Development Of Altered Hepatic Foci Research Articles

Inhibitory effect of tea polyphenols on hepatic preneoplastic foci in Wistar rats

Tea (Camellia sinensis) is one of the most widely used beverages worldwide and tea consumption has been shown to have an inverse correlation to the incidence of human cancers in epidemiological and experimental studies. In the present study, the protective effects of green tea polyphenols (GTP) and black tea polyphenols (BTP) in Wistar rats were assessed by medium-term bioassay, using altered hepatic foci (AHF) as end point. Animals were exposed to a single dose of diethylnitrosamine (DEN; 200 mg/kg body weight intraperitoneally), and GTP (1%) and BTP (1%) were then administered orally together with 0.05% 2-acetyl aminofluorene (2-AAF) crushed and mixed in the diet for 8 weeks. Numbers of AHF were scored and analyzed by quantitative stereology using the Image analysis system from frozen liver tissue sections. Tea polyphenol supplementation resulted in a significant protection against AHF induction in Wistar rats. In addition, levels of the positive biomarkers: gamma-glutamyl transpeptidase and glutathione-S-transferase (placental form) were reduced with GTP and BTP supplementation. Levels of the negative biomarkers adenosine triphosphatase and glucose-6-phosphatase were also restored by GTP and BTP administration. Thus, these results show the hepatoprotective effects of GTP and BTP against DEN- and 2-AAF-induced AHF development.

Inhibitory effect of celery seeds extract on chemically induced hepatocarcinogenesis: modulation of cell proliferation, metabolism and altered hepatic foci development

The chemopreventive activity of methanolic extract of Apium graveolens seeds (celery seeds) has been investigated against Solt Farber protocol of hepatocarcinogenesis, oxidative stress and induction of positive foci of γ-GT in the liver of Wistar rats. The prophylactic treatment of celery seeds extract protected dose dependently against diethylnitrosoamine (DEN)+2-acetylaminofluorine (AAF)+partial hepatectomy (PH) induced hepatocarcinogenesis and other related events such as induction of γ-GT positive foci ( P<0.001). 2-AAF administration in diet with PH in rats resulted in increased hepatic ornithine decarboxylase (ODC) activity and a consequent increase in the rate of DNA synthesis when compared to saline treated control group while pretreatment of rats with celery seeds extract resulted in inhibition of aforementioned parameters dose dependently. The augmentation of quinone reductase (QR), glutathione- S-transferase (GST) and serum γ-glutamyl transpeptidase (GGT) activities; and depletion of the tissue GSH content after 2-AAF (i.p. injection) for five consecutive days was prevented with the administration of celery seed extract. On the basis of the above results it can be said that A. graveolens is a potent plant against experimentally induced hepatocarcinogenesis in Wistar rats.

Chemopreventive Effect of Indole-3-Carbinol on Induction of Preneoplastic Altered Hepatic Foci

: Indole-3-carbinol (I3C) is a cleavage product of glucobrassicanin, a natural compound present in a wide variety of plant food substances including members of the family Cruciferae. I3C is known to possess cancer-chemopreventive potential in various animal models. The present study reveals the protective effect of I3C on the development of diethylnitrosamine (DEN)-initiated and 2-acetylaminofluorene (AAF)-promoted preneoplastic, altered hepatic foci (AHF) in Wistar rats. I3C was given at dose levels of 0.5 and 1 mg/kg body weight for five consecutive days along with DEN and AAF. AHF were scored and analyzed by quantitative stereology using the Image Analysis System from frozen liver sections stained for positive and negative biological markers of AHF, that is, glutathione S-transferase (GST-P), γ-glutamyl transpeptidase (GGT), glucose-6-phosphatase (G6Pase), adenosine triphosphatase (ATPase), and alkaline phosphatase (AlkPase). Results revealed the chemopreventive effect of I3C on the DEN-initiated AHF in Wistar rats. The expression of G6Pase, ATPase, and AlkPase was restored in the I3C-supplemented animal. Similarly the induced expression GST-P and GGT also decreased in the animals with I3C administration. The recovery of altered levels of these biomarkers was of comparatively higher magnitude in the animals given a higher dose of I3C (1 mg/kg body weight) in comparison with the animals given 0.5 mg/kg body weight dose of I3C, although no dose-dependence pattern was recorded in I3C-supplemented groups. These results thus suggest the chemopreventive effect of I3C in rat hepatocarcinogenesis by suppressing DEN- and AAF-induced AHF development.

Modulation of altered hepatic foci induction by diallyl sulphide in Wistar rats

Diallyl sulphide (DAS) is a sulphur-containing volatile compound present in garlic (Allium sativum). It has been shown to inhibit a number of chemically induced forms of cancer in experimental animals. The present study demonstrates the inhibitory effect of DAS on the development of diethylnitrosamine (DEN) initiated and 2-acetyl-aminofluorene (2-AAF) promoted preneoplastic altered hepatic foci (AHF) in Wistar rats. AHF were scored and analysed by quantitative stereology using the Image Analysis system from frozen liver sections stained for biological markers, namely glutathione S-transferase, placental form (GST-P), gamma-glutamyl transpeptidase (GGT), adenosine triphosphatase (ATPase), glucose-6-phosphatase (G6 Pase) and alkaline phosphatase (AlkPase). DAS-supplemented rats were found to restore the near-normal levels of enzymes GST-P and GGT when exposed to DEN and 2-AAF. DAS administration following DEN and 2-AAF exposure led to the restoration of enzymic activity of ATPase, G6 Pase and AlkPase, as evident by number and area of the foci. These findings suggest the protective role of DAS in rat hepatocarcinogenesis, by suppressing DEN- and 2-AAF-induced AHF development.

Enhancing effects of mustard oil on preneoplastic hepatic foci development in Wistar rats.

Dietary habits are known to be the major contributory factor in the development of cancer. Mustard oil, which is extensively used in India and elsewhere as a flying and cooking medium, is reported to induce an inflammatory response. The development of altered hepatic foci is an early carcinogenic change in rat liver in diethylnitrosamine (DEN)-induced hepatocarcinogenesis. In the present study, the development of preneoplastic lesions was observed following administration of mustard oil (0.5 mL/day for 8 weeks) in DEN-initiated and partially hepatomized Wistar rats. A significant decrease in the relative and absolute liver weight of mustard oil-exposed rats was recorded. The results revealed a significant increase in the number and area of placental glutathione-S-transferase (GST-P) and gamma-glutamyl transpeptidase (GGT)-positive foci in mustard oil-administered animals. The GST-P- and GGT-positive foci were more prominent in the animals given boiled (up to 300 degrees C for 3 hours) mustard oil in comparison to the animals given fresh mustard oil. These results indicate the possible tumourigenic risk associated with mustard oil consumption.

Suppression of Altered Hepatic Foci Development by Curcumin in Wistar Rats

Curcumin, a yellow pigment of turmeric (Curcuma longa), is a commonly used spice and a coloring agent in foods, drugs, and cosmetics. Curcumin is known to possess chemopreventive properties in various animal tumor models. In the present study the effect of curcumin on the development of altered hepatic foci (AHF), by using a medium term liver bioassay, has been evaluated. AHF were analyzed by quantitative stereology using the Leica Qwin Image Analysis system from frozen liver sections stained for g-glutamyl transferase, adenosine triphosphatase, glucose-6-phosphatase, alkaline phosphatase, and placental isozyme of glutathione S-transferase. A significant protection on diethylnitrosamine (DEN) initiated and 2-acetylaminofluorene (AAF) promoted AHF by curcumin was observed on these biological markers. The curcumin administration was found to restore the normal levels of the enzymes glutathione S-transferase and g-glutamyl transferase in rat liver following DEN-AAF exposure. Similarly, a significant protection was provided by curcumin in the enzyme-deficient foci for the adenosine triphosphatase-, alkaline phosphatase-, and glucose-6-phosphatase-treated groups in comparison to the DEN-AAF-treated group. These results show that curcumin can effectively suppress the DEN-induced development of AHF in rat liver.

Menhaden Oil Inhibited γ-Glutamyltransferase-Positive Altered Hepatic Foci in Female Sprague-Dawley Rats

Feeding menhaden oil, high in n-3 fatty acids, or a mixture of lard and corn oil with a polyunsaturated-to-monounsaturated fatty acid ratio of 1:1 was hypothesized to inhibit promotion of hepatocarcinogenesis in rats by decreasing hepatic prostaglandin (PG) levels. Ten-day-old female Sprague-Dawley rats were injected with diethylnitrosamine (DEN, 15 mg/kg body wt ip). At 4 wk of age, rats were fed fumonisin B1(50 mg/kg diet) for 5 wk in diets containing 14% lard + 6% corn oil, 10% lard + 10% corn oil, 14% menhaden oil + 6% corn oil, and 7% menhaden oil + 13% corn oil. Plasma alanine aminotransferase activity was 20% lower in rats fed 10% lard than in rats fed the other diets (P < 0.05). In menhaden oil-fed rats, total plasma cholesterol concentrations decreased 26% (P <5) and hepatic phospholipid C20:5n-3, C22:5n-3, and C22:6n-3 fatty acid concentrations increased compared with lard-fed rats. Hepatic n-3 fatty acids were threefold greater in rats fed 10% lard than in rats fed 14% lard. The liver-associated natural killer cell activity in rats fed menhaden oil was 58% lower than in rats fed lard (P <3). Rats fed lard had threefold (P <5) greater area of γ-glutamyltransferase-positive altered hepatic foci (AHF) than did rats fed menhaden oil. There was no significant difference in placental glutathione S-transferase-positive AHF among the groups. Hepatic PGF2αproduction was 60-80% greater in rats fed 14% lard than in rats fed the other diets (P <5). Hepatic PGE2was 48% less in rats fed 14% menhaden oil than in rats fed 14% lard (P <5). Although γ-glutamyltransferase-positive focal area was inhibited by menhaden oil, only 14% menhaden oil inhibited PGE2;. Feeding 10% lard inhibited PGF2α, but not the development of AHF. Therefore, decreased hepatic PGs did not explain the inhibition of carcinogenesis.

Suppression of Altered Hepatic Foci Development by a High Fish Oil Diet Compared With a High Corn Oil Diet in Rats

Effects of low corn oil, high corn oil, and high fish oil diets on altered hepatic foci development in female Sprague-Dawley rats were investigated. Rats assigned to Groups 1-4 were initiated with saline as the control and those assigned to Groups 5-7 were initiated with diethylnitrosamine (DEN 15 mg/kg) at 24 hours of age. After weaning, all rats, except those in Group 1, received 500 ppm phenobarbital (PB) in their diet as tumor promoter for three months. Altered hepatic foci development was significantly lower in DEN-initiated rats fed the high fish oil + PB diet than in DEN-initiated rats fed the high corn oil + PB diets. Liver weight and relative liver weight were significantly greater in rats fed the high fish oil + PB diet than in rats fed the other diets, and hepatic biotransformation/detoxification enzyme activities were greater in rats fed the fish oil + PB diets than in rats fed the other diets. These results suggest that the effect of a high fish oil diet on altered hepatic foci may occur through regulation of hepatic biotransformation/detoxification enzyme activities, leading to alteration in the tumor-promoting action of PB. Dietary lipid significantly affected the hepatic phospholipid fatty acid composition of rats. n-3 polyunsaturated fatty acids were incorporated into membrane phospholipid at the expense of n-6 polyunsaturated fatty acids. A high fish oil diet caused greater oxidative stress in rats, as measured by plasma vitamin E level, red blood cell glutathione status, liver lipid peroxidation, and hepatic glutathione reductase activity. Pearson's correlation analysis indicated that the foci number was negatively correlated to the liver thiobarbituric acid-reactive substance and 7-pentoxyresorufin O-dealkylase activity, and the foci area was negatively correlated to the liver thiobarbituric acid-reactive substance activity (p << 0.05) in rats of groups that developed foci. These results suggest that the type of dietary lipid is the more important determinant for γ-glutamyl transpeptidase-positive foci development than the amount of dietary lipid when rats consumed approximately the same amount of calories in all the dietary groups, and the underlying mechanisms may be partially ascribed to the antioxidant/oxidation status and biotransformation/detoxification system of rats.

Effect of the separate and combined administration of mestranol and phenobarbital on the development of altered hepatic foci expressing placental form of glutathione S-transferase in the rat.

The stages in the carcinogenesis process include initiation, promotion and progression. Although many characteristics of tumor promotion and promoting agents have been reported, relatively few studies on the effects of combinations of promoting agents have been detailed. For study of the combined effects of phenobarbital (PB) and mestranol (MS) in multistage rat hepatocarcinogenesis, an initiation-promotion protocol was developed. Female rats were injected i.p. at 5 days of age with either diethylnitrosamine (DEN) (10 mg/kg) or the solvent tricaprilyn. At weaning, approximately 10 rats from both the DEN-initiated and the solvent control groups were provided basal diet alone, PB (10, 100 or 500 mg/kg diet), MS (0.02 or 0.2 mg/kg diet) or various combinations of both PB and MS in the basal diet. At 8 months of age, the rats were killed and the livers removed, sectioned and fixed in ice-cold acetone. Sections of 5 microgram thickness were stained for placental glutathione S-transferase (PGST) expression, and the volume fraction of liver occupied by altered hepatic foci was determined by stereology. In addition, incorporation of bromodeoxyuridine into nuclei of PGST-expressing (focal) and non-PGST-expressing (non-focal) hepatocytes was determined. Administration of the highest dose of PB resulted in a significant decrease in non-focal hepatocyte labeling index, with a 4-fold differential between the focal and non-focal hepatocyte labeling index. Administration of 0.2 mg/kg diet MS resulted in effective promotion. The non-focal labeling index was increased and the focal labeling index was further enhanced (3-fold) relative to the non-focal index by this dose of MS. Combination of the lower MS dose with PB resulted in at least an additive promoting effect; however, a lower volume fraction was noted for the combination of low MS dose plus the highest PB dose. Combination of the higher MS dose with PB resulted in an elevation of volume fraction only for the middle PB dose. These findings indicate that the potency of promotion by mixtures is modulated by the dose of each component as well as by pharmacodynamic and pharmacokinetic properties of each component of the mixture.

Focal and non-focal hepatic expression of placental glutathione S-transferase in carcinogen-treated rats.

Carcinogenesis develops in stages that have been operationally defined as initiation, promotion and progression. Although morphological end points have been described for detection and quantitation of these stages, to date initiation has been assessed only in the context of clonal growth in response to certain promoting agents. Initiated cells are morphologically indistinguishable from surrounding cells and early changes at the cellular level during initiation have not been clarified. One commonly used end point for the detection of preneoplastic hepatic lesions i their aberrant expression of the placental isozyme of glutathione S-transferase (PGST). Because single hepatocytes expressing PGST have been detected in aged rats and in those administered hepatocarcinogens, it has been suggested that such cells constitute a population of putatively initiated hepatocytes. In order to further elucidate the characteristics of single PGST-positive hepatocytes, we analyzed the number of these cells 2 and 18 weeks after various doses (0-100 mg/kg) of diethylnitrosamine (DEN) and of dimethylbenz[a]anthracene (DMBA). When determined 14 days after carcinogen administration, the number of single hepatocytes expressing PGST was greater after DEN administration (ranging from 0.8 +/- 0.3 per cm2 transection of liver at 1 mg/kg to 33.0 +/- 4.7 at 100 mg/kg) than after DMBA administration (ranging from 0.25 +/- 0.14 at 10 mg/kg to 3.03 +/- 0.5 at 100 mg/kg); none were detected in control rats of the same age. Additional rats were maintained on a basal diet or a basal diet plus phenobarbital for a further 4 month period. Whereas individual PGST-positive hepatocytes were only sporadically detected in rats treated with DMBA and maintained on a basal diet for 18 weeks, those rats placed on phenobarbital for 16 weeks had an even higher number of such PGST-positive hepatocytes than at 2 weeks after DMBA administration. In contrast, the dose-response curve observed for DEN-treated rats 18 weeks after carcinogen administration was similar to that observed 2 weeks after carcinogen treatment for both phenobarbital- and non-phenobarbital-treated rats. In addition, the number of single PGST-positive hepatocytes detected at 2 weeks was directly parallel to the number of altered hepatic foci expressing PGST 18 weeks after DEN administration. The dose-dependent induction of PGST-positive single hepatocytes after treatment with two hepatocarcinogens, the dose-dependent growth of altered hepatic foci (AHF) expressing PGST with phenobarbital administration and the parallel dose-response curve of single hepatocytes expressing PGST and later of AHF expressing PGST argue strongly for a precursor role of single PGST-positive cells in the development of AHF expressing PGST.

The insecticide endosulfan and its two stereoisomers promote the growth of altered hepatic foci in rats.

The non-genotoxic, chlorinated cyclodiene insecticide endosulfan was studied for its ability to act as a tumour promoter in a two-stage, altered hepatic foci bioassay in male Sprague-Dawley rats. Two stereoisomers of endosulfan, alpha-endosulfan (ENDO alpha) and beta-endosulfan (ENDO beta) were used, as well as a commercially-occurring mixture of the alpha- and beta-isomers (ENDO alpha beta). The animals were initiated by intraperitoneal injection of nitrosodiethylamine 24 h after a two-thirds-partial hepatectomy. Five weeks later the animals were transferred to diets containing 30, 100 and 300 p.p.m. of either ENDO alpha beta, ENDO alpha or ENDO beta. The study was terminated 25 weeks after initiation and the development of foci of gamma-glutamyltranspeptidase-positive hepatocytes was evaluated by stereological methods. The results show that endosulfan and its two stereoisomers promote the development of altered hepatic foci, suggesting that endosulfan is a tumour-promoting agent acting by clonal expansion of initiated cells.

Relative Liver Tumour Promoting Activity and Toxicity of some Polychlorinated Dibeiizo‐p‐dioxin‐ and Dibenzofuran‐Congeners in Female Sprague‐Dawley Rats

The polychlorinated dibenzo-p-dioxins/dibenzofurans 1,2,3,7,8-pentachlorodibenzo-p-dioxin (PeCDD), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 2,3,4,7,8-pentachlorodibenzofuran (PeCDF) were studied for liver tumour promoting activity in a medium-term altered foci assay in nitrosamine-initiated female Sprague-Dawley rats. The congeners under study were administered by weekly subcutaneous injections at three dose levels for 20 weeks. Evaluation of gamma-glutamyltranspeptidase (GGT+), altered hepatic foci development, showed that all congeners studied acted as potent promoters of hepatocarcinogenesis. TCDD and PeCDD were virtually equipotent as enhancers of foci development while PeCDF displayed approximately ten per cent of the activity of the dioxins. Analysis of the dioxin- and furan-congeners by gas chromatography/mass spectroscopy (GC/MS) technique showed that the retention of PeCDD and PeCDF in liver tissue was approximately 7 and 20 times, respectively, as high as the retention of TCDD. Based on the concentration of the respective congener in liver tissue, PeCDD and PeCDF were 0.14 and 0.007 times as active as TCDD as promoters of foci development. The dose related enhancement of GGT+ foci development induced by the PCDD/PCDF congeners was accompanied by an increased incidence of histological changes in the liver.

Modulation of 2,3,7,8-tetrachlorodibenzo- p-dioxin and phenobarbital-induced promotion of hepatocarcinogenesis in rats by the type of diet and vitamin a deficiency

This study was undertaken to investigate the influence of dietary vitamin A deficiency and type of diet on tetrachlorodibenzo- p-dioxin (TCDD)- and phenobarbital-induced liver tumor promotion in rats. Female Sprague-Dawley rats were partially hepatectomized and subsequently initiated with nitrosodiethylamine. One week later the rats were allocated to five different dietary regimens for the duration of the study: three purified (casein-based) diets containing 200, 1200, and 10000 IU vitamin A per kilogram, respectively, and two conventional (cereal-based) rat diets containing 2000 and 14000 IU vitamin A per kilogram, respectively. After an additional 4 weeks, groups of rats on each dietary regimen were started on one of four different promoter treatments: 0.07 μg TCDD/kg/week (sc); 0.7 μg TCDD/kg/week (sc); 500 ppm phenobarbital in the drinking water or vehicle only (arachis oil, sc). The study was terminated after 15 weeks of promoter treatment. Sections of liver were stained for γ-glutamyltranspeptidase (GGT) activity and GGT-positive altered hepatic foci (AHF) were evaluated by stereological methods. All factors studied (TCDD, phenobarbital, dietary vitamin A content, and the type of diet) were shown to influence AHF development significantly. As expected, TCDD and phenobarbital enhanced foci development. Vitamin A deficiency enhanced foci development in its own right and increased the TCDD-induced response markedly. Dietary vitamin A content did not modulate phenobarbital promotion of AHF in the same manner. The enhancement of TCDD-induced effects on foci development by vitamin A deprivation was accompanied by an increased incidence of histological changes marking degeneration in the liver (e.g., oval cell hyperplasia) and accentuation of other TCDD-related toxic responses. In addition, the groups of rats maintained on the cereal-based diets and subjected to the various promoter/vitamin A regimens exhibited significantly higher AHF incidence as compared to correspondingly treated rats fed the purified, casein-based diets. In conclusion, vitamin A deficiency alone may promote hepatocarcinogenesis and enhance the promoting effect of TCDD treatment. However, TCDD-induced depletion of hepatic vitamin A stores was not implicated as a major cause of promotion by TCDD. Nevertheless, vitamin A deficiency brought about by TCDD alone may well act as a promotive stimulus concertedly with an as yet unidentified cellular mechanism in TCDD-induced liver tumor promotion. The differential effects of the two types of diets recorded in the study remain undocumented.

The effect of the format of administration and the total dose of phenobarbital on altered hepatic foci following initiation in female rats with diethylnitrosamine

The effect of changing the format of administration as well as the total dose of the promoting agent phenobarbital (PB) on the development of altered hepatic foci (AHF) was determined in an initiation-promotion protocol with female rats fed the purified AIN-76 diet. Effects on the total number of AHF and the volume percentage of liver occupied by AHF were determined for four histochemical markers, the placental form of glutathione S-transferase, gamma-glutamyl-transpeptidase canalicular ATPase, and glucose-6-phosphatase after 16 and 60 weeks of promotion with varying doses and formats of PB, as well as for a further 16-week period in which no PB was administered. At the 16-week point, animals fed 0.1% PB continuously exhibited the largest number and volume percentage of AHF, whereas rats fed 0.1% PB for 4 days followed by 10 days of no PB with continuous repetition of this pattern during the 16-week treatment period exhibited no increase in the number of AHF over control and only a slight increase in volume percentage. Rats fed a continuous repetition of 0.2% PB for 2 days followed by 12 days of no PB exhibited an intermediate increase in the number of AHF as well as the volume percentage fraction after 16 weeks of this regimen. After 60 weeks of feeding PB by these three different formats, the numbers of AHF observed in these groups were equivalent and had increased above those seen after 16 weeks of feeding. The volume percentage occupied by the AHF in these three groups was also similar, although animals receiving 0.2% PB intermittently showed a significantly lower volume percentage than animals receiving 0.1% PB continuously for 60 weeks. When animals were maintained for an additional 16 weeks without PB feeding, the numbers of AHF decreased dramatically, much more so in animals fed PB intermittently, whereas the volume percentage fraction of AHF in livers of animals receiving 0.1% PB continuously for 60 weeks almost doubled. In contrast, the volume percentage fraction of AHF in livers of animals receiving PB intermittently for 60 weeks followed by 16 weeks of no PB was slightly less. Examination of the individual size classes of AHF showed little change in their distribution at 16 and 60 weeks, but after 16 weeks of PB withdrawal (76 weeks total time), the distribution of AHF in animals that had received 0.1% PB continuously for 60 weeks exhibited a decidedly greater shift to larger AHF than animals receiving PB intermittently for the 60-week period.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of dietary vitamin E on the development of altered hepatic foci and hepatic tumors induced by the peroxisome proliferator ciprofibrate

The purpose of this study was to determine the effect of the dietary antioxidant vitamin E on hepatocarcinogenesis by peroxisome proliferators which, it is hypothesized, induce tumors by increased production of hydrogen peroxide or other oxygen radicals. Rats were fed diets containing the peroxisome proliferator ciprofibrate and one of three concentrations (10, 50, or 500 ppm) of alpha-tocopheryl acetate for 6 months or 21 months. The incidence of hepatic tumors and the number and volume of gamma-glutamyl-transpeptidase-positive, ATPase-negative, glucose-6-phosphatase-negative, and glucose-6-phosphatase-positive foci were quantified. No tumors or altered hepatic foci were seen at 6 months, but at 21 months the incidence of hepatic tumors and the number and volume of altered hepatic foci were increased in rats fed higher levels of vitamin E. Indices of oxidative damage--concentrations of malonaldehyde, conjugated dienes, and lipid-soluble fluorescence products--were not affected or were lower in rats fed higher amounts of vitamin E; the enhancing effect of vitamin E on the development of altered hepatic foci and hepatic tumors, therefore, was not related to the induction of cellular oxidative damage. Hepatic peroxisomal fatty acid beta-oxidation and vitamin C concentrations were not affected by vitamin E, whereas the glutathione concentration was decreased in rats fed higher amounts of vitamin E. This study shows that increasing the vitamin E content of the diet enhances ciprofibrate-induced hepatocarcinogenesis, but the mechanism of this effect is unclear.

Quantitative stereologic study of the effects of varying the time between initiation and promotion on four histochemical markers in rat liver during hepatocarcinogenesis

The effects of varying the interval of time between initiation with diethylnitrosamine (DEN) and promotion by phenobarbital (PB) on the development of altered hepatic foci (AHF) and hepatomas in female Fischer 344 rats was investigated. The intervals between DEN initiation after a 70% partial hepatectomy and a subsequent 6 month period of promotion by feeding of PB were 1 day, 1 week, 1 month, 2 months, 6 months and 11 months. The number and volume percentage occupied by AHF were determined by quantitative stereologic methods on serial frozen sections stained for the markers gamma-glutamyltranspeptidase (GGT), canalicular adenosine triphosphatase (ATPase), glucose-6-phosphatase (G6Pase) and the placental form of glutathione S-transferase (GST-pi). The number of AHF was greatest when the initiation-promotion interval was only 1 day, and there was a tendency for the number of AHF to decrease as the interval between initiation with DEN and the start of PB promotion was extended. An 11 month delay between initiation and promotion resulted in only 20% fewer AHF than when promotion was begun 1 day after initiation. On the other hand, the volume percentage fraction of AHF did not change when the initiation-promotion interval was increased from 1 day to 2 months. An interval of 6 months roughly doubled the volume percentage fraction, but an interval of 11 months led to a 7- to 8-fold increase in the volume percentage of AHF over that from a 1 day interval. The phenotypic distribution of AHF was significantly lower in relation to certain markers, especially GGT and GST-pi, in those animals only initiated with DEN compared with those initiated with DEN and promoted with PB. When no exogenous promotion was given, there was still a nearly linear increase in both the number and volume percentage occupied by AHF in the liver of rats initiated with DEN. On the other hand, rats subjected to a 1 week interval between DEN initiation and PB promotion exhibited the greatest number of hepatocellular carcinomas 14 months after initiation, compared with other groups. These studies demonstrated a gradually decreasing effectiveness of PB as a promoting agent to stimulate the growth of all AHF initiated by DEN as the interval between initiation and promotion was extended.(ABSTRACT TRUNCATED AT 400 WORDS)

Enhancement of altered hepatic foci development by 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) — Effects of vitamin A supplementation and the type of diet

In this study we investigated the modification of TCDD-induced enhancement of nitrosamine-initiated altered hepatic foci (AHF) development by dietary vitamin A supplementation and the type of diet. Vitamin A deficiency was shown to potentiate the effect of TCDD. Furthermore, severe vitamin A deficiency alone enhanced the development of AHF. A differential effect on AHF-development by the two types of diet used (purified vs cereal diet) was apparent. However, it was concluded that TCDD-induced depletion of hepatic stores of vitamin A is not a primary mechanism in liver tumour promotion induced by this compound. However, it may well be that TCDD-induced vitamin A deficiency acts as a tumour promoting stimulus in concert with an, as yet, unidentified mechanism of tumour promotion by TCDD.

Promotion of altered hepatic foci development in rat liver, cytochrome P450 enzyme induction and inhibition of cell-cell communication by DDT and some structurally related organohalogen pesticides

The organochlorine pesticide 1,1'-(2,2,2-trichloroethylidene) bis(4-chlorobenzene) (DDT) and four structural analogues (bromopropylate, chlorobenzilate, dicofol and fenarimol) were investigated for their ability to inhibit gap junctional intercellular communication both in the Chinese hamster V79 metabolic co-operation assay and in the scrape-loading/dye-transfer assay in WB-F344 rat liver epithelial cells. The pesticides were also studied for their ability to enhance the development of gamma-glutamyltranspeptidase-positive altered hepatic foci and induce cytochrome P450 monooxygenase isoenzymes in nitrosamine-initiated male Sprague-Dawley rats. The in vitro studies showed all organohalogens except fenarimol to be potent inhibitors of cell-cell communication in both test systems used. Concomitant results were recorded in the in vivo study. Thus, all potent inhibitors of intercellular communication were found to enhance significantly foci development and fenarimol was again without any significant effect. All pesticides studied were shown to be potent inducers of the phenobarbital-inducible cytochrome P450b isoenzyme and to cause hepatomegaly. Thus, no strict correlation between cytochrome P450b induction/liver growth and tumour promotion-related effects in vivo and in vitro was apparent for these organohalogen pesticides in the present study.

Effects of calcium glucarate on the promotion of diethylnitrosamine-initiated altered hepatic foci in rats

Calcium glucarate (CGT), an inhibitor of β-glucuronidase, is a potent inhibitor of chemically-induced tumors when administered orally. The present study was undertaken to determine the effects of CGT on the promotion of hepatocarcinogenesis by phenobarbital following initiation with diethylnitrosamine (DENA). Partially hepatectomized, DENA-initiated female Sprague-Dawley rats, previously maintained only on chow diet for 2 months, were supplemented with either 0.05% phenobarbital alone or 0.05% phenobarbital plus 4% dietary CGT, for varying time intervals up to 6 months. Histopathologic evaluation of the liver sections showed that CGT significantly delayed the development of altered hepatic foci (AHF). By the seventh month post-initiation, however, the frequency and severity of changes seen in the livers of experimental animals approximated those of the controls.

Sequential study on the synergistic effects of 2,2',4,4',5,5'-hexabromobiphenyl and 3,3',4,4',5,5'-hexabromobiphenyl on hepatic tumor promotion.

A sequential study was completed to determine the effect of polybrominated biphenyl (PBB) congeners on the enhancement of gamma-glutamyl transpeptidase (GGT)-positive altered hepatic foci (AHF) and the development of hepatic nodules (HN) and carcinomas. Female Sprague-Dawley rats were given a single dose of N-nitrosodiethylamine (NDEA) 24 h following a 70% partial hepatectomy. Thirty days later, rats were randomly assigned to groups and fed a basal diet or the basal diet containing 10 p.p.m. 2,2',4,4',5,5'-hexabromobiphenyl (245-HBB), 0.1 p.p.m. 3,3',4,4',5,5'-hexabromobiphenyl (345-HBB) or 10 p.p.m. 245-HBB plus 0.1 p.p.m. 345-HBB for 140 days followed by a basal diet for up to another 310 days. Rats from each group were killed 170, 240 or 480 days after partial hepatectomy. Dietary exposure to 245-HBB and 245-HBB plus 345-HBB enhanced the development of AHF and HN whereas 345-HBB alone did not. The combination of 245-HBB and 345-HBB caused a synergistic effect on the development of AHF and HN. The number of hepatocellular carcinomas was low and evenly distributed among the groups of rats fed diets containing PBB.