Protective potentials of a potentized homeopathic drug, Lycopodium-30, in ameliorating azo dye induced hepatocarcinogenesis in mice

The protective potentials of a potentized homeopathic drug, Lycopodium-30, prepared from extract of spores of a plant, Lyocopodium clavatum (Fam: Lycopodiaceae) and used as a remedy for various liver ailments, have been tested in mice chronically fed p-dimethyl amino azo benzene (p-DAB) – an initiator, and phenobarbital (PB) – a promoter of hepatic cancer, by using some cytogenetic endpoints like chromosome aberrations (CA), micronuclei (MN), mitotic index (MI) and sperm head abnormality (SHA), and toxicity biomarkers like acid and alkaline phosphatases (AcP and AlkP, respectively), alanine and aspartate amino transferases (ALT and AST, respectively) and lipid peroxidation (LPO) and reduced glutathione (GSH) activities. The effects of chronic treatment of the carcinogens were assessed at different intervals of fixation, namely, at day 7, 15, 30, 60, 90 and day 120, and compared with that of mice fed conjointly with the carcinogens and the homeopathic remedy. Both the assay systems indicated considerable protective potentials of the homeopathic remedy against p-DAB induced hepatocarcinogenesis in mice.     

Cytogenetical effects of sonication in mice and their modulations by actinomycin D and a homeopathic drug,Arnica 30

Experiments were designed to examine if Actinomycin D, an antibiotic, and Amica 30, a homeopathic drug used against shock and injury, can ameliorate cytogenetic damage induced by single or multiple exposures to ultrasonication. Separate sets of healthy mice were directly exposed to sonication for two minutes either once or they received multiple exposures at an interval of 20 days. The mice were then assessed at different intervals, against suitable controls, using parameters like chromosome aberrations (CA), mitotic index (MI), sperm head anomaly (SHA) and micronucleated erythrocytes (MNE). Separate groups of sonicated mice were either orally administered with Arnica 30 (alcohol 30 in control) or injected intramuscularly with Actinomycin-D (AMD). Elevated frequencies of CA, MI, MNE and SHA were noted in sonicated series. AMD had genotoxic effects of its own and also had additive effects on sonication induced genotoxicity. Sonicated mice fed with Arnica 30 showed appreciably reduced genotoxicity as against alcohol 30 and distilled water fed controls, thereby showing ameliorating effect which may have human application.     

Differential staining of preneoplastic foci in rat liver parenchyma during azo dye carcinogenesis

Summary In preneoplastic liver of rats fed the azo dye 4-dimethylaminoazobenzene, certain cellular populations are characterized by cytologic changes typical of tumor cells and appear as the sites of neoplastic transformation. With a basic dye such as toluidine blue, cytoplasmic RNA in preneoplastic foci and hepatomas stains more intensely than in surrounding tissue. In the present study, it was found that when a basic dye (hematoxylin) was combined with an acid dye (tartrazine), these areas stained differentially from the surrounding liver parenchyma. RNAse hydrolysis has shown that such staining properties might be due to the increased proportion of cytoplasmic RNA to components stainable with tartrazine in hyperbasophilic cells, while the surrounding parenchymal cells are probably distinguished by the opposite ratio.It is suggested that the increased basophilia in preneoplastic areas and hepatomas results from the presence of excess RNA and a corresponding decrease in cellular components stained with tartrazine. However, the present method does not permit us to determine whether hyperbasophilia is due to a normal type or an altered form of RNA present in excess.This work was supported by grants from The Quebec Medical Research Council and The René Hébert Foundation.     

On the promoting action of tamoxifen in a model of hepatocarcinogenesis induced by p-dimethylaminoazobenzene in CF1 mice

BACKGROUND AND AIMS: Tamoxifen (TMX) has proven to be an effective palliative treatment for advanced breast cancer with low reported incidence of side effects. TMX has been demonstrated to be an initiator and/or a promoter in the rat model of hepatocarcinogenesis. To document the long-term effect of TMX in mice treated with p-dimethylaminoazobenzene (DAB), we have investigated the time response action of these drugs on different biochemical parameters. METHODS: A group of animals was placed on dietary DAB (0.5%, w/w) during a period of 28 weeks. Control animals received a standard laboratory diet. Two other groups of non-treated and DAB-treated animals received TMX citrate (0.025%, w/w) in the diet since day 20. RESULTS: The activities of the enzymes involved in heme synthesis and degradation as evaluated in the DAB group was not further affected by TMX. DAB and/or TMX treatment significantly increased the content of total cytochrome P450 and also the activity of glutathione S-transferase indicating liver damage. In all treated groups oxidative stress and an adaptive response of the natural defense system (catalase and superoxide dismutase) were demonstrated. Histological and morphological studies revealed liver cell hyperplasia in DAB treated group; however, only in the DAB+TMX group solid, trabecular and acinar hepatocellular carcinoma was confirmed at the end of the experimental trial. CONCLUSION: We have demonstrated that TMX produced changes in hepatic enzyme activities which may be relevant for the metabolism and disposition of this and/or other drugs. Because liver tumors could be initiated and promoted by several agents which need to be activated, the possible hazard of TMX should be considered. This study reports that long-term treatment with TMX enhances hepatocarcinogenesis induced by DAB. The widespread use of TMX as an anticancer agent adds to the significance of this study.     

Alterations in turnover of labelled precursors incorporated into rat liver nuclear macromolecules during azo dye feeding

Continual feeding of either 4-dimethylaminoazobenzene (DAB) or 2-methyl-4-dimethylaminoazobenzene (2-MeDAB) to rats resulted in an increase in the uptake but a decrease in the turnover of [³H]lysine in all the nuclear proteins of rat liver. The pattern of lysine turnover in acidic nuclear proteins from DAB-fed animals was more similar to that of normal acidic nuclear proteins than that from the 2-MeDAB-fed animals. The histone fractions showed an increase in uptake after dye feeding which was greater in the lysine-rich fractions than in the arginine-rich fractions. During DAB feeding both the uptake and rate of turnover of [³H]thymidine were greatly increased, but with the noncarcinogenic 2-MeDAB the uptake of the precursor was lower and the rate of turnover slower than in normal animals. These differences in metabolism in response to azo dye feeding are discussed in relation to azo dye carcinogenesis.     

Detection of mRNAs present at low concentrations in rat liver by in situ hybridization: application to the study of metabolic regulation and azo dye hepatocarcinogenesis

In situ hybridization on tissue sections was used to detect mRNAs present at low concentrations during metabolic adaptation and azo dye carcinogenesis in rat liver. The method consisted of hybridizing the slices at relatively high stringency with [35S]-labeled single-stranded probes derived from cDNA insert clones into the M13 phage. L-pyruvate kinase mRNA was proved to be present at very low concentrations in hepatocytes of fasted rats and to be relatively abundant in all hepatocytes after 18 hr of refeeding on a carbohydrate-rich diet. Aldolase A mRNA concentrations have been previously shown to increase markedly in liver of 3'-methyl DAB-fed rats, with a maximum at the fourth week. We demonstrate here, using our in situ hybridization technique, that this phenomenon is not due to re-expression of this "fetal marker" in hepatocytes but to its abundancy in proliferating small cells (i.e., so-called oval and transitional cells). Small amounts were also detected in sinusoidal cells. In normal liver, aldolase A mRNAs were detected only in some sinusoidal cells.     

The histochemical demonstration of gamma-glutamyl transpeptidase activity in different populations of rat liver during azo dye carcinogenesis

To better assess the reliability of gamma-glutamyl transpeptidase (gamma-GTase) as a marker of preneoplastic liver lesions and hepatomas, the gamma-GTase activity of different cell populations was examined in liver sections from rats fed 4-dimethylaminoazobenzene. The results indicated that the biliary ductular cells in trabeculae of cirrhotic livers may exhibit appreciable gamma-GTase activity in addition to that shown by islands of regenerating parenchyma. At later stages of azo dye carcinogenesis, the epithelial cells of bile duct cysts and cholangiomas, as well as those of hepatomas, gave positive reactions for gamma-GTase. Thus biochemical data on liver gamma-GTase in different models of hepatocarcinogenesis cannot be translated directly in terms of alterations in a particular cell type unless such interpretation is justified by parallel histochemical investigations.     

Aerobic degradation of the azo dye acid red 151 in a sequencing batch biofilter

The azo dye acid red 151 (AR151) was aerobically biodegraded in a sequencing batch biofilter packed with a porous volcanic rock. AR151 was used as the sole source of carbon and energy for acclimated microorganisms. Acclimation was followed using the degradation time and the oxygen uptake rate. A maximal oxygen uptake rate of 0.5 mg O(2)/(lmin) was obtained. Mineralization studies showed that 73% (as carbon) of the initial azo dye was transformed to CO(2) by the consortia. A maximal substrate degradation rate of 247 mg AR151/(l(reactor)d) was obtained. Color removal was up to 99% using an initial concentration of 50 mg AR151/l. Anaerobic tests suggested that in the interior of the porous material, anaerobic biotransformations can occur, contributing from 14% to 16% of the decoloration of the azo dye.     

Plasmodium yoelii: characterization of a protective idiotype during malarial infection in mice

We have previously identified and characterized a monoclonal antibody (McAb 302) with potent passive protective activity in mice infected with Plasmodium yoelii, a murine malarial parasite which depends on antibodies for resolution. To further study the appearance and regulation of this antibody during infection, we prepared syngeneic monoclonal antibodies specific for idiotopes present on McAb 302. Three hybridomas were established which synthesized antibodies that bound only to the homologous idiotype but which did not recognize isotypic specificities. All three of these antibodies were found to recognize distinct 302 idiotopes and two of these were shown to be specific for determinants associated with the antibody combining site of McAb 302. One of these monoclonal anti-idiotypic antibodies was used to develop an enzyme-linked immunosorbent assay for the 302 idiotype. When serum samples taken at different times from mice undergoing a primary infection with P. yoelii were tested in this assay, the 302 idiotype could not be detected even though the host was mounting a significant humoral response to the 230-kDa antigen recognized by McAb 302. These studies suggest that the idiotype of the protective McAb 302 is not a predominant one involved in the resolution of a P. yoelii infection and that only some idiotypes of antibodies directed to relevant plasmodial antigens possess significant biological activity. Therefore, protective immunization with plasmodial antigens may require the elicitation of selected idiotypes. Attempts to alter the course of P. yoelii infection by preimmunization with monoclonal or polyclonal anti-idiotypic reagents were unsuccessful.     

Assessment of protective role of polyherbal preparation, Livina, against anti-tubercular drug induced liver dysfunction

The present study evaluated the possible protective role of Livina (a polyherbal preparation) against anti-tubercular therapy (ATT)-induced liver dysfunction in patients of pulmonary tuberculosis. Patients were given intensive phase treatment with 4-drugs (rifampicin, INH, pyrazinamide and ethambutol) used for anti-tubercular therapy for 2 months, followed by a 4-month continuous phase treatment with 2 drugs (rifampicin and INH) under clinical advice and supervision. Both qualitative and quantitative measures of liver function were assessed, at different time intervals, before and after ATT. Analysis of data showed that the incidence of qualitative manifestations of liver dysfunction were greater in the placebo treated group as compared to the test drug group. None of the patients of either group showed clinical jaundice. Most signific changes ant were observed in the SGOT and SGPT levels in the placebo group, wherein the levels of both enzymes were higher at 4 and 8 weeks post-ATT, as compared to the respective baseline (0 week) values. When Livina (2 capsules twice daily) was given with ATT drugs, incidence of qualitative manifestation of liver dysfunction was insignificant and SGOT and SGPT levels were also significantly lower than the placebo+AITT drugs treated group. These results indicate that the test drug (Livina) was efficacious, against ATT-induced hepatic dysfunction in patients of pulmonary tuberculosis.     

Lack of a relationship between immune function and chemically induced hepatocarcinogenesis in B6C3F1 mice

Summary The relationship between immune function and chemically induced hepatocarcinogenesis was studied employing an in vivo murine model. Neonatal B6C3F₁ mice were given a single carcinogenic dose of diethylnitrosamine (DEN) and the time-response kinetics for the early (foci of alteration) and late (adenomas/carcinomas) phases of hepatocellular carcinogenesis were compared to changes in hematopoiesis and immune functions associated with immune surveillance and natural resistance. Increases in hematopoiesis occurred just prior to or concurrent with the appearance of hepatocellular carcinomas, while increased macrophage and natural killer cell cytotoxicity and suppression of cell-mediated immunity occurred following tumor appearance and progressed with increasing tumor burden. Neither immunological nor hematopoietic changes were associated with early phases of hepatocarcinogenesis, as monitored by the appearance of altered hepatocellular foci. Although changes in hematopoiesis may represent an early indicator for hepatocarcinogenesis in the mouse tumor model, the data suggest that altered immune surveillance and natural resistance are not factors in the development of chemically induced hepatocellular tumors, and the changes in immune function are probably secondary to tumor development.     

Evaluation of the anti-inflammatory drug indomethacin, for its genotoxicity in mice

Indomethacin, a non-steroidal anti-inflammatory drug was assessed for its genotoxicity in Swiss male mice employing bone marrow micronucleus induction, abnormal sperm formation and the induction of meiotic chromosome anomalies in spermatocytes as the test parameters. The results showed that the test compound was toxic to the genetic material in the dose range of 12-36 mg/kg body weight.     

Effect of redox mediator, AQDS, on the decolourisation of a reactive azo dye containing triazine group in a thermophilic anaerobic EGSB reactor

The feasibility of thermophilic (55 °C) anaerobic treatment applied to colour removal of a triazine contained reactive azo dye was investigated in two 0.53 l expanded granular sludge blanket (EGSB) reactors in parallel at a hydraulic retention time (HRT) of 10 h. Generally, this group of azo dyes shows the lowest decolourisation rates during mesophilic anaerobic treatment. The impact of the redox mediator addition on colour removal rates was also evaluated. Reactive Red 2 (RR2) and anthraquinone-2,6-disulfonate (AQDS) were selected as model compounds for azo dye and redox mediator, respectively. The reactors achieved excellent colour removal efficiencies with a high stability, even when high loading rates of RR2 were applied (2.7 g RR2 l⁻¹ per day). Although AQDS addition at catalytic concentrations improved the decolourisation rates, the impact of AQDS on colour removal was less apparent than expected. Results show that the AQDS-free reactor R2 achieved excellent colour removal rates with efficiencies around 91%, compared with the efficiencies around 95% for the AQDS-supplied reactor R1. Batch experiments confirmed that the decolourisation rates were co-substrate dependent, in which the volatile fatty acids (VFA) mixture was the least efficient co-substrate. The highest decolourisation rate was achieved in the presence of either hydrogen or formate, although the presence of glucose had a significant impact on the colour removal rates.     

Evidence for centrophenoxine as a protective drug in aluminium induced behavioral and biochemical alteration in rat brain

The aim of the present work was to study the effects of an unilateral ischaemic-reperfusion injury on Na⁺, K⁺-ATPase activity, α₁ and β₁ subunits protein and mRNA abundance and ATP content in cortical and medullary tissues from postischaemic and contralateral kidneys. Right renal artery was clamped for 40 min followed by 24 and 48 h of reperfusion. Postischaemic and contralateral renal function was studied cannulating the ureter of each kidney. Postischaemic kidneys after 24 (IR24) and 48 (IR48) hours of reperfusion presented a significant dysfunction. Na⁺, K⁺-ATPase α₁ subunit abundance increased in IR24 and IR48 cortical tissue and β₁ subunit decreased in IR48. In IR24 medullary tissue, α₁ abundance increased and returned to control values in IR48 while β₁ abundance was decreased in both periods. Forty minutes of ischaemia without reperfusion (I40) promoted an increment in α₁ mRNA in cortex and medulla that normalised after 24 h of reperfusion. β₁ mRNA was decreased in IR24 medullas. No changes were observed in contralateral kidneys. This work provides evidences that after an ischaemic insult α₁ and β₁ protein subunit abundance and mRNA levels are independently regulated. After ischaemic-reperfusion injury, cortical and medullary tissue showed a different pattern of response. Although ATP and Na⁺, K⁺-ATPase activity returned to control values, postischemic kidney showed an abnormal function after 48 h of reflow.