 10 weeks diminished hepatocellular carcinoma (HCC) induction compared to that observed in rats given AFB1 alone at weeks 60-82. No animals in a control group developed HCC lesions, while only 1 of 22 rats treated with P. berghei alone developed a neoplastic nodule at week 82. These data suggest a reducing effect of P. berghei on the development of liver tumors induced by AFB1 in male Buffalo rats.     

Promotion mechanism of phenobarbital and partial hepatectomy in DENA hepatocarcinogenesis cell kinetics effect

Diethylnitrosamine (DENA, 10 mg kg-1 per day) was fed to rats for 2, 4 and 6 weeks. One week after the cessation of DENA, animals were submitted either to partial hepatectomy or to phenobarbital administration. Partial hepatectomy did not promote neoplastic transformation, except after a 6-week DENA treatment. A minimum of phenobarbital was required to reach a significant promoting effect in DENA carcinogenesis. A too-limited treatment was ineffectual but could be compensated for by prolonged DENA administration. The phenobarbital treatment became unnecessary when neoplastic nodules were present. Phenobarbital continuously given after the carcinogen administration promoted neoplastic transformation even after a subcarcinogenic DENA treatment (2 weeks). It accelerated the pathological evolution and increased the tumour incidence. In these conditions, phenobarbital increased the proliferation advantage of preneoplastic cells over normal cells. In the different experimental modalities, the promoting effect was associated with the induction of chronic cell proliferation, the inhibition of the rapid response to the 2/3 partial hepatectomy and the mitotic circadian rhythm normally present during liver regeneration. It is concluded that the promotion mechanism could consist in disturbing the mitotic control in order to maintain, for a long time, a chronic low level of cell proliferation permitting the selective growth of preneoplastic cells and their subsequent transformation.     

CCT亚基γ基因在AFB_1诱发大鼠肝癌过程中表达的变化

目的探讨AFB1诱发肝癌过程中CCT亚基γ基因蛋白的表达及其意义。方法 45只Wistar大鼠随机分为AFB1组和对照组,AFB1组以200μg/kg体重腹腔注射AFB1,第13周、第33周和第53周对大鼠进行肝活检,第73周处死全部动物并取肝组织。用免疫组化法检测AFB1诱发肝癌过程中不同时期CCT亚基γ基因蛋白的表达情况。结果在第73周AFB1组CCT亚基γ基因蛋白表达水平显著高于对照组(P<0.05)。同一组不同时段比较,随着诱癌的进行,CCT亚基γ基因蛋白表达上调,第73周AFB1组CCT亚基γ基因蛋白的表达水平不仅明显高于第13周,而且亦高于第33周(P<0.01)。其余各组和同组不同时段之间CCT亚基γ基因蛋白的表达差异无统计学意义(P>0.05)。AFB1组中发生肝癌的动物肝组织CCT亚基γ基因蛋白表达率为100%(10/10),显著高于同期对照组CCT亚基γ基因蛋白的表达率30%(3/10)(P<0.05)。结论 AFB1诱癌过程中CCT亚基γ基因蛋白表达上调,CCT亚基γ基因蛋白有可能参与了肝细胞癌的发生和发展。     

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)