Influence of dietary fat and selenium in initiation and promotion of aflatoxin B1-induced preneoplastic foci in rat liver

Aflatoxin B₁-induced hepatic -glutamyl transpeptidase-positivefoci were quantified in rats fed different levels of fat andselenium during either initiation or early promotion. Male Sprague-Dawleyrats were divided into 12 groups. One of six experimental dietswere fed to groups 1–6 prior to and during aflatoxin B₁exposure (initiation, weeks 1–4.5) and to groups 7–12during weeks 4.5–15 (promotion). The six experimentaldiets contained 2 or 20% corn oil, each with either <0.02,0.15 or 2.5 (or 1.9) p.p.m. selenium. When not fed the experimentaldiets, rats were fed a modified MN 76A diet. In groups 1–6,0.03% phenobarbital was added as a promoter to the AIN-76A diet.Individual and interactive effects of selenium and fat weredependent on the stage of carcinogenesis. High dietary fat fedwith either <0.02 or 0.15 p.p.m. selenium during initiationresulted in a significant increase in the number and size offoci when compared with low fat groups. In rats fed 20% fatand 2.5 p.p.m. selenium during initiation, preneoplastic developmentwas reduced below all low fat groups. In contrast, seleniumstatus but not dietary fat level influenced focal formationduring promotion. Rats fed <0.02 p.p.m. selenium had a significantlygreater percentage of liver section occupied by foci than ratsfed either 0.15 or 1.9 p.p.m. seleniwn. Feeding 1.9 p.p.m. seleniumduring promotion did not afford greater protection above the0.15 p.p.m. level. Hepatic glutathione peroxidase activity atweek 15 was significantly diminished in animals fed <0.02p.p.m. selenium during promotion. Feeding 1.9 p.p.m. seleniumwhen compared with 0.15 p.p.m. did not result in a consistentincrease in enzyme activity. Although differences were observedin growth due to dietary treatment, there were no significantcorrelations between preneoplastic foci and body weight, foodconsumption or food effidency. These findings indicate an interactionbetween dietary fat and selenium during initiation, but notduring early promotion. Furthermore, dietary selenium and fatmay function by different mechan isms at different stages ofcarcinogenesis.     

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.     

Dietary protein intervention during the postdosing phase of aflatoxin B1-induced hepatic preneoplastic lesion development

The effects of high and low dietary protein intervention on the growth and development of hepatic preneoplastic lesions were examined. Inbred male F344 rats fed a semipurified (AIN-76) 20% casein diet were given doses orally of aflatoxin B1 (10 doses, each 250 micrograms/kg). One week after the last dose, animals were fed diets containing either 5 or 20% casein. Animals fed a 5% casein diet throughout the 12-week postdosing period had a marked reduction in development of gamma-glutamyltransferase-positive foci. Animals fed a 20% casein diet throughout the same period had the highest response. Groups fed the 5% casein diet for half of the postdosing period and 20% for the other half had intermediate responses. The data show that the high response observed in animals fed a high-protein diet can be inhibited by the postinitiation intervention of a low-protein diet. Likewise, the low response observed in the animals fed a low-protein diet was increased by the introduction of a high-protein diet late in the experiment.     

SHORT COMMUNICATION: Evaluation of the post-initiation effects of oltipraz on aflatoxin B1-induced preneoplastic foci in a rat model of hepatic tumorigenesis

Previous studies have demonstrated that ingestion of 5-(2-pyimmyl)-4-methyl-1,2-dithiole-3-thione (oltipraz) during the aflatoxin B¹ (AFB₁)treatment phase completely prevented hepatic cancer. In thisstudy we evaluated the effect of feeding oltipraz during thepost-AFB₁ treatment phase. Fifty-five male F344 rats were dividedinto five groups. All rats were gavaged with 25 µg AFB₁/rat,five times a week for two successive weeks. The rats were fedthe oltipraz-supplemented diet according to three differentfeeding regimes: during the AFB₁ treatment phase (1 week priorto, during and 1 week after the last gavage with AFBj); duringthe post-treatment phase; or throughout the entire time of theexperiment. Phenobarbital-supplemented diet was fed during post-treatmentphase to one group and this was used as a positive control forthe promotion of AFB₁-induced focal growth. The burden of putative,preneoplastk, hepatic glutathione S-transferase P-positive fociwas evaluated at 13 weeks after the AFB₁ treatment phase. Asseen previously, oltipraz fed during the AFB₁ treatment phasesignificantly inhibited focal development, i.e. the volume percentof the liver occupied with foci was reduced by 87%. Oltiprazwhen fed during the post-treatment phase neither inhibited norenhanced focal development.     

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.     

Cell phenotype instability in preneoplastic foci of rat liver

Carcinogenesis is a multi-step process in which geneticphenotypicinstability and sequential selection of preneoplastic cellsfor increased growth capacity and other neoplastic characteristicsare essential phenomena. During chemical carcinogenesis in ratliver, the development of enzyme-deficient foci, their clonalorigin and their relationship to tumour formation are known.We report the results of four carcinogenesis protocols consistingof one or two cycles of diethylnitrosamine and phenobarbital.Histochemistry of three enzymes on serial sections has revealedseven different kinds of homogeneous liver foci, resulting fromsimple and combined enzyme deficiencies, and also heterogeneousfoci showing smaller foci inside. We consider such secondaryfoci as subclones originating from cells already modified thathave developed an additional phenotypic change. Some of suchfoci develop after the first cycle if the promotion phase isas long as 57 weeks. Comparing the number of foci per surfacearea of liver section with the number of secondary foci persurface area of focus section, it seems clear that cells alreadymodified are less stable than other hepatocytes, showing a highertendency to develop secondary changes.     

Sequential histological and histochemical study of the rat liver during aflatoxin B1-induced carcinogenesis.

Male Wistar rats were given 50 mug of aflatoxin B1 twice a week for 4 weeks, and thereafter 75 mug twice a week for 10 weeks. Their livers were investigated histologically and histochemically for glycogen, RNA, fat, alkaline and acid phosphatases, adenosine triphosphatase, 5'-nucleotidase, glucose-6-phosphatase, glucose-6-phosphate dehydrogenase, succinic dehydrogenase, and alkaline and acid nucleases. No significant lesions occurred before 15 weeks. During this period, the liver was histochemically unchanged except for a periportal decrease of alkaline phosphatase and adenosine triphosphatase. Scattered hepatocytes with a strong glucose-6-phosphatase activity appeared. These changes represent toxic effects of aflatoxin B1 and are irrelevant to carcinogenesis. From 15 weeks onward, three types of liver cell hyperplastic foci and nodules developed. Histologically, and with respect to glycogen, fat, and RNA content, only two of these types were considered as potential precursors of hepatocarcinomas. However, all types exhibited a decrease or absence of the enzymes studied. Both histological and histochemical changes stressed the complex heterogeneity existing between and within hepatic foci and nodules. From 11 months on, hepatocarcinomas developed. The tumors disclosed similar histochemical changes. This similarity further supports the "precarcinomatous" nature of hyperplastic foci and nodules. It appears that focal changes in surface as well as in cytoplasmic and nuclear enzymes are intimately and very early linked to the carcinogenic process. Whether they are fundamental or only represent an epiphenomenon remains unclear.     

Effect of dietary protein quality on development of aflatoxin B1-induced hepatic preneoplastic lesions

The effect of the quality of dietary protein on the post-initiation development of aflatoxin B1-initiated putatively preneoplastic foci in Fischer 344 rat liver was compared with the effect of the quantity of dietary protein. Feeding wheat gluten, a low-quality protein, during the postinitiation period (between the end of aflatoxin B1 dosing and the death of the rats) inhibited the development of gamma-glutamyltransferase-positive foci when compared with that in animals fed high-quality protein (casein) diets during the same period. Lysine supplementation of wheat gluten during the postinitiation period enhanced the gamma-glutamyltransferase-positive response to a level comparable with that of the high-quality protein. These results suggest that one can inhibit the development of foci either by decreasing the quantity of protein intake and holding the quality of the protein constant or by decreasing the quality and holding the quantity constant.     

Inhibition of aflatoxin B1-induced gamma-glutamyltranspeptidase positive (GGT+) hepatic preneoplastic foci and tumors by low protein diets: evidence that altered GGT+ foci indicate neoplastic potential.

Previous studies in this laboratory with young Fischer 344 male rats have shown that the post-initiation development of aflatoxin B1 (AFB1)-induced gamma-glutamyltranspeptidase positive (GGT+) hepatic foci was markedly inhibited by low protein feeding, even though the energy intake was greater. This dietary effect, however, did not necessarily apply to hepatic tumor development. Thus, the present investigation was undertaken to examine this dietary effect upon the development of hepatic tumors and, is so doing, to determine the correlation of foci development with tumor development. Following AFB1 dosing (15 daily doses of 0.3 mg/kg each), animals were fed diets containing 6, 14 or 22% casein (5.2, 12.2, 19.1% protein) for 6, 12, 40, 58 and 100 weeks. Foci at 12 weeks and tumors at 40, 58 and 100 weeks developed dose-dependently to protein intake. Foci development, tumor incidence, tumor size and the number of tumors per animal were markedly reduced while the time to tumor emergence was increased with low protein feeding. Non-hepatic tumor incidence also was lower in the animals fed the lowest protein diet. Foci development indices (foci number, per cent liver volume occupied) were highly correlated with tumor incidence at 58 and 100 weeks (r = 0.90-1.00). Tumor and foci inhibition occurred in spite of the greater energy intake.     

Biochemical studies during aflatoxin B1-induced liver damage in rats fed different levels of dietary protein

Aflatoxin B₁ has been fed for 6 months to young rats in low or high protein diets. In agreement with previous research, it was found that the high protein diet was associated with hyperplastic activity in the liver, of a type similar to that usually found in rats developing aflatoxin B₁-induced hepatoma. During the same period, negligible precancerous-like changes were seen in the rats fed the low protein diet with aflatoxin B₁. This model has been used to test the sensitivity of various liver function tests to the dietary-induced difference in liver reaction to aflatoxin B₁ feeding. The serum enzymes lactic dehydrogenase and alkaline phosphatase were considerably elevated in rats with precancerous-like lesions, but the former was the most sensitive enzyme. Serum glutamate-oxalate transaminase and serum glutamate-pyruvate transaminase were both raised in the rats with precancerous-like lesions, but much less so than lactic dehydrogenase and alkaline phosphatase. Urinary aflatoxin metabolites were also measured. Aflatoxin M₁ and aflatoxin P₁ were both found after feeding aflatoxin B₁. During the feeding period, aflatoxin P₁ excretion steadily increased, while aflatoxin M₁ increased between the second and fourth months and then fell. In the first 4 months, rats fed a low protein diet tended to produce a lower ratio of aflatoxin M₁ to aflatoxin P₁ compared to the rats fed a high protein diet. At 6 months, the ratio decreased markedly, especially in the rats with the precancerous-like lesions. It was concluded that lactic dehydrogenase, alkaline phosphatase, and the ratio of urinary excretion of aflatoxin M₁ to aflatoxin P₁ could be useful tests for inclusion in a diagnostic procedure for aflatoxin B₁-induced precancerous liver changes that might be expected in human studies.     

Natural chlorophyll inhibits aflatoxin B1-induced multi-organ carcinogenesis in the rat

Chemoprevention by chlorophyll (Chl) was investigated in a rat multi-organ carcinogenesis model. Twenty-one male F344 rats in three gavage groups (N = 7 rats each) received five daily doses of 250 microg/kg [(3)H]-aflatoxin B(1) ([(3)H]-AFB(1)) alone, or with 250 mg/kg chlorophyllin (CHL), or an equimolar amount (300 mg/kg) of Chl. CHL and Chl reduced hepatic DNA adduction by 42% (P = 0.031) and 55% (P = 0.008), respectively, AFB(1)-albumin adducts by 65% (P < 0.001) and 71% (P < 0.001), respectively, and the major AFB-N(7)-guanine urinary adduct by 90% (P = 0.0047) and 92% (P = 0.0029), respectively. To explore mechanisms, fluorescence quenching experiments established formation of a non-covalent complex in vitro between AFB(1) and Chl (K(d) = 1.22 +/- 0.05 microM, stoichiometry = 1Chl:1AFB(1)) as well as CHL (K(d) = 3.05 +/- 0.04 microM; stoichiometry = 1CHL:1AFB(1)). The feces of CHL and Chl co-gavaged rats contained 137% (P = 0.0003) and 412% (P = 0.0048) more AFB(1) equivalents, respectively, than control feces, indicating CHL and Chl inhibited AFB(1) uptake. However, CHL or Chl treatment in vivo did not induce hepatic quinone reductase (NAD(P)H:quinone oxidoreductase) or glutathione S-transferase (GST) above control levels. These results are consistent with a mechanism involving complex-mediated reduction of carcinogen uptake, and do not support a role for phase II enzyme induction in vivo under these conditions. In a second study, 30 rats in three experimental groups were dosed as in study 1, but for 10 days. At 18 weeks, CHL and Chl had reduced the volume percent of liver occupied by GST placental form-positive foci by 74% (P < 0.001) and 77% (P < 0.001), respectively compared with control livers. CHL and Chl reduced the mean number of aberrant crypt foci per colon by 63% (P = 0.0026) and 75% (P = 0.0004), respectively. These results show Chl and CHL provide potent chemoprotection against early biochemical and late pathophysiological biomarkers of AFB(1) carcinogenesis in the rat liver and colon.     

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.     

DNA damage triggers imbalance of proliferation and apoptosis during development of preneoplastic foci in the liver of Long-Evans Cinnamon rats

The mutant strain Long-Evans Cinnamon (LEC) rat accumulates copper, resulting in spontaneous hepatitis and subsequent development of hepatocellular carcinomas (HCCs) in the liver, providing a promising model for investigation of the relationship between hepatitis induced by oxidative stress and hepatocarcinogenesis. We examined DNA strand breaks in peripheral blood cells and p53 expression in livers during acute and chronic hepatitis in LEC rats, along with preneoplastic lesions, and cell proliferation and apoptosis in non-cancerous portions of livers from LEC rats aged 7-115 weeks. Immunohistochemistry using antibodies against glutathione S-transferase placental-form (GST-P), proliferating cell nuclear antigen (PCNA), and in situ DNA nick labeling (TUNEL) were used. Long-Evans Agouti (LEA) rats, a sibling line of the LEC strain, were used as controls. In the LEC rats, DNA strand breaks and expression of p53 were significantly higher than that of LEA rats at 24 weeks of age. The number of GST-P-positive (GST-P+) foci/cm2 increased and peaked at 48 weeks old, and the areas rapidly expanded thereafter. The level of cell proliferation increased with the development of hepatitis and was highest at about 48 weeks old. The induction of apoptosis in LEC rats was transiently higher than that in LEA rats during the period from 24 to 34 weeks of age. However, the ratio of PCNA-positive cells to the apoptotic index showed a growth imbalance in favor of cell proliferation, supporting sustained net growth in LEC rats. These findings suggest that DNA damage, reflected in DNA strand breaks, plays a critical role in the development of hepatocellular preneoplastic foci, with an imbalance between high proliferation and relatively low apoptosis.     

Aflatoxin B1-induced DNA damage and its repair

Aflatoxin B(1) (AFB(1))-N(7)-guanine is the predominant adduct formed upon the reaction of AFB(1)-8,9-exo-epoxide with guanine residues in DNA. AFB(1)-N(7)-guanine can convert to the ring-opened formamidopyrimidine, or the adducted strand can undergo depurination. AFB(1)-N(7)-guanine and AFB(1)-formamidopyrimidine are thought to be predominantly repaired by nucleotide excision repair in bacteria, yeast and mammals. Although AFB(1)-formamidopyrimidine is removed less efficiently than AFB(1)-N(7)-guanine in mammals, both lesions are repaired with equal efficiencies in bacteria, reflecting differences in damage recognition between bacterial and mammalian repair systems. Furthermore, DNA repair activity and modulation of repair by AFB(1) seem to be major determinants of susceptibility to AFB(1)-induced carcinogenesis.     

Ethoxyquin alone induces preneoplastic changes in rat kidney whilst preventing induction of such lesions in liver by aflatoxin B1

Pretreatment of Fischer 344 rats with the antioxidant ethoxy-quin(EQ), followed by administration of aflatoxin B₁, (AFB) in thecontinuing presence of EQ was used to examine the effect ofthe antioxidant on liver and kidney. EQ (0.5% in diet) completelyprevented the formation of AFB₁-induced preneoplastic liverlesions as judged by morphological alteration, or by markerssuch as gamma glutamyl transpeptidase, glutathione S-transferaseP or J1, an unknown membrane-bound antigen. While protectionwas afforded to the liver, EQ alone caused severe damage tothe kidney. Many changes were those of chronic glomerulonephrosis,such that EQ appeared to accelerate the ageing process. In addition,many hyperplastic and putative preneoplastic tubules were visible,suggesting that EQ may be exerting a carcinogenic effect inthe kidney.     

Immunohistochemical demonstration of a mRNA-transport protein in rat liver putative preneoplastic foci

A 65K protein, known for promoting nucleocytoplasmic mRNA transport in a cell-free system, was previously found in fetal and tumor cells of the rat. The primary objective of this study was to show specificity of immunohistochemical staining for the 65K protein in the livers of rats subjected to a hepatocarcinogenesis protocol. Altered hepatic foci were induced by feeding male weanling Sprague-Dawley rats 2-acetylaminofluorene (AAF) followed by a phenobarbital (PB) diet. It was shown, using polyclonal antibodies produced in rabbits, that the 65K protein was present in the cells of rat liver putative preneoplastic foci, with little or none being detected in the surrounding cells.     

Effect of aflatoxin B1 on DNA enzymatic methylation and secondary structures of the rat liver

Methylation and secondary structure of DNA in rat liver versus time of aflatoxin B1 (AfB1) treatment were studied. A 50% drop in 5-methylcytosine level in liver DNA as compared with control was observed long before tumor development. Also, certain changes in DNA secondary structure were seen. The DNA changes were more pronounced in the F1 progeny of rats inoculated with AfB1 at the terminal stage of gestation.     

金蒲抑瘤片在实验诱发大鼠肝癌过程中的作用

目的探讨金蒲抑瘤片对黄曲霉毒素B1(aflatoxin B1,AFB1)致肝癌作用的影响。方法实验动物随机分为高剂量、低剂量和对照组。用AFB1处理各组动物,高、低剂量组大鼠在接受AFB1期间分别喂含量为9.3和2.3g/kg的金蒲抑瘤片混合饲料,对照组喂基础饲料。8周后处死动物,观察各组动物肝组织内γ-谷氨酰转肽酶阳性肝细胞增生(γ-glu-tamyltranspeptidase-positive hyperplastic livercell,γ-GT)灶的数量和大小。结果高、低剂量金蒲抑瘤片均能减少AFB1诱发的γ-GT灶的数量和大小高、低剂量组的数量分别为0.90和3.72个/cm2,均低于对照组6.10个/cm2,抑制率分别为85%和39%,高剂量组与对照组相比差异有统计学意义,t=2.597,P=0.028。高、低剂量组的大小分别为0.24和1.94mm2/个,均低于对照组2.36mm2/个,抑制率分别为90%和17%,但差异无统计学意义,P>0.05。结论金蒲抑瘤片有减少AFB1诱发大鼠肝γ-GT灶的数量和大小的作用,而高剂量金蒲抑瘤片的减少趋势更强。     

Glucose alters rat liver S9-mediated mutagenesis, metabolism and DNA-binding of aflatoxin B1

The administration of 30% glucose in drinking water to rats for 48 h caused a significant increase in the hepatic S9-mediated mutagenicity of aflatoxin B1, in Salmonella typhimurium TA100 and in the binding of alfatoxin B1, to calf thymus DNA in vitro. These effects correlated with a reduction in the metabolism and detoxification of aflatoxin B1, by S9 from glucose-treated rats and suggest that the oral intake of sugar may affect the hepatocarcinogenicity of aflatoxin B1.