Modulatory effects of metanil yellow on immunity in rodents.

Pathomorphological and immunological studies were carried out on rodents following oral administration of 0, 0.1, 0.25 and 0.5% (w/w) metanil yellow, mixed in diet, for 30 days. No significant change in hematologic parameters and histologic architecture of liver, kidney, mesenteric lymph node, thymus and urinary bladder was observed except for mild desquamation of intestinal villi and moderate changes in Peyer's patches of small intestine with higher doses. Among immunological parameters, significant enhancement in the primary humoral immune response (anti-SRBC IgM plaque forming cells of spleen) was observed with the lowest dose of metanil yellow while higher doses produced opposing effects. An elevated cutaneous delayed type hypersensitivity (DTH) reaction to SRBC was seen in 0.1% metanil yellow treated animals but higher doses did not influence the reaction. The treatment also caused changes in functional capabilities of macrophages. Although these immune alterations could hardly influence the local immunity of gut, as measured by the capacity of animals to cause rejection of Nippostrongylus brasiliensis parasite, the potential to modulate the immunity in general by metanil yellow however assumes considerable biological significance.     

Effect of DNA synthesis on induction of preneoplastic and neoplastic lesions in rat liver by a single dose of methylazoxymethanol acetate

A single intravenous injection of methylazoxymethanol (MAM) acetate in doses of either 20 or 35 mg/kg body weight to male Sprague-Dawley rats induced altered liver cell foci and later, liver neoplasms in a dose related manner. Sequential observations in the rats given 35 mg/kg and thereafter fed an iron-loading diet revealed that the number of iron-excluding foci/cm2 increased with time. Partial hepatectomy (PH) before the high dose of MAM acetate resulted in 100% lethality while hepatectomy before the low dose carcinogen exposure lead to a higher incidence of neoplasms than in rats that received carcinogen alone. PH after either high or low dose carcinogen exposure did not result in a greater occurrence of liver neoplasms.     

Effect of rosuvastatin on cholestasis‐induced hepatic injury in rat livers

Recent studies reported that 3‐hydroxy‐3‐methyl‐glutaryl coenzyme A (HMG‐CoA) reductase inhibitors have pleotropic effects independent of their lipid‐lowering properties. The present study was undertaken to determine whether treatment with rosuvastatin (RO) would be beneficial in a rat model of bile duct ligation (BDL). Animals were divided into three groups: a sham group (group I), a BDL group treated with vehicle (group II), and a BDL group treated with RO (10 mg/kg) (group III). Serum levels of total bilirubin, γ‐glutamyl transpeptidase, alanine aminotransferase, and aspartate aminotransferase decreased significantly in group III when compared to group II. Lipid peroxides and NO levels of group III were found to be significantly lower than those of group II. Antioxidant enzymes (superoxide dismutase, glutathione‐S‐transferase, and catalase) activity in liver tissues markedly decreased in group II, whereas treatment with RO preserved antioxidant enzyme activity. DT‐diaphorase activity in group II was significantly higher than that in group III. The histopathological results showed multiple numbers of newly formed bile ductules with inflammatory cells infiltration in group II. These pathological changes were improved in group III. Our data indicate that RO ameliorates hepatic injury, inflammation, lipid peroxidation and increases antioxidant enzymes activity in rats subjected to BDL. RO may have a beneficial effect on treatment of cholestatic liver diseases. © 2010 Wiley Periodicals, Inc. J Biochem Mol Toxicol 24:89–94, 2010; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20315     

The distribution of acid and alkaline ribonuclease activities in preneoplastic and neoplastic rat livers.

Ribonuclease (RNase) activities revealed by the substrate film method were compared with reactions for acid and alkaline RNase obtained by lead precipitation technique in serial sections of preneoplastic livers and hepatomas. The preneoplastic parenchymal tissue giving positive reactions with ribonucleic acid films showed both acid and alkaline RNase activities by lead precipitation technique, and the area of hyperplastic nodules nonreactive against substrate films were deficient in acid and alkaline RNase activities. Preneoplastic hyperbasophilic foci and hepatoma gave weak or negative reactions by either method, but necrotic areas and stromal tissue showed appreciable RNase activities. Thus a good correlation was observed in these tissues between the RNase activities revealed by the film method and those demonstrated by lead precipitation.     

DNA hypomethylation in ethionine-induced rat preneoplastic hepatocyte nodules

DNA from hepatocyte nodules induced in rats with dietary DL-ethionine and from the surrounding non-nodular liver contained less 5-methyldeoxycytidine per deoxycytidine when compared with that from normal adult liver. The degree of apparent hypomethylation, 37% in nodules and 20% in the surrounding liver, decreased somewhat (29% and 16% respectively) at 2 weeks after terminating the exposure to ethionine. Nodules and surrounding liver, like normal liver, responded to partial hepatectomy with a decrease in the 5-methyldeoxycytidine level at 24 hrs and a return to the level at the time of partial hepatectomy by 38 hrs. These findings indicate the need for careful control of cell proliferation in comparing the levels of a post-replicative DNA modification, methylation, in proliferating and non-proliferating cell populations. These findings also suggest that a portion of the hypomethylation in preneoplastic nodules may be due to a bona fide decrease in the level of cytosine methylation in the parental strand of DNA. This hypomethylation could be one basis for the altered gene expression in hepatocyte nodules, possible precursors for liver cancer.     

Histochemical comparison of focal losses of RNase and ATPase activities in preneoplastic rat livers.

To better assess the significance of enzyme-deficient foci as putative premalignant lesions, parallel histochemical analyses of RNase and ATPase activities were carried out in serial sections of livers from rats fed 4-dimethylaminoazobenzene. The results showed that focal losses of RNase and canalicular ATPase activities occur simultaneously in congruent areas of liver parenchyma at early stages of carcinogenesis. Such foci presumably represent altered cells capable of progressing to neoplasia since the changes observed in this new cell population persist in developing tumors.     

Metabolic disposition of [14C] metanil yellow in rats

The absorption, metabolism and excretion of [14C] metanil yellow was studied in rats. Following administration of a single oral dose of 5 mg dye (7.6 microCi)/kg body weight, 80.5% of the dose was excreted in the urine and faeces within 96 hr, with the majority being accounted for in the faeces. Liver, kidney, spleen and testis retained no count whereas 13.6% of the radioactivity was retained by gastrointestinal tract. Analysis of urine and faeces detected two azo-reduction metabolites of metanil yellow which were characterized by TLC and IR, NMR and mass spectroscopic studies as metanilic acid and p-aminodiphenylamine.     

Time-dependent onset of Interferon-alpha2b-induced apoptosis in isolated hepatocytes from preneoplastic rat livers

We have already demonstrated that interferon alfa-2b (IFN-alpha2b) induces apoptosis in isolated hepatocytes from preneoplastic rat livers via the secretion of transforming growth factor beta(1) (TGF-beta(1)), and this process is accompanied by caspase-3 activation. The aim of this study was to further investigate the mechanism of this activation. Isolated hepatocytes from preneoplastic livers induced DNA fragmentation in response to IFN-alpha2b, which was completely blocked when anti-TGF-beta(1) was added to the culture media. IFN-alpha2b mediated radical oxygen species (ROS) production that preceded the loss of mitochondrial transmembrane potential (DeltaPsi), release of cytochrome c, and activation of caspase-3. Bax levels increased in a time-dependent fashion, and Bcl-x(L) was down-regulated in the early hours of IFN-alpha2b treatment. The delayed translocation of Bid into the mitochondria was in concordance with late caspase-8 activation. In conclusion, endogenous TGF-beta(1) secreted under IFN-alpha2b stimulus seems to induce cytochrome c release through a mechanism related to Bcl-2 family members and loss of mitochondrial DeltaPsi. Bax protein could be responsible of the release of cytochrome c during the initial hours of IFN-alpha2b-induced apoptosis via TGF-beta(1). Activated Bid by caspases could amplificate the mitochondrial events, enhancing the release of cytochrome c.     

Immunogenic chromatin proteins of rat hepatoma induced by N-nitrosodiethylamine]

Immunoglobulins G against chromatin of N-nitrosodiethylamine-induced hepatoma of rats have been obtained. They help determining in the hepatoma tumour-associated chromatin proteins and proteins common for chromatins of the hepatoma and normal liver. Both tissue-specific and tissue-nonspecific proteins compose immunogenic chromatin proteins of the hepatoma.     

Chemopreventive effect of orange oil on the development of hepatic preneoplastic lesions induced by N-nitrosodiethylamine in rats: an ultrastructural study

Orange peel oil is used extensively as an approved flavour enhancer in fruit drinks, carbonated beverages and as a scenting agent in soaps and cosmetics. Limonene, which is a monocyclic monoterpene is present in orange peel oil from 90 to 95% (w/w). Monoterpenes have been shown to be very effective chemopreventive agents against several rodent tumors and are currently in clinical trials. However, not much information is available regarding the ultrastructural changes associated with the chemopreventive effects of the monoterpenes. The effect of orange oil on the suppression of preneoplastic hepatic lesions during N-nitrosodiethylamine (DEN) induced hepatocarcinogenesis was studied electron microscopically. Rats were administered 200 ppm DEN through drinking water for a period of 1 month. After an interval of 2 weeks, the animals were administered orange oil by gavage for a period of 5 1/2 months. The chemopreventive effect of orange oil was monitored on the basis of liver weight profile, histological pattern by light microscopy and ultrastructural alterations by electronmicroscopy. Orange oil administration following DEN treatment showed decreased liver weights, increased intercellular gap junctional complexes, cell density and polarity when compared with only the DEN treated rats. In the present study chemopreventive effect of orange oil on DEN-induced hepatic preneoplasia in rats which is associated with the restoration of the normal phenotype and upregulation of junctional complexes has been demonstrated.     

Protective role of Vitamin E pre-treatment on N-nitrosodiethylamine induced oxidative stress in rat liver

Nitrosamine compounds are known hepatic carcinogens. In the metabolism of nitrosamines, such as N-nitrosodiethylamine (NDEA), there is evidence of the formation of reactive oxygen species (ROS) resulting in oxidative stress, which may be one of the factors in the etiology of cancer. The formation of ROS may alter the antioxidant system, while the presence of Vitamin E may counteract NDEA induced oxidative stress. This study was planned to determine whether pre-treatment with Vitamin E (40 mg/kg body weight, i.p., twice a week for 4 weeks) to NDEA induced rats provides protection against oxidative stress in liver caused by the carcinogen. A single necrogenic dose of NDEA (200mg/kg body weight) was administered i.p. to the male albino rats with or without Vitamin E pre-treatment and the animals were sacrificed on Days 7, 14 or 21 after the administration of NDEA. The result showed enhanced levels of hepatic lipid peroxidation (LPO) and conjugated dienes of NDEA treated rats as the indices of oxidative stress, however, Vitamin E pre-treated rats administered NDEA showed decreased LPO and conjugated dienes (Day 21). Superoxide dismutase (SOD) activity in liver was not altered significantly in NDEA treated rats with or without Vitamin E pre-treatment. Catalase (CAT) activity was inhibited with NDEA treatment, however, Vitamin E pre-treatment showed recovery in hepatic CAT activity (Days 14 and 21). Total and Se-glutathione peroxidase (GSH-Px) activities and glutathione-S-transferase (GST) activity in liver increased in NDEA treated rats irrespective of Vitamin E pre-treatment. Glutathione reductase (GSH-R) activity as well as total glutathione (GSH) content in liver decreased in NDEA treated animals, both of which were recovered in Vitamin E pre-treated rats administered NDEA. Activities of serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) were increased significantly following NDEA treatment to rats with or without Vitamin E pre-treatment. The activities of AST and ALT enzymes were significantly reduced on Days 14 and 21 and ALP activity was reduced on Day 21 in NDEA+Vitamin E treated animals when compared to NDEA treated alone. LDH enzyme activity was normalized on Day 14 in Vitamin E pre-treated animals administered NDEA. However, the AST, ALT and ALP enzyme activities remained high in all treatment groups as compared to control group. Normal control and Vitamin E treated alone rats revealed normal histology of liver. On the other hand, NDEA treated animals showed alterations in normal hepatic histoarchitecture, which comprised of necrosis and vacuolization of the cells. However, the rats treated with Vitamin E+NDEA showed that the liver cells were normal, with very little necrosis (Day 21). This study concludes that the pre-treatment with Vitamin E prior to the administration of NDEA, reduced the degree of oxidative stress, although this vitamin produced only slight changes in the hepatic injury, in a time-dependent manner.     

Long-term low-dose mutation studies in human cells: metanil yellow and orange II

We tested the mutagenic effects of two commonly used fold colors, metanil yellow and orange II, in AHH-1 human lymphoblast cells. The cell line, which is competent for oxidative metabolism of various chemicals, was exposed to both compounds in high-dose x short-term (3 day) or high-dose x long-term (10-day) and low-dose x long-term (20-day) treatments. Concentrations of metanil yellow and orange II as low as 22 nM and 12 nM, respectively, were sufficient to induce mutation rates which were equal to twice the spontaneous mutation rate at the HPRT locus in AHH-1 cells.     

NaCl induced ornithine decarboxylase and DNA synthesis in rat stomach mucosa

A sensitive method is described that detects an alteration in the structure of tRNA that is caused by cadmium but not by magnesium or zinc ions. The chromatographic system, RPC-5, separates Drosophila tyrosyl-tRNA into two fractions. These two isoacceptors differ by a single position in the anticodon where either a guanosine or queuine resides. Cadmium ions apparently interact with the tRNA and prevent the chromatographic separation. This is the first instance where cadmium is shown to cause a selective change in nucleic acid structure. The RPC-5 system seems to be uniquely useful in detecting such a change.     

Neurotensin enhances estradiol induced DNA synthesis in immature rat uterus

Systemic administration of Neurotensin, a tridecapeptide, in immature rats treated with estradiol benzoate significantly enhances uterine DNA synthesis as reflected by the incorporation of 3H-thymidine. The peptide may have a direct action on the uterus. Substance P, a related peptide, had no effect on uterine DNA synthesis.     

Effect of testosterone on RNA and DNA synthesis in rat perineal muscle

Nucleid acid metabolism in the LAM of the rat, both before and after castration, and during testosterone treatment, was investigated. RNA synthesis was increased by testosterone treatment, to varying degree, in adult and in prepubertal castrated rats, and was not merely dependent on the degree of hypertrophy of the LAM. The DNA content and the incorporation rate of formate-14C into DNA showed a characteristic profile under the same conditions: the atrophy of LAM following castration and its subsequent restoration appeared to be accompanied by variations in nuclei number. The possible role of testosterone in DNA duplication and in cell mitosis is hypothesized here; further investigation must be integrated by careful morphometric observation.     

Effect of colchicine and vincristine on DNA synthesis in regenerating rat liver

The increases in the activities of hepatic thymidylate synthetase and thymidine kinase were significantly suppressed at 24 h after 70% partial hepatectomy in rats which had been administered a microtubule disrupter, colchicine or vincristine. The decrease of these enzymic activities was accompanied by a reduction of DNA content in 24 h regenerating liver. The immunoblotting assay showed that the depression of the thymidylate synthetase activity by the injection of colchicine or vincristine was due to the decrease of the enzyme protein. These results indicate that colchicine and vincristine inhibit the DNA synthesis during liver regeneration by inhibiting the induction of the key enzyme in DNA synthesis.     

Effect of urethan on polyamine and DNA synthesis in the regenerating rat liver

When a single dose of urethan was injected into the peritoneal cavity of rats immediately after partial hepatectomy, DNA synthesis was delayed by 12 h. The induction of ornithine decarboxylase which was induced biphasically following partial hepatectomy was also reduced and delayed by 14–15 h by the administration of urethan. S-Adenosylmethionine decarboxylase activity in urethan-treated rat liver at 20 h and 29 h after operation was significantly lower than that of untreated animals. This enzyme activity was shown to increase thereafter, reaching a higher level than in untreated rats at 37–42 h. Hepatic spermidine content changed biphasically in a manner similar to DNA synthesis. These results suggest that the activities of ornithine decarboxylase and S-adenosylmethionine decarboxylase may correlate with DNA synthesis and that an increase of spermidine concentration is necessary to DNA synthesis.