Body Weight Of Diethylnitrosamine Research Articles

Effect of Selected Fruit Wastes on Hematological Parameters in DEN-Induced Hepatic Carcinoma in Rats

Fruits and processed food demand has greatly increased due to which a huge amount of fruit waste is generated. In this study, the effect of the ethanolic extract of waste material Punica granatum (pomegranate) peel and Vitis vinifera (grapes) seeds was evaluated on hematological parameters in Wistar rats with diethylnitrosamine (DEN) induced hepatocellular carcinoma. The treatments were divided into four groups: Group 1 was untreated while Groups 2-4 received 200 mg/kg body weight of DEN by single intraperitoneal administration. Groups 3 and 4 received DEN and co-treated with 400 mg/kg pomegranate peel extract and 400 mg/kg grape seed extract, respectively. There was a significant decrease in the body weight of animals in Group 2, while Group 3 and 4 animals were found to have a significant rise in body weight. The weight of the liver was significantly increased in the cancer-bearing group and its size was significantly reverted in the treated groups. The kidney and spleen showed a significant decrease in size of cancer-induced groups, and these organs significantly increased in treated groups. Hemoglobin, red blood cells (RBC), neutrophil, packed cell volume (PCV), Mean corpuscular volume (MCV) and mean corpuscular haemoglobin (MCH) were significantly reduced in Group 2 animals compared to Group 1. The levels of white blood cells (WBC) and erythrocyte sedimentation rate were increased significantly in Group 2 animals relating to Group 1. Both in Group 3 and 4 animals, these changes were reversed. Results reveal that selected fruit wastes alleviate vital hematological parameters in hepatocellular carcinoma-induced rats.

Knockout of Putative Tumor Suppressor Aldh1l1 in Mice Reprograms Metabolism to Accelerate Growth of Tumors in a Diethylnitrosamine (DEN) Model of Liver Carcinogenesis.

Simple SummaryCancers often loose the enzyme of folate metabolism ALDH1L1. We proposed that such loss is advantageous for the malignant tumor growth and tested this hypothesis in mice proficient or deficient (gene knockout) in ALDH1L1 expression. Liver cancer in both groups was induced by injection of chemical carcinogen diethylnitrosamine. While the number of tumors observed in ALDH1L1 proficient and deficient mice was similar, tumors grew faster and to a larger size in the knockout mice. We conclude that the ALDH1L1 loss promotes liver tumor growth without affecting tumor initiation or multiplicity. Accelerated growth of tumors lacking the enzyme was linked to several metabolic pathways, which are beneficial for rapid proliferation.Cytosolic 10-formyltetrahydrofolate dehydrogenase (ALDH1L1) is commonly downregulated in human cancers through promoter methylation. We proposed that ALDH1L1 loss promotes malignant tumor growth. Here, we investigated the effect of the Aldh1l1 mouse knockout (Aldh1l1−/−) on hepatocellular carcinoma using a chemical carcinogenesis model. Fifteen-day-old male Aldh1l1 knockout mice and their wild-type littermate controls (Aldh1l1+/+) were injected intraperitoneally with 20 μg/g body weight of DEN (diethylnitrosamine). Mice were sacrificed 10, 20, 28, and 36 weeks post-DEN injection, and livers were examined for tumor multiplicity and size. We observed that while tumor multiplicity did not differ between Aldh1l1−/− and Aldh1l1+/+ animals, larger tumors grew in Aldh1l1−/− compared to Aldh1l1+/+ mice at 28 and 36 weeks. Profound differences between Aldh1l1−/− and Aldh1l1+/+ mice in the expression of inflammation-related genes were seen at 10 and 20 weeks. Of note, large tumors from wild-type mice showed a strong decrease of ALDH1L1 protein at 36 weeks. Metabolomic analysis of liver tissues at 20 weeks showed stronger differences in Aldh1l1+/+ versus Aldh1l1−/− metabotypes than at 10 weeks, which underscores metabolic pathways that respond to DEN in an ALDH1L1-dependent manner. Our study indicates that Aldh1l1 knockout promoted liver tumor growth without affecting tumor initiation or multiplicity.

THERAPEUTIC EFFECT OF SILVER NANOPARTICLES AGAINST DIETHYL NITROSAMIN AND CARBON TETTRACHLORIDE-INDUCED HEPATOCELLULAR CARCINOMA IN RATS

Objective: Hepatic cancer is known as primary liver cancer and hepatocellular carcinoma (HCC). Newly silver nanoparticles gained importance due to its advantages and multiple potential such as molecular imaging agent, antimicrobial, wound healing, anti-inflammatory and anticancer activity. The current study deals to assess therapeutic property silver nanoparticles (AgNPs) against diethylnitrosamine (DENA), and carbon tetrachloride (CCL4) induced hepatic cancer.
 Methods: Thirty male albino rats (200-250g) were distributed into four groups and hepatic cancer was induced with a single intraperitoneal dose of 200 mg/kg body weight of DENA. Two weeks later, animals received subcutaneous injections of CCl4 once a week in a dose of 3 ml/kg body weight for 6weeks. Serum biomarkers, antioxidants enzymes, inflammatory markers were evaluated to find the anti-proliferative potential of silver nanoparticles. Histological evaluation and microscopic reports were also done to document the results of the current work.
 Results: AgNPs significantly recover the serum marker enzymes of hepatic parameter AST, ALT, ALP, and total bilirubin and also decreased the levels of NO, IL-6 and TNF-α. Histopathological features also exhibited recovery of a hepatic architecture in cancer-induced rats. Moreover, the immunohistochemical investigation demonstrated that the levels of PCNA, and Caspase-3, which are hepatocarcinogenic markers, were significantly improved by AgNPs.
 Conclusion: These results concluded that AgNPs showed promising curing effects on hepatocellular ailments.

A129: Analysis of Igf axis in development of HCC

IGF signaling pathway plays an important role in the regulation of cell growth, proliferation, differentiation, apoptosis and survival. Deregulation of this pathway has been frequently identified in the development of hepatocellular carcinoma (HCC). IGF signaling pathway consists of IGF ligands (IGF-I and IGF-II), IGF binding proteins (IGFBP 1-7), and membrane-bound IGF receptors (IGF-1R, IGF-II/M6PR, and IGF-2R). The insulin-like growth factor binding proteins (IGFBPs) have several functions, such as IGF transporting, accumulation of IGF at the specific cell pools, inhibition and activation of ligand–receptor interaction. Furthermore, the Igfbps may act independently of IGF-pathway.Diethylnitrosamine (DEN) was used for induction of hepatic tumorigenesis. The male mice, 2weeks old, were injectedintraperitoneally with 20mg/kg body weight of DEN. The liver tissue samples were collected 4, 6, 8, 12months after injection. We performed a comprehensive histological analysis of all kinds of liver samples: tumors, tumor surrounding tissue and normal tissue. Dysplasia was observed 4months after injection, at hepatocellular adenomas were identified 6–8months after injection. In the experimental group, 100% of mice had multifocal HCC 12months after injection. Evaluation of samples from the control group showed normal liver architecture and histology.Western blot analysis showed the presence the HCC tumor marker alfa-fetoprotein only in tumor samples. High levels of Igf-1R and FoxO3 proteins were observed in tumor tissue in contrast to the surrounding and normal tissues 12months after injection. The expression profiles of Igfbp-1,-2,-3,-4,-5,-6,-7, Igf-1R, FoxO3 were analyzed by real time PCR. The most significant results were observed 8 and 12months after DEN injection. RT-PCR showed the dynamic increase in Igf-1R expression in tumor. Expression pattern of Igfbps has shown a high level of Igf-1,-2,-5,-6,-7 mRNA in surrounding tissue and Igfbp-1,-3,-5 in tumor compared to control. The expression of Igfbp-2,-4,-5,-6,-7 was up-regulated, whereas expression of Igfbp-1,-3 was down-regulated in surrounding tissue. The expression of Igfbp-4 was decreased in tumor samples. The expression levels of Igfbp-3,-4 in surrounding tissue and Igfbp-7 in tumor were the same as in the control. We detected high level of mRNA of FoxO3 in tumor.In conclusion, these data confirm previously existing assumption about paracrine effect of Igfbps on tumor growth and progression. Phosphorylation of FoxO3 protein may play an important role in survival of cancer cells. Our results showed up-regulation of Igfbp-2 and Igfbp-5 in tumor and tumor surrounding tissue. Igfbp-2 and Igfbp-5, well known activators of Igfs, can be pro-tumorigenic factors, which are important for hepatocarcinogenesis. Hence, therapeutic targeting of these proteins may offer options for intervention in human HCC.Project supported by Russian Foundation for Basic Research (Russia) No. (113-04-01459).

Enhancement of preneoplastic lesion yield by Chios Mastic Gum in a rat liver medium-term carcinogenesis bioassay

The mastic ( Pistacia lentiscus var. chia) tree is native throughout the Mediterranean region and has long proved a source of food additives and medical treatments. To investigate the modifying effects of Chios Mastic Gum on rat liver carcinogenesis, 6-week-old male F344 rats were subjected to the established rat liver medium-term carcinogenesis bioassay (Ito-test). At the commencement, rats (groups 1–4) were intraperitoneally injected with 200 mg/kg body weight of diethylnitrosamine (DEN). After two weeks, mastic was added to CRF (Charles River Formula)-1 powdered basal diet at doses of 0, 0.01, 0.1 and 1% in groups 1–4, respectively. At week 3, all rats were underwent two-thirds partial hepatectomy. The experiment was terminated at week 8. As results show, liver weights were significantly increased in a mastic dose-dependent manner among groups 1–4. The numbers (/cm 2) and the areas (mm 2/cm 2) of glutathione S-transferase placental form (GST-P)-positive cell foci (≥ 0.2 mm in diameter) were significantly increased in the DEN-1% group compared to the DEN-alone group, along with the average areas per foci and larger-sized foci (≥ 0.4 mm). 5-Bromo-2′-deoxyuridine (BrdU) + GST-P double-immunohistochemistry showed the highest BrdU-labeling indices within GST-P foci in the DEN-1% group. 8-hydroxydeoxyguanosine (8-OHdG) levels in liver DNA did not vary, while real-time quantitative polymerase chain reaction (PCR) analysis of livers revealed many up- or down-regulated genes in the DEN-1% group. In conclusion, this is the first report to display a promotion potential of Chios Mastic Gum on the formation of preneoplastic lesions in the established rat liver medium-term carcinogenesis bioassay.

Enhancement of hepatocarcinogenesis initiated with diethylnitrosamine or N-nitrosobis(2-hydroxypropyl)amine by a choline-deficient, L-amino acid-defined diet administered prior to the carcinogen exposure in rats

Effects of pre-administration of a choline-deficient, L-amino acid-defined (CDAA) diet on hepatocarcinogenesis initiated with diethylnitrosamine (DEN) or N-nitrosobis(2-hydroxypropyl)amine (BHP) in rats were investigated. A pre-administrating period was set as 1 week, because CDAA diet induces liver injuries by this time-point. In a time-course study, male Fischer 344 rats, 6 weeks old, received a 1-week pre-administration of choline-supplemented, L-amino acid-defined (CSAA) or CDAA diet, DEN at a dose of 100 mg/kg body weight by a single intraperitoneal injection, then CSAA or CDAA diet for up to 8 weeks, and were sacrificed 4, 6 and 8 weeks after DEN. CDAA diet administered only after DEN significantly increased the numbers of glutathione S-transferase placental form (GST-P)-positive lesions 4, 6 and 8 weeks after DEN and their sizes 6 and 8 weeks after DEN. CDAA diet administered both before and after DEN similarly increased the numbers and sizes of GST-P-positive lesions, but with a significantly greater degree than obtained by the diet administered only after DEN. In a dose response study, rats received vechicle or DEN, at a dose of 0.001, 0.01, 0.1, 1, 10, 20, 50, 100 or 200 mg/kg body weight, 1 week after the commencement of CSAA or CDAA diet, and sacrificed 8 weeks after vehicle or DEN. The significant increases of the numbers of GST-P-positive lesions were obtained after 50-200 mg/kg body weight of DEN under the CSAA diet administration, whereas those were detected after 10-200 mg/kg under CDAA diet administration. Sizes became significantly larger with only 200 mg/kg body weight of DEN in the CSAA case but with 50-200 mg/kg in the CDAA case. Male Wistar rats received a 1-week pre-administration of CSAA or CDAA diet, vehicle or BHP, at a dose of 600 or 1200 mg/kg body weight, by a single intraperitoneal injection, then CSAA or CDAA diet for 8 weeks, and were then sacrificed. The numbers of GST-P-positive lesions demonstrated significant increment with 1200 mg/kg body weight of BHP by CDAA diet administered only after BHP and, to a significantly greater degree, by the diet administered both before and after BHP. While CDAA diet administered only after BHP did not alter the sizes of GST-P-positive lesions, the diet administered both before and after 600 and 1200 mg/kg body weight of BHP significantly increased the sizes of the lesions. These results indicate that the pre- plus post-administration of CDAA diet enhances hepatocarcinogenesis initiated with DEN or BHP, more than the post-administration only, thus providing a sensitive model to detect weak liver carcinogenic potency of environmental chemicals.

Strong promoting activity of phenylethyl isothiocyanate and benzyl isothiocyanate on urinary bladder carcinogenesis in F344 male rats.

Post-initiation effects of phenylethyl isothiocyanate (PEITC) and benzyl isothiocyanate (BITC) on hepatocarcinogenesis and urinary bladder carcinogenesis were examined in rats pretreated with diethylnitrosamine (DEN) and N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN). Groups of 21 rats received a single intraperitoneal injection of 200 mg/kg body weight of DEN. Starting 2 days thereafter, they were administered 0.05% BBN in the drinking water for 4 weeks. Three days after completion of the carcinogen treatment, they were placed on a diet containing PEITC or BITC at a dose of 0.1%, or a basal diet alone for 32 weeks and then killed for autopsy. Further groups of 6 rats each were similarly treated with PEITC, BITC or basal diet alone for 32 weeks without prior DEN and BBN exposure. In the liver, although the incidences of liver tumors were not significantly affected, the multiplicity of foci larger than 0.5 cm in diameter was slightly increased by PEITC. In the urinary bladder, the incidences of papillary or nodular (PN) hyperplasias and carcinomas were significantly elevated by PEITC or BITC after DEN and BBN initiation. In the groups without initiation, PN hyperplasia was found in all rats of both PEITC and BITC groups, along with papillomas and carcinomas in some animals. Tumors and PN hyperplasias in the groups treated with PEITC and BITC are characterized by downward growth. Our results thus showed PEITC and BITC to be strong promoters of urinary bladder carcinogenesis with some complete carcinogenic potential.

Inhibition by green tea extract of diethylnitrosamine-initiated but not choline-deficient, L-amino acid-defined diet-associated development of putative preneoplastic, glutathione S-transferase placental form-positive lesions in rat liver.

The effects of green tea extract (GTE) on exogenous and endogenous models of rat liver carcinogenesis using diethylnitrosamine (DEN) and a choline–deficient, L–amino acid–defined (CDAA) diet were studied. For the exogenous carcinogenesis study, male Fischer 344 rats, 6 weeks old, were given a single intraperitoneal dose of 200 mg/Kg body weight of DEN, partially hepatectomized at week 3, and administered GTE at doses of 0, 0.01 and 0.1% in the drinking water from week 2 for 10 weeks. For the endogenous carcinogenesis study, rate were fed the CDAA diet and simultaneously given GTE for 12 weeks. All rats were killed at the end of week 12. After DEN–initiation, the apparent numbers of glutathione S–transferase placental form–positive foci, assayed as putative preneoplastic lesions, were decreased by the administration of GTE, though their sizes were not altered. In contrast, GTE did not significantly reduce the numbers of the lesions induced by the CDAA diet or affect their sizes. While the levels of 8–hydroxyguanine, a parameter of oxidative DNA damage, were reduced by the GTE administration in both experimental models, GTE did not protect against the CDAA–diet–associated liver tissue damage in terms of either histology or plasma marker enzyme levels. We conclude that, while GTE may be a possible chemopreventive agent for nitrosamine–initiated hepato–carcinogenesis in the absence of chronic hepatocyte damage, it does not significantly inhibit lesion development in hepatocarcinogenesis associated with the CDAA diet, a cirrhosis–associated model.

Development of altered hepatocyte foci by separate and combined treatments with radiation and diethylnitrosamine in neonatal rats.

To establish an in vivo radiation carcinogenesis model using glutathione S-transferase placental form positive (GST-P+) hepatic foci, newborn rats were irradiated once by 0.5 Gy and 2 Gy of gamma ray or 0.15 Gy and 0.6 Gy of neutron with or without 0.05% phenobarbital (PB). When the rats were sacrificed at the 12th or 21st week, the incidence of GST-P+ foci induction by radiation alone was very low. The neutron was more sensitive than the gamma ray at week 12 and the reverse phenomenon was observed in the groups at week 21. PB combination showed an increased incidence of GST-P+ foci in gamma ray irradiated groups. The neutron irradiation combined with PB did not show any significant difference compared with the corresponding PB untreated groups. We also investigated the combined effect of diethylnitrosamine (DEN) and 0.75 Gy of gamma ray irradiation. Intraperitoneal injection of 0.15 mumol/g body weight of DEN at 1 hour after gamma ray irradiation showed significantly increased the number and area of GST-P+ foci compared with those of DEN alone or DEN at 1 hour before gamma radiation (P < 0.001). From these data, after more defined experiments, an in vivo radiation carcinogenesis model will be established by radiation alone or a combination of radiation and carcinogens.

Immunocytochemical studies of subtypes of pulmonary endocrine cells in diethylnitrosamine-treated rabbits

New Zealand White rabbits were injected subcutaneously with 20 mg/kg body weight of diethylnitrosamine (DEN), twice per week, starting when they were 1 week old. The animals were sacrificed 6 to 12 months after the first injection and lung tissues were processed for light microscopy. Using serotonin (5HT) and neuron specific enolase (NSE) as markers for the endocrine cells, tissue sections were stained immunocytochemically by the avidin-biotin complex method. Numerous neuroepithelial bodies (NEBs) positive for 5HT, but negative for NSE, were seen in the alveolar duct regions of DEN-treated rabbits. On the other hand, an increased number of solitary endocrine cells immunoreactive for NSE was found in bronchial or bronchiolar epithelia. The results indicate that DEN induced increases in two distinct types of endocrine cells: the component cells of NEBs are positive for 5HT and solitary cells are positive for NSE.

SCANNING ELECTRON MICROSCOPIC OBSERVATIONS ON PRENEOPLASTIC AND NEOPLASTIC LESIONS OF THE TRACHEA IN SYRIAN GOLDEN HAMSTERS INDUCED BY DIETHYLNITROSAMINE

Ultrastructural observations by scanning electron microscope (SEM) of pre- or neoplastic tracheal lesions in Syrian golden hamsters induced by diethylnitrosamine (DEN) were investigated. Twenty five, six-week-old, male Syrian golden hamsters were injected subcutaneously twice a week for 12 weeks with 18mg/kg body weight of DEN. Six hamsters were injected only vehicle and served as control. At weeks 2, 4, 8, 12, and 16, 5 hamsters of DEN treated group were sacrificed. At weeks 2, 8, and 12, hamsters of vehicle control group were also sacrificed. Sequential changes of tracheal tumors were investigated by ultrastructural and histopathological observations. At week 2, a focal loss of cilia from surface cells of the trachea of DEN treated hamster were already detected by SEM. At week 4, decilinated changes of the surface became more markedly, and at week 8, various kinds of preneoplastic and neoplastic changes were observed in trachea of DEN treated hamster. Those changes could be detected by microscopy as well as SEM. By week 16, papillary tumors were found in all animals treated with DEN. No changes were observed in trachea of control animals. These results showed that scanning observations can detect the very early lesions of trachea and it is a very useful method especially for detecting the changes of tracheal lesions on hamster tracheal carcinogenesis.

Modulation of diethylnitrosamine-initiated placental glutathione S-transferase positive preneoplastic and neoplastic lesions by clofibrate, a hepatic peroxisome proliferator.

The effect of clofibrate (CF) on proliferation of diethylnitrosamine (DEN)-initiated glutathione S-transferase placental form (GST-P)-positive preneoplastic and neoplastic lesions as studied in male F344 rats. Animals were given a single i.p. injection of 200 mg/kg body weight of DEN, and then from 2 weeks later were given a diet containing 0.3% CF (group 1), or no supplement (group 2) until week 64. Group 3 received an injection of 0.9% NaCl instead of DEN and then a diet containing 0.3% CF, like group 1. Animals in all groups were subjected to partial hepatectomy at week 3 and killed at weeks 8, 20, 32, 49 or 64. The results showed that development of GST-P-positive lesions was significantly less in group 1 than in group 2 from week 8 (P less than 0.05). However, in group 1, morphologically distinguishable GST-P-negative preneoplastic lesions increased from week 20 (P less than 0.05), and the total number of GST-P-positive and -negative lesions was significantly greater than that in group 2 from week 32 (P less than 0.05). The induction of hepatocellular carcinoma (HCC) was greater in group 1 than in group 2 from week 49. All the HCCs induced in group 2 were GST-P-positive, whereas 38.9% (7/18) of those in group 1 were GST-P-negative. In group 3, only a few GST-P-positive and/or -negative preneoplastic lesions developed by week 64. These results suggest that CF has tumor-promoting activity, and that GST-P-positive cells induced by DEN changed to GST-P-negative cells on subsequent treatment with CF.

Stable phenotypic expression of glutathione S-transferase placental type and unstable phenotypic expression of gamma-glutamyltransferase in rat liver preneoplastic and neoplastic lesions.

Using immunohistochemical demonstration of glutathione S-transferase placental type (GST-P) and histochemical demonstration of gamma-glutamyltransferase (gamma-GT), the long-term development of preneoplastic and neoplastic lesions was followed in rats over a 50-week period. Rats were given a single i.p. injection of 200 mg/kg body weight of diethylnitrosamine (DEN), and then 2 weeks later were administered 0.02% 2-acetylaminofluorene (2-AAF) (group 1), 0.05% phenobarbital (PB) (group 2), 2.0% butylated hydroxyanisole (BHA) (group 3) or no supplement (group 4) in their diet for 6 weeks, all rats being subjected to partial hepatectomy at week 3. Hepatocellular proliferated lesions were classified as foci, nodules and hepatocellular carcinomas. Development of foci, nodules and hepatocellular carcinomas was enhanced strongly by 2-AAF and weakly by PB, and inhibited by BHA. Almost all foci and nodules were GST-P positive, although 5-10% of the GST-P-positive foci were gamma-GT negative. The areas of GST-P-positive foci and nodules increased with time in all groups. In contrast, while the areas of gamma-GT-positive lesions also increased with time in groups 2-4, they decreased from week 12 in group 1. As the percentage gamma-GT-positive area in GST-P-positive foci significantly decreased with time in all groups, the rate of phenotypic reversion of gamma-GT in foci in group 1 was revealed to be larger than the focus growing rate, whereas that in groups 2-4 was smaller. Gamma-GT-negative and GST-P-positive micro-nodules of altered morphology appeared within gamma-GT- and GST-P-positive nodules in later stages. All hepatocellular carcinomas found in this experiment consisted of GST-P-positive cells. In contrast, 37% (13/35) of the hepatocellular carcinomas were negative for gamma-GT. The results indicate GST-P to be the most accurate marker enzyme for detection of initiated cells during liver carcinogenesis and gamma-GT to be more appropriate for indicating changes of phenotypic expression in each lesion type.

Relative merits of immunohistochemical demonstrations of placental, A, B and C forms of glutathione S-transferase and histochemical demonstration of gamma-glutamyl transferase as markers of altered foci during liver carcinogenesis in rats.

The values of the immunohistochemical demonstrations of glutathione S-transferases (GSTs) A, B, C and P and histochemical demonstrations of gamma-glutamyl transpeptidase (gamma-GT) for detection of enzyme altered foci in F344 rat liver were compared. Rats were given a single i.p. injection of 200 mg/kg body weight of diethylnitrosamine (DENA), from 2 weeks later they were given 0.02% N-2-fluorenylacetamide (2-FAA), phenobarbital (PB), butylated hydroxyanisole (BHA) or butylated hydroxytoluene (BHT) in their diet for 6 weeks and then they were given basal diet and tap water for 4 weeks. They were subjected to partial hepatectomy at the end of week 3. Results showed that immunohistochemical demonstration of GSTs A, B and C for detection of foci were only effective when the administration of 2-FAA, PB, BHA or BHT in the diet was discontinued, because these GSTs were induced in surrounding hepatocytes by these compounds in the diet. gamma-GT was induced in periportal hepatocytes strongly by BHA and BHT and slightly by PB, and gamma-GT positive foci in periportal areas were not distinguishable from gamma-GT positive periportal hepatocytes. GST-P was also induced moderately by BHA and slightly by BHT in periportal hepatocytes, but all GST-P positive foci were clearly distinguishable. In addition, almost all gamma-GT positive foci gave a positive reaction for GST-P, but 5-10% of the GST-P positive foci were not gamma-GT positive.

Promoting activities of butylated hydroxyanisole and butylated hydroxytoluene on 2-stage urinary bladder carcinogenesis and inhibition of gamma-glutamyl transpeptidase-positive foci development in the liver of rats.

Butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) were evaluated for possible promoting activity for urinary bladder carcinogenesis in male F344 rats initiated by N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN). The rats were treated with 0.01 or 0.05% BBN in the drinking water for 4 weeks and then administered 2% BHA or 1% BHT in the diet for 32 weeks. Surviving rats were killed at the end of week 36 of the experiment. The incidences of cancer and papilloma and the average number of cancers, papillomas and papillary or nodular hyperplasias (PN hyperplasias) per 10 cm of basement membrane were significantly increased in the group receiving BHA following initiation by 0.05% BBN compared with the group given BBN only. BHT also significantly increased these lesions of the bladder, but not the average number of cancers, in rats treated with 0.05% BBN. The ability of four antioxidants, BHA, BHT, sodium L-ascorbate (ascorbate) and ethoxyquin, to promote the induction of gamma-glutamyltranspeptidase (gamma-GT)-positive foci initiated by diethylnitrosamine (DENA) in the liver of F344 rats was tested. Rats were given a single i.p. injection of 200 mg/kg body weight of DENA, and 2 weeks later the animals were exposed to 2% BHA, 1% BHT, 5% ascorbate or 1% ethoxyquin, respectively, in the diet for 6 weeks. All animals were subjected to partial hepatectomy at the end of week 3. The number of gamma-GT-positive foci in the groups fed either BHA, BHT or ethoxyquin after DENA were significantly decreased compared with the control group. These findings show that BHA and BHT are promoters for the urinary bladder carcinogenesis initiated by BBN, but that these and other antioxidants significantly inhibit the induction of gamma-GT-positive foci in the liver.

A sequential quantitative study of the reversibility or irreversibility of liver hyperplastic nodules in rats exposed to hepatocarcinogens.

The behavior of hyperplastic nodules following an in vivo short-term screening test for hepatocarcinogens was studied. Rats were injected ip with 200 mg/kg body weight of diethylnitrosamine (DEN), given basal diet containing 200 ppm of N-2-fluorenylacetamide (2-FAA) (group 1), 1000 ppm of the alpha-isomer of 1,2,3,4,5,6,-hexachlorocyclohexane (alpha-BHC) (group 2) or basal diet (group 3) from week 3 to week 8, and then given basal diet and tap water. They were subjected to partial hepatectomy at the end of week 3. Animals were killed at weeks 4, 6, 8, 10, 20, 30, 40, and 50. A significant disappearance of hyperplastic nodules following the cessation of carcinogen treatment was observed in group 1, but was not evident in groups 2 and 3. With gamma-glutamyltranspeptidase (GGTase) as a positive marker and adenosine triphosphatase (ATPase) as a negative marker, hyperplastic nodules were classified into 3 different phenotypic categories, i.e., (1) GGTase-positive and ATPase-negative, (2) GGTase-positive, and (3) ATPase-negative. The percentages of GGTase-positive and ATPase-negative hyperplastic nodules were almost 80 approximately 90% in group 1 and 70 approximately 80% in groups 2 and 3. Some of the hyperplastic nodules were necrotic from week 8 in groups 1 and 2, and from week 20 in group 3. Subsequently, the numbers of necrotic hyperplastic nodules increased with time. Hepatocellular carcinomas were found at weeks 30, 40, and 50 in group 1, and at weeks 40 and 50 in group 2. Significantly higher incidences of cancer were found in group 1 than in group 2. The hepatocellular carcinomas were also classified enzyme-histochemically into 3 different phenotypic categories as for hyperplastic nodules, but the percentage (20%) of GGTase-positive and ATPase-negative hepatocellular carcinomas was significantly lower than that (70 approximately 90%) of GGTase-positive and ATPase-negative hyperplastic nodules in each group.

Sequential quantitative studies on hyperplastic nodules in the liver of rats treated with carcinogenic chemicals.

Sequential quantitative analyses were made of hyperplastic liver nodules induced by treating male Fischer rats first with diethylnitrosamine (DEN) and then with N-(2-fluorenyl)acetamide (2-FAA), alpha-isomer of 1,2-3,4,5,6-hexachlorocyclohexane (alpha-BHC), or phenobarbital. The test rats were injected ip with 200 mg/kg body weight of DEN, given basal diet containing 2-FAA, alpha-BHC, or phenobarbital from week 2 to 12, and subjected to partial hepatectomy at the end of week 3. From week 6, significantly higher percentage areas of hyperplastic nodules per unit area of liver were found in all experimental groups than in the control group. From week 8, significantly more hyperplastic nodules were found in all experimental groups than in the control group. Continuous administration of alpha-BHC or phenobarbital for 12 weeks with partial hepatectomy did not induce hyperplastic nodules. Continuous administration of 2-FAA did induce hyperplastic nodules, but the percentage areas and number of these nodules were significantly lower than in rats injected with DEN and then given 2-FAA orally, with partial hepatectomy.