Helianthin induces antiproliferative effect on human glioblastoma cells in vitro.

A major focus of brain cancer research today is to translate understanding of glioma biology into advances in treatment, by exploring the potential of target therapy. Here we investigated the ability of three compounds belonging to the chemical class of azo dyes (methyl red, methyl yellow, and helianthin) to inhibit glioblastoma (GB) cell growth in vitro. Our results showed that helianthin induced cytotoxicity in two GB cell cultures, cell lines 18 and 38, whereas methyl red and methyl yellow were not cytotoxic. The effect of helianthin on EGFR, IGF-1R, and their common intracellular signaling via PI3-K and ERK1/2 was also analyzed. Treatment with helianthin down-regulated EGFR and IGF-1R activity in both cell lines. Helianthin treatment blocked ERK1/2 phosphorylation without affecting PI3K activity in cell line 18 and reduced both PI3K and ERK1/2 in GB 38 cell line. The cell death was accompanied by degradation of PARP without affecting BCL2 expression in both GB cell cultures. Because of the genetic heterogeneity of malignant gliomas, we tested the effect of helianthin on other two primary GB lines (11 and 15) and two early-passage GB cultures (BT1GB and BT2GB), to assess the general nature of the anti-tumor effect of the drug in GB cells. We found that helianthin treatment induced cell death in all the GB cell cultures analyzed. To our knowledge, this is the first report indicating that helianthin can reduce GB cell growth.

Cytochemical localization of catalase in leaf microbodies (peroxisomes).

Segments of mature tobacco leaves were fixed in glutaraldehyde, incubated in medium containing 3,3'-diaminobenzidine (DAB) and hydrogen peroxide, and postfixed in osmium tetroxide. Electron microscopic observation of treated tissues revealed pronounced deposition of a highly electron-opaque material in microbodies but not in other organelles. The coarsely granular reaction product is presumably osmium black formed by reaction of oxidized DAB with osmium tetroxide. Reaction of the microbodies with DAB was completely inhibited by 0.02 M 3-amino-1,2,4-triazole and was considerably reduced by 0.01 M potassium cyanide. These results, when considered in light of recent biochemical studies, strongly suggest that catalase is responsible for the reaction. Sharp localization of this enzyme in microbodies establishes that they are identical to the catalase-rich "peroxisomes" recently isolated from leaf cell homogenates. A browning reaction that occurred in leaves during the incubation step was inhibited by cyanide but not by aminotriazole and therefore could not have been caused by the same enzyme. This reaction and a slight deposition of dense material within primary and secondary walls are ascribed to oxidation of DAB by soluble and wall-localized peroxidases.

Bicarbonate dependence of glutamate receptor activation by beta-N-methylamino-L-alanine: channel recording and study with related compounds.

beta-N-methylamino-L-alanine (BMAA) is a neurotoxic glutamate agonist possibly responsible for the neuronal degeneration found in the Guam amyotrophic lateral sclerosis-Parkinsonism-dementia complex. The basis for glutamate receptor activation by BMAA has been unclear, as BMAA lacks the omega electronegative moiety characteristic of other excitatory amino acids. We recently reported that the neuroexcitatory and neurotoxic effects of BMAA depend strongly on the presence of bicarbonate ions and proposed that an interaction between bicarbonate and the beta amino group of BMAA produces a molecular configuration appropriate for activating glutamate receptors. We now report that bicarbonate potentiates the ability of BMAA to open NMDA receptor-activated channels in isolated membrane patches. Furthermore, the neurotoxic and neuroexcitatory effects of two structural analogs of BMAA, DL-2,4-diaminobutyrate and DL-2,3-diaminopropionate, were also potentiated by bicarbonate. These findings support the bicarbonate cofactor hypothesis for BMAA action and provide direct evidence that it may be generalizable to certain other compounds.

Aminoazo dye-protein-adduct enhances inhibitory effect on digestibility and damages to Gastro-Duodenal-Hepatic axis.

4-Dimethylaminoazobenzene (DAB, methyl yellow, or butter yellow), a human carcinogen, has been banned for use in foods since 1988. In 2014, DAB adulteration in Tofu occurred in Taiwan. We hypothesize that DAB can form [DAB•SBP]adduct adduct with soybean protein (SBP) which could damage Gastro-Duodenal-Hepatic axis. Sprague-Dawley rats gavage fed [DAB•SBP]adduct adduct revealed severely reduced body weight and damaged duodenum, liver, hepatic mitochondria, and spleen. Hepatic levels of glutathione and ATP were severely reduced. Serum GOT and GPT were substantially elevated. Analysis by the adsorption isotherm clearly revealed DAB formed very stable [DAB•SBP]adduct adduct at 1:1 molar ration (Phase A). The equilibrium constant of this colloidal adduct [DAB•SBP]adduct was KeqA = ∝, behaving as the most stable and toxic species. At higher protein concentration (Phase C) it formed conjugate [DAB×SBPgross]conjugate, with KeqC = 3.23×10-2 mg/mL, implicating a moderately strong adsorption. The in vitro pepsin digestibility test showed apparently reduced digestibility by 27% (by Ninhydrin assay) or 8% (by Bradford assay). Conclusively, this is the first report indicating that [DAB•SBP]adduct potentially is capable to damage the Gastro-Duodenal-Hepatic axis.

Influence of complexation with cyclodextrins on photo-induced free radical production by the common sunscreen agents octyl-dimethylaminobenzoate and octyl-methoxycinnamate.

The influence of complexation with hydroxypropyl-beta-cyclodextrin (HP-beta-CD) or beta-cyclodextrin (beta-CD) on the photo-induced production of free radicals by the sunscreen agents octyl-dimethylaminobenzoate (ODAB), oxybenzone (OB) and octyl-methoxycinnamate (OMC) was investigated. The formation of radical species during irradiation was detected by spin-trapping electron paramagnetic resonance (EPR) spectroscopy. 2,2,6,6-tetramethylpiperidine-1-oxyl, nitroxide radical (TEMPO) and 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) were used a spin-traps. Following the 4-h illumination with simulated sunlight, OB did not generate radicals. On the other hand, photoexcitation of solutions containing ODAB or OMC produced a marked decrease (>40%) of the TEMPO signal intensity, demonstrating the formation of carbon-centred radicals. In addition, the results obtained on irradiation of ODAB solutions containing DMPO as spin-trap indicated the generation of oxygen-centred radicals. Complexation of ODAB with HP-beta-CD and OMC with beta-CD markedly inhibited (>64%) the formation of free radicals generated by the sunscreens on exposure to simulated sunlight. Therefore, inclusion of ODAB and OMC into the cyclodextrin cavities minimizes their photosensitising potential.

A comparison of the effects of 3'-methyl-4-dimethylaminoazobenzene, 2-methyl-4-dimethylaminoazobenzene, and 2-acetylaminofluorene on rat liver DNA stability and new synthesis.

The objective of the present study was to define early biochemical changes occuring in livers of rats that were fed various chemical carcinogens. Rats were subjected to partial hepatectomy and subsequently given multiple injections of radioactive thymidine to prelabel DNA in their liver. Following a 4-week recovery period the rats were placed on either basal diets or diets containing either 0.05% 3'-methyl-4-dimethylaminoazobenzene (3'-MeDAB), 0.028% 2-acetylaminofluorene, or 0.05% 2-methyl-4-dimethylaminoazobenzene for various periods. After 5 weeks 3'-MeDAB had caused a dose-dependent loss of prelabeled DNA demonstrating the cytotoxicity of this carcinogen. The comparatively noncarcinogenic 2-methyl-4-dimethylaminoazobenzene caused only a small loss of prelabeled DNA. In contrast, the hepatocarcinogen 2-acetylaminofluorene did not cause a loss of prelabeled DNA, demonstrating its low cytotoxicity. Autoradiography and histology revealed that the loss of prelabeled DNA in livers of rats fed 3'-MeDAB was largely due to parenchymal cell death. Experiments designed to separate liver regenerative hyperplasia from neoplastic hyperplasia revealed the presence of both an early and a delayed elevation of thymidine incorporation into liver DNA in rats fed 0.05% 3'-MeDAB. An "early" elevation of incorporation occurred during and shortly after 3'-MeDAB feeding, and a "delayed" elevation of incorporation occurred some weeks after the dye was discontinued. Autoradiography revealed that parenchymal cells were largely responsible for the increased incorporation. Feeding of 2-methyl-4-dimethylaminoazobenzene depressed thymidine incorproation. A direct comparison of the effects of isomolar levels of 3'-MeDAB and 2-acetylaminogluorene on hepatic hyperplasia indicated that both carcinogens caused comparable increases in thymidine incorporation, which returned to control levels upon feeding of carcinogen-free diet. The differences and similarities between the responses to the three compounds are discussed and considered with regard to initiation and promotion of hepatoma formation.

A study of alpha1-fetoprotein levels during exposure to 3'-methyl-4-dimethylaminoazobenzene and its analogs.

The serum concentration of alpha1-fetoprotein (alpha1F) was determined in rats following exposure to the hepatocarcinogen, 3'-methyl-4-dimethylaminoazobenzene, and its analogs. Small quantities of the carcinogen caused a rapid and significant elevation of alpha1F. Neither 2-methyl-4-dimethylaminoazobenzene nor p-aminoazobenzene resulted in any elevation of alpha1F. Further, under circumstances wherein 2-methyl-4-dimeth-laminoazobenzene is reported to become carcinogenic, i.e., when administered at the time of 70 percent hepatectomy, neither elevation of alpha1F nor histological alteration of the liver was noted. The increase in alpha1F after 3'-methyl-4-dimethylaminoazobenzene exposure is the result of a highly selective interaction. The possible contribution of hepatocyte mitosis to the elecation of alpha1F seen during chemical carcinogenesis is emphasized.

Analysis of lipophilic carcinogen-membrane interactions using a model human erythrocyte membrane system.

Human erythrocytes have been used as a model for evaluating the chemical carcinogen-plasma membrane interaction. The carcinogenic aromatic amines 2-acetylaminofluorene, dimethylaminoazobenzene, and 3'-methyldimethylaminoazobene stabilize erythrocytes against lysis in hypotonic solution. In general, the stabilization potential of these compounds reflects their oil:water partition coefficients and may be related to both their extracellular distribution and ultimate capacity for penetration of target cells. The polycyclic aromatic hydrocarbons, 3-methylcholanthrene and benz[a]anthracene, confer little protection against hemolysis and simultaneous incubation of nonprotective 3-methylcholanthrene and protective 3'-methyldimethylaminoazobenzene slightly alters the stabilization afforded by the latter. 7,12-dimethylbenz[a]anthracene exhibits greater protective capacity than does benz[a]anthracene. Polycyclic aromatic hydrocarbons manifested considerably higher degrees of absolute binding to erythrocytes in isotonic solution than did aromatic amines. The difference in erythrocyte binding and stabilization exhibited by the 2 classes of carcinogens suggest distinct mechanisms of membrane association that may relate to their metabolic disposition.

Characteristics and significance of albumin-positive hepatocytes in analbuminemic rats.

Analbuminemic rats (NAR) are a mutant strain in which splicing of the albumin mRNA is blocked due to a seven-base-pair deletion in an intron of the albumin gene. NAR liver contains a few hepatocytes that react with anti-rat albumin antibody (Alb+ hepatocytes), and these cells increase in number during aging and on treatment with hepatocarcinogens. To characterize these Alb+ hepatocytes, we examine their albumin mRNA, the biochemical specificity of their albumin, and its intracellular distribution. Signals of albumin mRNA were observed in a few hepatocytes by in situ hybridization. Moreover, a small amount of cytoplasmic albumin mRNA was detected by RNA blot analysis in the liver of aged NAR and NAR treated with 3'-methyl-4-diaminoazobenzene (DAB). Immunoelectron microscopic examination revealed the cisternae of the rough and smooth endoplasmic reticula, Golgi complexes, and secretory vesicles of the Alb+ hepatocyte of NAR being filled with material that reacted with anti-rat albumin antibody. These facts suggested that albumin was gradually synthesized in Alb+ hepatocytes but that its secretion was disturbed. The albumin-like proteins of NAR were shown by Western blot analysis to consist of three species of 68 kDa, 50 kDa, and 25 kDa proteins. The 50 kDa albumin was thought to be formed by exon-skipping splicing of the albumin mRNA precursor, which was recently reported by Shalaby and Shafritz (Proc. Natl. Acad. Sci. USA 87, 2652-2656 (1990)). The 25 kDa protein was suspected to be formed by fragmentation of the 50 kDa protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation and partial purification of mitochondrial and cytosolic rhodanese from liver of normal and p-dimethylaminoazobenzene treated mice.

Rhodanese (thiosulfate:cyanide sulfurtransferase, E.C. 2.8.1.1), an enzyme involved in heme regulation, showed distinctive mitochondrial and cytoplasmic activities in several models of tumorigenesis. To investigate the basis for these differences, the enzyme was partly purified and characterized from the mitochondrial and cytosolic liver fraction of mice treated with the carcinogen p-dimethyl-aminoazobenzene (DAB). A linear relationship between incubation time and specific activity was observed up to about 30 min for cytosolic enzyme and 15 min for mitochondrial enzyme irrespective of whether or not the enzyme was derived from treated or untreated animals. The optimum incubation temperature was 3 degrees C for the enzyme of both fractions in control animals and 30 degrees C for treated animals in both cases. In control and DAB treated animals the cytoplasmic rhodanese exhibited a maximum at a lower pH than for the mitochondrial enzyme. The enzyme showed typical Michaelis-Menten behavior with cyanide inhibition at concentrations higher than 25 mM for controls and 10 mM for treated animals for both fractions and thiosulfate inhibition at concentrations higher than 100 mM in all cases studied. Km values of 190 and 65.66 mM were obtained for thiosulfate and 6.37 and 9.79 mM for cyanide for both mitochondrial and cytosolic fractions of control animals; while Km values of 31.75 and 4.58 mM were obtained for thiosulfate and 0.61 and 1.11 mM for cyanide in both fractions of treated animals. We demonstrated differences in the kinetics for rhodanese derived from mitochondrial and cytoplasmic fractions of livers taken from tumor bearing mice. These differences might provide an explanation for the abnormalities of heme synthesis previously reported during hepatocarcinogenesis.

Cell cycle arrest and modulation of HO-1 expression induced by acetyl salicylic acid in hepatocarcinogenesis.

BACKGROUND AND AIMS: Control of cell proliferation is important for cancer prevention since cell proliferation has an essential role in carcinogenesis. In rodent carcinogenesis models, antioxidant agents suppress carcinogen-induced cellular hyper proliferation in the target organs. Strict control of cell division is an essential process to ensure that DNA synthesis and mitotic division are accurately and coordinately executed. We studied the interplay between cell cycle and heme oxygenase-1 (HO-1) and the effect of the acetylsalicylic acid (ASA) in hepatic carcinogenesis. METHODS: Male CF1 mice pre-treated with dietary p-dimethylaminoazobenzene (DAB; 0.5%, w/w) were fed with ASA (0.16%, w/w). We investigated the hepatic expression of cyclin D1, cyclin E, Cdk2, Cdk4, p21, p27, p53; the level of bcl-2, an antiapoptotic protein and of heme oxygenase-1 (HO-1), a marker of oxidative stress, by Western blot analysis. RESULTS: The treatment with ASA produced an important attenuation in the induction of cyclin E and cyclin D1 provoked by DAB. p21 and p27 levels were increased when animals received both drugs. The administration of ASA to DAB treated animals induced Cdk2 (29%). HO-1 induction (65%) provoked by DAB was diminished by ASA administration reaching lower induction levels (23%). CONCLUSION: The deregulation of cyclin/CDK expression and the up-regulation of p21 and p27 with the administration of ASA, post-treatment of the carcinogen administration, would block the pass through out to the G0/G1 check point to permit the cells to repair their DNA and HO-1 protected the liver from reactive oxygen species produced from DAB.

Aging changes in the beta-endorphin neuronal system in the preoptic area of the C57BL/6J mouse: ultrastructural analysis.

In hypothalami of aging rodents, beta-endorphin (beta-EP) neuron number and content are reduced. The objectives of this study were: first, to analyze ultrastructurally the population of neuronal elements in a selected region of the preoptic area (POA) in young and old mice; second, to study the beta-EP neuronal system in the same region to determine whether or not this population remains stable with age. Vibratome sections from the most caudal POA through the diagonal band of Broca were examined by light microscopy and immunocytochemistry in mature, cycling (5-6 months old) and old, acyclic, disease-free (24-26 months old) mice. A subset of beta-EP-like perikarya and associated structures was observed in the periventricular POA. When this subregion was examined at the ultrastructural level, there was a significant decrease in the number of recipient dendrites [3.78 +/- 0.04 SEM/micron 2 young vs. 0.82 +/- 0.03/micron 2 old; p < 0.007, analysis of variance (ANOVA)], but a significant increase in the number of nonmyelinated axons (20.0 +/- 2.6/micron 2 young vs. 26.8 +/- 0.7/micron 2 old; p < 0.05). Immunolabeled terminals that contained a synapse comprised 2.56 +/- 0.08% of all terminals with synapses in young mice but only 0.34 +/- 0.04% in old ones when corrected for surface area examined (p < 0.03). A significant age-related loss was also observed in the nonmyelinated beta-EP-labeled axon population (1.50 +/- 0.10% young vs. 0.40 +/- 0.01% old; p < 0.009, ANOVA). We conclude that there are critical changes in the microenvironment of the POA in old, noncycling female mice that are likely to affect neuron function.

Increased synaptic input to gonadotropin-releasing hormone neurons in aged, virgin, male Sprague-Dawley rats.

Using double-label ultrastructural immunocytochemistry, we found the synaptic input to gonadotropin-releasing hormone (GnRH) neurons in the preoptic area of aged (20 months old), virgin, male Sprague-Dawley rats to be denser than that in young adults (3 months old). These results confirmed earlier observations on F-344 virgin male rats. The aging F-344 rat, however, is prone to testicular tumor and so it was essential to see if the phenomenon was reproducible in another rat strain. In the first study, a portion of the increase in synaptic density was due to an increase in the proportion of synapses containing pleiomorphic vesicles, frequently associated with the neurotransmitter GABA. We tested the possibility directly using a double-label protocol for GnRH and glutamic acid decarboxylase (GAD). However, in the present study the density of input by GABA did not change with age. This inhibitory amino acid represented about 10% of the total innervation in young animals; but, in aged animals, because the total synaptic input was greater, GABA represented only about 4% of the innervation. Synaptic vesicles within GAD-immunoreactive terminals were uniformly clear and spherical, suggesting that pleiomorphic vesicle shape is not an appropriate criterion for GABAergic innervation.

The use of the peroxidase reaction to obliterate staining of eosinophils by fluorescein-labelled conjugates.

The staining of eosinophils by fluorescein conjugates can be obliterated by utilising the peroxidase activity of the eosinophil. The brown reaction product of 3,4,3',4'-tetraaminobiphenyl hydrochloride (DAB) with hydrogen peroxide was more effective than Lendrum's stain. A satisfactory alternative to the possibly carcinogenic DAB was 2,7-fluorenediamine.

Detection of hepatoma associated embryonic antigen in tumour-bearer serum.

Embryonic antigen associated with an aminoazo dye-induced rat hepatoma was identified in the serum from rats bearing progressively growing tumours. Antigenic activity in serum samples was detected by their capacity to neutralize multiparous rat serum antibody reacting with surface embryonic antigens expressed upon viable hepatoma cells as assessed with use of the indirect membrane immunofluorescence test. Serum taken at various states of tumour growth from hepatoma-bearing rats was separated by Sephadex G-150 gel filtration column chromatography at pH 7.3 and pH 2.8 with use of procedures designed to identify free circulating antigen and antigen derived from immune complexes. Hepatoma-associated embryonic antigen was demonstrable in tumour-bearer serum in a free form most markedly in the later stages after implantation of tumour cells (from the end of the 2nd week to the 5th week of tumour growth). Antigenic activity in fractions derived from immune complexes was detected earlier during tumour development (from day 8 after tumour induction), and this was present in all serum samples taken up to the 5th week after tumour cell inoculation.

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.

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.

Mutagenicity of carcinogenic azo dyes and their derivatives.

The mutagenicity of N,N-dimethyl-4-aminoazobenzene and N-methyl-4-aminoazobenzene and their derivatives was shown on Salmonella typhimurium TA100 and TA98. S-9 Mix, obtained from rat liver after injection of polychlorinated biphenyl, was abligatory for their mutagenic action. N-Acetoxy-N-methyl-4-aminoazobenzene and N-benzoyloxy-N-methyl-4-aminoazobenzene and their 4'-methoxycarbonyl derivatives were also mutagenic on TA100 and TA98 and did not require metabolic activation by S-9 Mix. It is suggested that the carcinogenic effects of azo dyes may involve modification of DNA.

Polyamine concentration, activity of the ribosomal dissociation factor and proportion of ribosomes active in protein synthesis in the 4-dimethylaminoazobenzene-induced rat hepatoma.

4-Dimethylaminoazobenzene (DAB) induced liver tumour tissue showed a reduced proportion of ribosomes active in protein synthesis compared with control liver. Tumour cell extracts caused an increased association of ribosome subunits into inactive 80 S monomers, compared with the dissociation into active subunits caused by normal liver extracts. These findings may be explained, at least in part, by the increased proportion of spermidine to putrescine found in tumour tissue which would predispose towards association of the subunits.

Analysis of cytological changes in hepatocytes from rats dosed with 3'-methyl-4-dimethylaminoazobenzene: initial response appears to involve cytokinesis of binucleated cells.

The reduction in the ratio of tetraploid (4N + 2 X 2N) to diploid (2N) hepatocytes in the adult rat after treatment with the hepatocarcinogen 3'-methyl-4-dimethylaminoazobenzene (3'M) has been investigated. Analysis of isolated hepatocytes 18-28 days after treatment has confirmed that initially some of the 2 X 2N hepatocytes are converted into 2N cells by cytokinesis, and that there is no DNA synthesis during this process. Shortly afterwards nonpolyploidizing growth commences by proliferation of some 2N cells.