Effects of certain food dyes on chromosomes of Allium cepa

The effects of 4 permitted food dyes, i.e., fast green FCF, indigo carmine, orange G and tartrazine, and the non-permitted dye metanil yellow on chromosomes of Allium cepa are reported. A significant increase in polyploid cells was observed in all cases. High doses of these dyes induced chromosome breaks and micronucleus formation. Although all dyes produced mitotic aberrations, metanil yellow and fast green FCF showed comparatively stronger clastogenic activity.     

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.     

Differential dichrome staining of tissue culture monolayers: alternate dyes and possible mechanism.

A polyacid-dependent dichrome has been devised which will differentiate epithelial from mesenchymal cells in young dividing primary cultures. Epithelial cells and colonies and nuclei are stained with metanil yellow, the stain is fixed and differentiated with phosphotungstic acid, and the mesenchymal elements are stained with toluidine blue. Several other dyes are tested for substitution in this method. Biebrich scarlet and aniline blue could be substituted for the metanil yellow; Bismarck brown T, Janus green B, crystal violet, and neutral red could be substituted for the basic dye.     

Assay for gene mutation in a human lymphoblast line, AHH-1, competent for xenobiotic metabolism

A novel quantitative gene-locus mutation assay has been developed using a line of human lymphoblast cells, designated AHH-1, competent in oxidative xenobiotic metabolism. AHH-1 cells are sensitive to the mutagenic action of both chemically reactive mutagens and mutagens which require oxidative metabolism to exert their mutagenicity. These cells are readily mutated by direct exposure to ethyl methanesulfonate, ICR-191, 2-acetoaminofluorene, aflatoxin B1, benzo[a]pyrene (BP), cyclopenta[c, d]pyrene, dimethylnitrosamine, lasiocarpine, and 1-methylphenanthrene.     

Differential Dichrome Staining of Tissue Culture Monolayers: Alternate Dyes and Possible Mechanism

A polyacid-dependent dichrome has been devised which will differentiate epithelial from mesenchymal cells in young dividing primary cultures. Epithelial cells and colonies and nuclei are stained with metanil yellow, the stain is fixed and differentiated with phosphotungstic acid, and the mesenchymal elements are stained with toluidine blue. Several other dyes are tested for substitution in this method. Biebrich scarlet and aniline blue could be substituted for the metanil yellow; Bismarck brown T, Janus green B, crystal violet, and neutral red could be substituted for the basic dye.     

Decolorization of Fast red by metabolizing cells of <Emphasis Type="Italic">Oenococcus oeni</Emphasis> ML34

Three different azo dyes such as Fast red, metanil yellow and Fast orange were examined for their decolorization by O. oeni ML34. Fast red (FR) was decolorized by 68%, whereas the other dyes were removed by only about 30%. The effects of glucose addition, substrate (dye) concentration and environmental factors (temperature, pH) on decolorization were investigated by two-level factorial design. The statistical analyses revealed that glucose specifically increases the extent of FR decolorization. A glucose level of 5 g/l was the optimum concentration for removal of, FR reaching a decolorization percentage of up to 93%.     

Mutagenicity of 1,2-dichloroethane and 1,2-dibromoethane in two human lymphoblastoid cell lines

1,2-Dichloroethane (DCE) and 1,2-dibromoethane (DBE) were tested for the ability to induce gene mutations in two human lymphoblastoid cell lines, designated AHH-1 and TK6. Both chemicals were 'direct-acting' mutagens in both cell lines. DBE was essentially equally mutagenic in TK6 cells and AHH-1 cells. In contrast, DCE was 25-fold more mutagenic in the AHH-1 cell line than in the TK6 cell line. This differential sensitivity between AHH-1 cells and TK6 cells was related to the levels of glutathione S-transferase activity in these two cell lines.     

P53 integrity in the genetically engineered mammalian cell lines AHH-1 and MCL-5

Recently, a C to T transition mutation in exon 8 of the p53 gene has been identified in a subculture of the genetically engineered human lymphoblastoid cell line AHH-1 and this mutation was proposed to cause a loss of function of the p53 suppressor protein and may limit the use of this cell culture in genotoxicology test assays. This led us to investigate early passage cultures of AHH-1 and its derivative MCL-5 to determine the distribution of the mutation. In order to characterise the presence of mutations at the p53 locus, exon 8 was analysed using restriction enzyme analysis and automated sequencing to locate possible changes of sequence. Mutations were identified at codon 282, and treatment with the Msp1 restriction enzyme led to incomplete digestion suggesting the presence of heterozygosity at the site which was confirmed by sequencing. Our results indicate that the p53 gene is heterozygous at the interface between the codons 281 and 282 in both AHH-1 and MCL-5. An Annexin V labeling study was carried out and both AHH-1 and MCL-5 cell lines were shown to undergo DNA damage induced cell death after a 1-h exposure to MNNG.     

Xenobiotic metabolism and mutation in a human lymphoblastoid cell line

Aryl hydrocarbon hydroxylase-1 (AHH-1) cells are a human lymphoblastoid cell line competent in some aspects of xenobiotic metabolism. This cell line contains stable mixed function oxidase activity which is inducible by polycyclic aromatic hydrocarbons (PAHs) but not by phenobarbital or Arochlor 1254. Two substrates for the cellular mixed function oxidase activity, benzo[a]pyrene (B[a]P) and 7-ethoxyresorufin, have been examined. The basal and induced activities have different kinetic parameters toward these two substrates. In contrast, basal and induced activities had similar sensitivities to two cytochrome P-450 suicide substrates. B[a]P metabolism and mutagenicity were studied in this cell line. AHH-1 cells were found to produce predominantly B[a]P phenols and quinones. The major phenol metabolite cochromatographed with authentic 9-hydroxy B[a]P. AHH-1 cells were capable of forming glucuronic acid conjugates of B[a]P phenols; the major product after hydrolysis cochromatographed with 3-hydroxy B[a]P standard. AHH-1 cells did not contain detectable epoxide hydrolase activity using B[a]P-4,5-oxide as substrate. This observation is consistent with the absence of trans-dihydrodiol B[a]P metabolites in the metabolic profile. B[a]P-induced mutagenicity at the hypoxanthine guanine phosphoribosyl transferase (hgprt) locus in AHH-1 cells was found to be linearly related to phenol production during treatment and inhibited by alpha-naphthoflavone (ANF).     

A novel explant outgrowth culture model for mouse pancreatic acinar cells with long-term maintenance of secretory phenotype

The development of in vitro models able to support the long-term viability and function of acinar cells is critical for exploring pancreatic pathophysiology. Despite considerable efforts, no long-term culture models for non-transformed pancreatic acini exist. Our aim was to develop and validate culture conditions for this purpose. An explant outgrowth culture design was established in which mouse pancreatic explants were cultured at the gas-liquid interphase. An enriched culture medium, pH 7.8, was employed to promote the selective outgrowth of acinar cells and to support their differentiated phenotype. After 7 days, the outgrown primary acinar cells were subcultured and maintained up to an additional 7 days as secondary monolayers on tissue culture plastic. Measurements of basal and caerulein-induced amylase secretion, phase-contrast microscopy and immunohistochemical analyses were used to characterize the cultures. Explants retained their pancreatic cytoarchitecture for 2 days in vitro. A triphasic dose response to caerulein was detected in 7-day primary cultures. The maximal rate of secretion was 1.2-fold versus basal (p=0.009) and 1.7-fold versus 1 pM caerulein (p=0.014). In secondary cultures the response was biphasic with maximal rates of secretion being 1.9-fold in 3- to 4-day cultures at 0.01 nM (p=0.049) and 2-fold in 6- to 7-day cultures at 0.1 nM (p=0.003). The present culture model provides a means to obtain functionally competent normal mouse acinar cells for long-term in vitro experimentation.     

Reactive oxygen species, but not mitochondrial membrane potential, is associated with radiation-induced apoptosis of AHH-1 human lymphoblastoid cells

The aim of the study was to investigate the sensitivity of AHH-1 human lymphoblastoid cells to radiation and its relevance to intracellular events, specifically alteration in cellular energy-producing systems. AHH-1 human lymphoblastoid cells were irradiated with 6 Gy of gamma radiation, and then were collected at the indicated time points. Parallel studies were conducted to assess the effects of radiation on the cell proliferation and apoptotic index. Mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) production were monitored. A marked decrease of cell viability was observed as early as 12 h postirradiation and fraction of apoptotic cells was highest at 24 h. Intracellular ROS generation measured with 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) appeared to be highest as early as 30 min postirradiation and resumed to normal level at 6 h. Unexpectedly, the fluorescence intensity of Rhodamine 123 for measuring MMP did not change during the first 3h after radiation and exhibited an aberrant increase at 6 h. The results suggest that AHH-1 cells are sensitive to radiation-induced apoptosis and ROS generation is an early phase in the apoptosis process. Moreover, the results might cast doubts on those studies using Rhodamine 123 which hypothesized that the fall in MMP is one of the early events of apoptosis.     

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.     

Angiotensin II elevates nitric oxide synthase 3 expression and nitric oxide production via a mitogen-activated protein kinase cascade in ovine fetoplacental artery endothelial cells

Normal pregnancy is associated with high angiotensin II (ANG II) concentrations in the maternal and fetal circulation. These high levels of ANG II may promote production vasodilators such as nitric oxide (NO). ANG II receptors are expressed in ovine fetoplacental artery endothelial (OFPAE) cells and mediate ANG II-stimulated OFPAE cell proliferation. Herein, we tested whether ANG II stimulated NO synthase 3 (NOS3, also known as eNOS) expression and total NO (NO(x)) production via activation of mitogen-activated protein kinase 3/1 (MAPK3/1, also known as ERK1/2) in OFPAE cells. ANG II elevated (P < 0.05) eNOS protein, but not mRNA levels with a maximum effect at 10 nM. ANG II also dose dependently increased (P < 0.05) NO(x) production with a maximal effect at doses of 1-100 nM. Activation of ERK1/2 by ANG II was determined by immunocytochemistry and Western blot analysis. ANG II rapidly induced positive staining for phosphorylated ERK1/2, appearing in cytosol after 1-5 min of ANG II treatment, accumulating in nuclei after 10 min, and disappearing at 15 min. ANG II increased (P < 0.05) phosphorylated ERK1/2 protein levels. Activation of ERK1/2 was confirmed by an immunocomplex kinase assay using ELK1 as a substrate. PD98059 significantly inhibited ANG II-induced ERK1/2 activation, and the ANG II-elevated eNOS protein levels but only partially reduced ANG II-increased NO(x) production. Thus, in OFPAE cells, the ANG II increased NO(x) production is associated with elevated eNOS protein expression, which is mediated at least in part via activation of the mitogen-activated protein kinase kinase1 and kinase2 (MAP2K1 and MAP2K2, known also as MEK1/2)/ERK1/2 cascade. Together with our previous observation that ANG II stimulates OFPAE cell proliferation, these data suggest that ANG II is a key regulator for both vasodilation and angiogenesis in the ovine fetoplacenta.     

Biogeochemistry and community composition of iron- and sulfur-precipitating microbial mats at the Chefren mud volcano (Nile Deep Sea Fan, Eastern Mediterranean)

In this study we determined the composition and biogeochemistry of novel, brightly colored, white and orange microbial mats at the surface of a brine seep at the outer rim of the Chefren mud volcano. These mats were interspersed with one another, but their underlying sediment biogeochemistries differed considerably. Microscopy revealed that the white mats were granules composed of elemental S filaments, similar to those produced by the sulfide-oxidizing epsilonproteobacterium "Candidatus Arcobacter sulfidicus." Fluorescence in situ hybridization indicated that microorganisms targeted by a "Ca. Arcobacter sulfidicus"-specific oligonucleotide probe constituted up to 24% of the total the cells within these mats. Several 16S rRNA gene sequences from organisms closely related to "Ca. Arcobacter sulfidicus" were identified. In contrast, the orange mat consisted mostly of bright orange flakes composed of empty Fe(III) (hydr)oxide-coated microbial sheaths, similar to those produced by the neutrophilic Fe(II)-oxidizing betaproteobacterium Leptothrix ochracea. None of the 16S rRNA gene sequences obtained from these samples were closely related to sequences of known neutrophilic aerobic Fe(II)-oxidizing bacteria. The sediments below both types of mats showed relatively high sulfate reduction rates (300 nmol x cm(-3) x day(-1)) partially fueled by the anaerobic oxidation of methane (10 to 20 nmol x cm(-3) x day(-1)). Free sulfide produced below the white mat was depleted by sulfide oxidation within the mat itself. Below the orange mat free Fe(II) reached the surface layer and was depleted in part by microbial Fe(II) oxidation. Both mats and the sediments underneath them hosted very diverse microbial communities and contained mineral precipitates, most likely due to differences in fluid flow patterns.     

Release of prostaglandins and thromboxanes by guinea pig isolated type II pneumocytes

Lung cells have been isolated by enzymatic digestion of guinea pig lungs and mechanical dispersion to obtain a suspension of viable cells (approximately 500 X 10(6) cells). Type II pneumocytes have been purified to approximately 92% by centrifugal elutriation (2000 rpm, 15 ml/min) followed by a plating in plastic dishes coated with guinea pig IgG (500 micrograms/ml). We have investigated the arachidonic acid metabolism through the cyclooxygenase pathway in this freshly isolated type II cells (2 x 10(6) cells/ml). Purified type II pneumocytes produced thromboxane B2 (TxB2) predominantly and to a smaller extent the 6-keto prostaglandin PGF1 alpha (6-keto-PGF1 alpha) and prostaglandin E2 (PGE2) after incubation with 10 microM arachidonic acid. The stimulation of pneumocytes with 2 microM calcium ionophore A23187 released less eicosanoids than were produced when cells were incubated with 10 microM arachidonic acid. There was no additive effect when the cells were treated with both arachidonic acid and the ionophore A23187. Guinea pig type II pneumocytes failed to release significant amounts of TxB2, 6-keto-PGF1 alpha and PGE2 after stimulation with 10 nM leukotriene B4, 10 nM leukotriene D4, 10 nM platelet-activating factor, 5 microM formyl-methionyl-leucyl-phenylalanine, 0.2 microM bradykinin and 10 nM phorbol myristate acetate. Our findings indicate that guinea pig type II pneunomocytes possess the enzymatic machinery necessary to convert arachidonic acid to specific cyclooxygenase products, which may suggest a role for these cells in lung inflammatory processes.     

Effects of pH indicators on various activities of chromatophroes of Rhodospirillum rubrum.

1. The effects of pH indicators on activities for ATP hydrolysis in the dark and ATP-Pi exchange in the dark were examined with chromatophores from Rhodospirillum rubrum. Of thirty-one pH indicators tested, eleven (metanil yellow, 2, 4-dinitrophenol, ethyl orange, bromocresol green, resazurin, neutral red, bromthymol blue, alpha-naphtholphthalein, o-cresolphthalein, phenolphthalein, and alizarin yellow G) almost completely inhibited the activities for ATP formation and ATP-Pi exchange at concentrations of 1 mM, and were studied in detail. 2. Of the eleven pH indicators, those other than alpha-naptholphthalein, o-cresolphthalein and phenolphthalein, when assayed at appropriate concentrations, inhibited ATP-Pi exchange, but not ATP hydrolysis. In ATP-Pi exchange, these eight pH indicators at the concentrations described above were competitive against Pi, and non-competitive against ATP. The remaining three kinds of pH indicators were non-competitive against either Pi or ATP, when assayed at concentrations of the dyes that inhibited both activities. 3. The amounts of pH indicators bound with chromatophores were measured. No correlation was found between the amounts of the bound dyes and the extents of their inhibition of either ATP formation or ATP-Pi exchange. 4. Ethyl orange (pKa=4.1) and 2, 4-dinitrophenol (pKa=3.9) stimulated ATP hydrolysis to the greatest extent. The latter dye was hardly bound with chromatophores. 5. The stimulatory effects of pH indicators on ATP hydrolysis were hardly affected by extraction of quinones from chromatophores. 6. Most of the pH indicators stimulated both succinate-cytochrome c2 and NADH-cytochrome c2 reductions in the dark. 7. The mechanism of uncoupling of the electron transfer system and the phosphorylation system by pH indicators and the mechanism of the coupling are discussed.     

Three classes of epidermal growth factor receptors on HeLa cells

The kinetics of 125I-labeled epidermal growth factor (EGF) binding to receptors on HeLa cells were investigated. Scatchard analysis revealed the presence of 22,000 high affinity receptors (Kd = 0.12 nM) and 25,000 low affinity receptors per cell (Kd = 9.2 nM). The kinetic analysis of EGF binding to high affinity receptors was performed with cells pretreated with the monoclonal antibody 2E9, which prevents specifically EGF binding to low affinity receptors. The study of EGF binding to only low affinity receptors was performed with cells pretreated with the phorbol ester phorbol 12-myristate 13-acetate, which induces a conversion of high affinity receptors to low affinity receptors. This kinetic analysis of EGF binding to HeLa cells revealed the presence of three types of receptors. High affinity receptors were found to consist of one receptor type (type I) with a kinetic association constant (kass) of 6.2 x 10(5) M-1.s-1 and a kinetic dissociation constant (kdis) of 3.5 x 10(-4) s-1. The low affinity receptors were found to consist of two kinetic distinguishable sites: type II or fast sites with kass = 3.3 x 10(6) M-1.s-1 and kdis = 8.1 x 10(-3) s-1 and the type III or slow sites with kass = 3.2 x 10(4) M-1.s-1 and kdis = 1.6 x 10(-4) s-1. The regulatory mechanism which may determine the EGF binding characteristics is discussed.     

Manganese Oxidation by Bacterial Isolates from the Indian Ridge System

The abundance and activity of culturable manganese-oxidizing bacteria were assessed from near-bottom water samples of the tectonically active Carlsberg Ridge. Retrievable counts as colony forming units (CFU) on dilute nutrient agar medium (dilNA = 2 gm l⁻¹ nutrient broth+2% agar) and on dilNA supplemented with 1, 2 and 3 mM MnCl₂·4H₂O were in the order of 10⁶ CFU l⁻¹. Retrievability of heterotrophs ranged from non-detectable levels (ND) to 2.82 × 10⁶ CFU l⁻¹. The retrievable counts on Mn amended dilNA ranged from ND to 3.21× 10⁶, 1.47 × 10⁶ and 1.45 × 10⁶ CFU l⁻¹ on 1, 2 and 3 mM, respectively. About 87% of the Mn tolerant isolates (n = 39) showed taxonomic affinities to Pseudomonas I and II sp. Two representative strains CR35 and CR48 (CR–Carlsberg Ridge) isolated on manganese-supplemented media were tested for their ability to tolerate a range of Mn amendments from 1 nM to 100 mM in terms of growth and respiration. CR35 represents 66% of the total CFU (3.04 × 10⁶ CFU l⁻¹), while CR48 represented only 6% of the total CFU (1.05 × 10⁶ CFU l⁻¹). The colonies of these two isolates were dark brown in color suggesting precipitation of Mn as oxide. Tests for the effect on growth and respiration were conducted in media simulating heterotrophic (amended with 0.01% glucose) and lithotrophic (unamended) conditions. Maximum stimulation in growth and respiration of CR35 occurred at 100 μM Mn both in unamended and amended media. At levels of Mn greater than 100 μM the counts decreased steadily. Total respiring cells of CR48 were stimulated to a maximum at 1 μM Mn in unamended medium and 1 nM in amended medium. Total cells counts for the same decreased beyond 100 μM Mn in unamended and 1 nM in amended medium. The isolates were tested for their ability to oxidize Mn ammendments from 1 μM to 10 mM Mn. At the end of a 76-day incubation period, there was evidence of manganese oxide precipitation at high Mn concentrations (≥1 mM) as a dark brown coloration on the sides of culture tubes. Highest Mn oxidation rates were observed at 10 mM Mn(II) concentration with CR35 oxidizing 27 and 25 μM Mn day⁻¹ in unamended and amended condition, respectively. CR48 oxidized Mn at the rate of 26 μM Mn day⁻¹ in unamended medium and 35 μM Mn day⁻¹ in amended medium. Scanning electron microscope (SEM) observations of both isolates revealed free-living cells in clustered matrices ≈2 μm diameter. Energy dispersive spectrum of the cell matrix of CR35 cultured in 1 mM Mn detected 30% Mn, while the cell aggregates of CR48 harbored 7–10% Mn. The relatively high specific activity of these mixotrophic bacteria under relatively oligotrophic conditions suggests that they may be responsible for scavenging dissolved Mn from the Carlsberg Ridge waters and could potentially participate in oxidation.     

Bcl-xL overexpression restricts gamma-radiation-induced apoptosis

Bcl-xL belongs to a family of proteins which inhibit apoptosis in a number of stimuli including ionizing radiation. To better understand the effects and mechanisms of Bcl-xL on the apoptosis of lymphocytes and provide experimental basis to treat immune injury induced by radiation, we used normal human lymphoblastoid AHH-1 cells that were engineered to overexpress Bcl-xL proteins. Our results showed that overexpressed Bcl-xL reduced time-dependent increase of apoptosis induced by ionizing radiation. Reactive oxygen species (ROS) generation and Bax protein expression in the transfected AHH1-Bcl-xL cells were also lower compared to parental AHH-1 cells. Unexpectedly, the fluorescence intensity of Rhodomine 123 (Rh 123) for measuring mitochondrial membrane potential (MMP) did not change at all detected time points. These results possess a vital significance for insights into a new strategy for gene therapy of radiation-induced immune injury.     

Determination of ACC-induced cell-programmed death in roots of Vicia faba ssp. minor seedlings by acridine orange and ethidium bromide staining

Fluorescence staining with acridine orange (AO) and ethidium bromide (EB) showed that nuclei of cortex root cells of 1-aminocyclopropane-1-carboxylic acid (ACC)-treated Vicia faba ssp. minor seedlings differed in color. Measurement of resultant fluorescence intensity (RFI) showed that it increased when the color of nuclear chromatin was changed from green to red, indicating that EB moved to the nuclei via the cell membrane which lost its integrity and stained nuclei red. AO/EB staining showed that changes in color of the nuclear chromatin were accompanied by DNA condensation, nuclei fragmentation, and chromatin degradation which were also shown after 4,6-diamidino-2-phenylindol staining. These results indicate that ACC induced programmed cell death. The increasing values of RFI together with the corresponding morphological changes of nuclear chromatin were the basis to prepare the standard curve; cells with green unchanged nuclear chromatin were alive while those with dark orange and bright red nuclei were dead. The cells with nuclei with green–yellow, yellow–orange, and bright orange chromatin with or without their condensation and fragmentation chromatin were dying. The prepared curve has became the basis to draw up the digital method for detection and determination of the number of living, dying, and dead cells in an in planta system and revealed that ACC induced death in about 20% of root cortex cells. This process was accompanied by increase in ion leakage, shortening of cells and whole roots, as well as by increase in weight and width of the apical part of roots and appearance of few aerenchymatic spaces while not by internucleosomal DNA degradation.