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PubMed2005

In vitro toxicity of selected pesticides on RTG-2 and RTL-W1 fish cell lines.

The rainbow trout fish cell lines RTG-2 and RTL-W1 were used to determine the cytotoxic effects of the pesticides bifenthrin, cypermethrin, cyhalothrin, lambda-cyhalothrin, quinalphos and chlorpyrifos. Cytotoxicity was measured by EROD and beta-Gal enzymatic activities, the neutral red (NR) uptake assay, and the FRAME KB protein (KBP) assay. The beta-Gal activity was unaffected by the pesticide exposure. The EROD activity was induced by cyhalothrin and lambda-cyhalothrin (RTG-2 and RTL-W1) and by bifenthrin (RTL-W1). Dose dependent inhibition responses were observed for EROD activity in cells exposed to quinalphos (RTL-W1) and chlorpyrifos (RTG-2 and RTL-W1). RTL-W1 offered a better response for EROD induction. The EC50 values on EROD endpoint were more sensitive than NR and KBP. The acute fish toxicity of chlorpyrifos and quinalphos depends highly on the species; the species sensitivity distributions cover several orders of magnitude and the values obtained for EROS were within the lowest part of the reported ranges.

Animals↗
PubMed1993

An in vitro model of cyclosporine-induced nephrotoxicity.

The predominant hypothesis for cyclosporine-induced acute renal failure is postulated to be prerenal vasoconstriction with concomitant hemodynamic changes; an alternate hypothesis, however, may be that cyclosporine (CsA) affects intrarenal processes, i.e., direct renal parenchymal cell injury. However, reports on this direct effect of CsA on renal parenchymal cells are contradictory. Therefore, the purpose of this study was to address whether CsA is directly toxic to renal parenchymal cells in a primary culture system of rat renal cortical epithelial cells. The cytotoxicity of Sandimmune, the commercial form of CsA in a polyoxyethylated castor oil vehicle (Cremophor), CsA without vehicle, and the Cremophor vehicle was assessed by plasma membrane integrity (lactate dehydrogenase leakage), mitochondrial metabolic activity [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction], and gross morphology (phase-contrast microscopy). The cytotoxicity of Sandimmune was also assessed by lysosomal activity (neutral red uptake), by proximal tubular enzyme activity (alkaline phosphatase), and by three fluorescent probes using a multiwell scanner. The three fluorescent probes were propidium iodide which stains nuclei of nonviable cells; bis-carboxyethyl-carboxyfluorescein which is retained by viable cells; and rhodamine 123, which assesses mitochondrial membrane potential. The results of this study demonstrated that Sandimmune caused dose- (10, 25, and 50 microM) and time- (12, 24, and 48 hr) dependent cytotoxicity, while Cremophor caused cytotoxicity only at high concentrations and long incubations. We conclude that (1) CsA is directly toxic to renal parenchymal cells in vitro and this system potentially represents a sensitive model for further mechanistic studies; (2) CsA plus vehicle (Sandimmune) was more cytotoxic to renal cells than CsA alone (without the polyoxyethylated castor oil vehicle).

Alkaline Phosphatase↗
PubMed1988

The feline oculomotor nucleus: morphological subdivisions and projection to the cerebellar cortex and nuclei.

The cytoarchitecture of the feline oculomotor nucleus was examined in sections stained with thionin and neutral red. Five different subdivisions (caudal central, paramedian, ventral, dorsomedial and dorsolateral divisions) can be identified on each side of the midline. This observation is discussed, and our findings are compared to previous studies of the cytoarchitecture or central muscular representation of the oculomotor nucleus in which different subgroups have been distinguished. Implants or injections of the wheat germ agglutinin-horseradish peroxidase complex have revealed that all five subdivisions project to different parts of the cerebellar cortex and nuclei. Retrogradely labelled cells were found in the oculomotor nucleus in 18 cases following deposition of tracer in the fastigial and interposed nuclei and certain regions of the anterior, posterior and flocculonodular lobes. The projection is bilateral and appears to have its main termination in flocculus. It originates from small neurons, especially from those located along the dorsal border of the oculomotor nucleus.

Afferent Pathways↗
PubMed1975

Photodynamic inactivation in experimental herpetic keratitis.

The effect of photodynamic inactivation on experimental herpes simplex keratitis in rabbits was investigated using neutral red as a photosensitizing dye followed by exposure to light at 425nm. Combined dye application and light exposure early in the disease (two days following infection) reduced to a minimal extent the severity and duration of the acute epithelial infection. The effect on well-established keratitis (three days postinfection) was negligible as evaluated by clinical grading, viral recovery, and histopathological study. In initial experiments, it was found that the dye and light did not have any observable deleterious effect on intact corneas or cause any noticeable delay in healing of injured cornias. Further, when light or dye were utilized alone, neither changed the severity or duration of the keratitis. In vitro treatment of the virus with light and dye destroys its ability to produce experimental keratitis.

Animals↗
PubMedDate not supplied

Measuring lysosomal stability as an effective tool for marine coastal environmental monitoring.

The use of lysosomal stability in the mussel, Mytilus galloprovincialis, as a potential biomarker of environmental contamination has been evaluated along the Portuguese coast. To this end, the neutral red retention (NRR) time was measured in mussel haemocytes gathered from nine different locations reflecting different degrees of anthropogenic contamination. Mussels collected in the vicinity of industrial and urban areas showed the lowest lysosomal stability. Additionally, no significant seasonal variability (winter-spring/summer) for NRR time was observed. In order to further support the usefulness of this method as an integrated tool for monitoring marine coastal environments, we compared the levels of xenobiotics in mussel tissues with the obtained NRR values. The results highlighted a consistent pattern, with the lowest lysosomal stability intimately correlated with the higher contaminant concentrations. In summary, this integrated approach further demonstrated that the NRR assay can provide useful and objective indications of the real health status of organisms subjected to different stress agents, being a valid option for environmental monitoring.

Animals↗
PubMed1990

Studies of collateral perfusion to canine middle cerebral artery territory.

Previous studies of intracranial collateral circulation have not distinguished between true "collateral" blood flow (flow to a region that occurs only when a primary artery is occluded) and "overlap" flow (flow to a region that is present under both normal and demand conditions). These experiments had three purposes: 1) to identify tissues that were truly collateral dependent, 2) to determine potential for true collateral flow in the absence of overlap flow, and 3) to determine whether an anatomical basis for overlap flow could be demonstrated. Branches (700-900 microns) of the dog middle cerebral artery (MCA) were perfused with autologous blood. The perfused region, which was the area at risk, was identified by intravenous injection of neutral red dye. Microspheres were used to measure regional cerebral blood flow (rCBF). Overlap flow was determined by perfusion of the artery with microsphere-free blood. True collateral flow (total rCBF minus overlap flow) was determined by analysis of rCBF to the risk area after cessation of vessel perfusion. Most of the risk area had substantial levels of overlap flow (about one-third of base line). In the center of the area at risk, the true collateral-dependent area was identified [mean overlap flow 4 +/- 1 (mean +/- SE) ml.min-1.100 g-1], which had high levels of perfusion from collateral vessels (102 +/- 14) within 30 s of vascular occlusion. Microfil injection into two adjacent MCA branches showed discrete borders between vascular territories, with no overlapping vessels.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1997

Toxicity assessment of 16 inorganic environmental pollutants by six bioassays.

The relative toxicity of 16 environmental pollutants, such as inorganic elements (Ba, Cd, Co, Cr, Cu, Fe, Ge, Hg, Mn, Nb, Pb, Sb, Sn, Ti, V, and Zn), is evaluated on the L-929 established cell line of murine fibroblasts, with five bioassays [RNA synthesis rate assay (RNA), MTT reduction assay (MTT), neutral red incorporation assay (NRI), Coomassie blue assay, and cellular growth rate assay], and on the ciliated protozoa Tetrahymena pyriformis GL [doubling time of T. pyriformis GL population assay (DTP)]. For each inorganic substance, the six bioassays allowed the toxicological index IC50 ("inhibitory concentration 50%") to be calculated. The IC50 values are useful to rank the tested elements and to compare the features of the six bioassays. The most sensitive assays were the RNA, MTT, NRI, and DTP assays. Moreover, the in vitro IC50 values correlated with the in vivo LD50 values; these results were close to those obtained with established lines of human, murine, or fish cells. The sensitivity and the complementarity of these bioassays would be in favor of their incorporation in a "battery" of tests used for toxicological screening studies of xenobiotics.

Animals↗
PubMedDate not supplied

Biochemical and cytogenetical study of the mycoplasmal antigen and of the cyclophosphamide action in mammalians, in vivo. The action of some immunomodulatory antioxidants.

It was proved spectrophotometrically that Mycoplasma agalactiae antigen inoculated in vivo in sheep modifies the corresponding erythrocyte lysates reactivity toward methylene blue and neutral red and induces several types of chromosomal rearrangements. The treatment in vivo of sheep with an original preparation obtained from the Phaseolus vulgaris pods restores the erythrocyte lysates reactivity toward the two redox dyes and reduces the chromosomal abnormalities frequency induced by the mycoplasmal antigen. It was also demonstrated by optical and electronical microscopy that the Smise line mouse meiocytes exhibit chromosomal abnormalities induced by the cyclophosphamide treatment in vivo. In the case of concomitant treatment with the cyclophosphamide and C vitamin the same frequency of abnormalities was recorded as in the simple treatment with the drug.

Adjuvants, Immunologic↗
PubMed1999

Influence of uranium(VI) speciation for the evaluation of in vitro uranium cytotoxicity on LLC-PK1 cells.

Very few data are available concerning the in vitro toxicity of uranium. In this work, we have determined the experimental chemical conditions permitting the observation of uranium(VI) cytotoxicity on LLC-PK1 cells. Uranium solutions made either by dissolving uranyl acetate or nitrate crystals, or by complexing uranium with bicarbonate, phosphate or citrate ligands, were prepared and tested. Experiments demonstrated that only uranium solutions containing citrate and bicarbonate ligands concentrations tenfold higher than the metal, were soluble in the cell culture medium. Cytotoxicity studies of all these uranium compounds were performed on LLC-PK1 cells and compared using LDH release, neutral red uptake and MTT assays. Dose dependent cytotoxicity curves were only obtained with uranium-bicarbonate medium. This study has revealed a toxicity of uranium-bicarbonate complexes for 24 h expositions and for concentrations ranging from 7 x 10(-4)-10(-3) M, under these conditions, the CI50 (cytotoxicity index) was evaluated between 8.5 and 9 x 10(-4) M. In contrast, we noticed a lack of cytotoxicity response for uranium(VI)-citrate complexes. Electron transmission microscopy studies revealed, when LLC-PK1 cells were exposed to the uranium-bicarbonate system, that uranium penetrated and precipitated within the cytoplasmic compartment. Morphological studies conducted with citrate complexes did not show any cellular intake of uranium.

Animals↗
PubMed1999

Protective effects of polyphenols against cadmium-induced glomerular mesangial cell myocontracture.

The main objective of this work was to determine the ability of polyphenols (procyanidoloic oligomers; PCO) to diminish the contracture of CdCl2-induced mesangial cells (smooth muscle cell type). Glomeruli were isolated by passing rat renal cortex pulp through calibrated sieves followed by a culture step for outgrowth of cells. PCO lethality was measured by microassay (Neutral Red uptake). This study has revealed an absence of PCO toxicity during exposure for 24 h and for concentrations ranging from 0.031 to 1% (w/v) on rat renal mesangial cells. We observed a lack of cytotoxicity response for the PCO mixture dissolved in medium. Quantitative assessments of the planar cell surface area (PCSA) were performed with an accurate automatized image analyser. The use of isolated cultured mesangial cells permits us to evaluate by quantitative morphometric analysis the contracture elicited either with CdCl2 salts alone or by previous incubation with a non-lethal dose of PCO. When renal mesangial cells were exposed for 10 min to the PCO mixture, the Cd-mediated myocontracturant response of the mesangial cells was totally abolished. These results suggest that polyphenols could be effective against renal damages induced by cadmium.

Animals↗
PubMed1998

Chemical and biological studies of a new cigarette that primarily heats tobacco. Part 3. In vitro toxicity of whole smoke.

Mainstream smoke from Kentucky reference low "tar" (1R4F) and ultra-low "tar" (1R5F) cigarettes and a test cigarette (TOB-HT), that primarily heats tobacco, was compared for cytotoxic and genotoxic potential using cellular smoke exposure technology (CSET). CSET includes a computer controlled 30-port AMESA/Battelle-Geneva smoke generator which exposes cultured mammalian Chinese hamster ovary cells (CHO) to whole smoke. Cytotoxicity was assessed using the neutral red assay and genotoxicity was assessed using the sister chromatid exchange (SCE) assay. Compared on a per cigarette basis, mainstream smoke from 1R5F and the TOB-HT cigarette was significantly less cytotoxic and genotoxic than the smoke from the 1R4F cigarette. The cytotoxic and genotoxic activity of smoke from the TOB-HT cigarettes was slightly greater than the smoke from the ultra-low "tar" Kentucky 1R5F reference cigarettes. In conclusion, in these assays mainstream whole smoke of the TOB-HT cigarette had slightly greater cytotoxic and genotoxic potential compared with an ultra-low "tar" 1R5F Kentucky reference cigarette and significantly less activity compared with the whole mainstream smoke from a low "tar" 1R4F Kentucky reference cigarette, representative of the US market average cigarette for FTC yields of "tar", CO and nicotine.

Animals↗
PubMed2004

Genotoxicity of the isoflavones genistein, daidzein and equol in V79 cells.

Hormonally active chemicals in the human diet, such as man-made estrogenic chemicals or plant-derived compounds (phytoestrogens), have become a matter of public concern. A significant part of human exposure to phytoestrogens is attributable to soy isoflavones. Besides their estrogenic properties, soy isoflavones also exert genotoxic actions. In this paper, the micronucleus (MN) assay in V79 cells was used to study chromosomal genotoxicity. Genistein caused a clear dose-related induction of MN within the range of 5-25 microM; MN rates were declining at higher genistein concentrations. This was probably due to cytotoxicity of genistein since reduced neutral red uptake and MTT formation with an IC(50) of about 75 microM occurred. Daidzein induced a comparatively shallow increase in the number of MN between 25 and 100 microM. In contrast, the daidzein metabolite equol caused an increase in the number of MN up to 25 microM with no further increase at higher concentrations. Additional staining with anti-kinetochore (CREST) antibodies served to determine if the micronuclei contain whole chromosomes or acentric fragments. Genistein induced mostly CREST(-) micronuclei, i.e. MN with chromosomal fragments, thus indicative of a clastogenic mode of action. MN induced by high concentrations of daidzein were partly CREST(+) and CREST(-), whilst equol induced mostly CREST(+) micronuclei indicative of an aneugenic action. These results point to a differential genotoxicity of phytoestrogens.

Animals↗
PubMed2004

A simple assay for determining antiviral activity against Crimean-Congo hemorrhagic fever virus.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne virus that is emerging as a significant human pathogen in many regions of the world, including Africa, Asia, and Europe. In this report, we describe a simple screening method for discovering new antiviral compounds directed against CCHFV. Antiviral activity was determined by assaying infected SW-13 cells (human adrenal gland carcinoma) for protection from cytopathic effect (CPE). By using an in vitro neutral red uptake assay, we were able to quantitatively measure CPE induced by CCHFV. As a proof of concept, we used this method to evaluate the antiviral activity of ribavirin and a series of structural analogs (ribamidine, 6-azauridine, selenazofurin, and tiazofurin) against four geographically diverse strains of CCHFV. Ribavirin inhibited the replication of CCHFV as reported previously using plaque reduction assays. One drug, ribamidine, showed antiviral activity that was 4.5- to 8-fold less than that of ribavirin, and the other three drugs (6-azauridine, selenazofurin, and tiazofurin) did not show significant antiviral activity. There were no significant differences in drug sensitivities among the CCHFV strains. Development of this simple and reliable assay will potentially allow high-throughput screening for discovering additional antiviral drugs to combat this important public health threat.

Animals↗
PubMedDate not supplied

Cocaethylene toxicity in rat primary myocardial cell cultures.

Cocaethylene is a unique cocaine metabolite formed in the presence of ethanol by the liver. Neither acute nor chronic cardiotoxic effects of this metabolite have been investigated. The purpose of this study was to establish a time- and dose-dependent toxicity profile for cocaethylene in primary myocardial cell cultures established from 3-5-day-old Sprague-Dawley rats. Alterations in lactate dehydrogenase (LDH) release, lysosomal neutral red (NR) retention, thiobarbituric acid-reactive substances (TBARS), morphology, and beating activity were evaluated after treatment of cultures with cocaethylene doses ranging from 1.0 x 10(-3) to 1.0 x 10(-9) M from 1 to 24 h. LDH release was significantly elevated after 24 h only with those cultures exposed to the highest dose of cocaethylene (1.0 x 10(-3) M). The highest dose of cocaethylene also significantly depressed NR retention. While all doses of cocaethylene depressed contractile activity and altered cellular morphology by 24 h, there were no TBARS formed up to 15 h. Thus, both low and high doses of cocaethylene are injurious to the cellular integrity and contractility of myocardial cell cultures. Future studies are warranted to determine mechanisms of cocaethylene toxicity in this in vitro model of spontaneously contracting myocardial cells.

Animals↗
PubMed1997

[Cytotoxicity of chemicals used in household products: estimation of eye irritating potency of 25 chemicals tested during 1991-1996].

Cytotoxicity potential of chemicals was evaluated by determining the concentrations inducing 50% reduction of neutral red (NR) uptake into Chinese hamster fibroblast V79 cells compared with control culture (IC50). The results of cytotoxicity test for surfactants with the data produced by the in vivo Draize eye and skin irritation test were compared. There was a good correlation between cytotoxicity and eye irritation score obtained from the Draize test. In contrast, no correlation was observed between Draize skin irritation score and cytotoxic potential of chemicals. Therefore, the NR cytotoxicity test was regarded as a possible in vitro model for predicting eye irritation. Based on the IC50 values in the NR cytotoxicity test, the eye irritation classification (weak, moderate and strong) for each chemical used in household products has been established. We evaluated the cytotoxicity of 25 chemicals used for antimicrobial, rubber accelerator, rubber antioxidant, ultraviolet absorber etc. in household products, and estimated the eye irritating potency of these test chemicals according to the criterion.

Animals↗
PubMed2005

In vitro toxicity evaluation in the development of new anticancer drugs-genistein glycosides.

In vitro cytotoxicity tests currently in use applied in the developmental stages of anticancer drug discovery are able to select the most potent compounds, but are not predictive of their potential toxicity. In this study, we have demonstrated the applicability of neutral red uptake assay using mouse fibroblasts Balb/c 3T3 cell line (3T3 NRU assay) for in vitro toxicity testing of newly synthesized genistein glycosides, the compounds that appear to show anticancer activity. We have also proven the compatibility of in-house 3T3 NRU assay with the prediction model for acute rodent oral toxicity testing, endorsed by NIEHS-ICCVAM workshop. The combined results from the cytotoxicity and the in vitro toxicity tests facilitated the selection of the most promising genistein derivatives, compounds G21 and G23, which were the most active and selective towards cancer cells. The comparison of predicted LD50 values revealed that almost all genistein derivatives are at least two-fold less toxic than the chemotherapeutics currently used in cancer therapy, which is very promising for this new group of compounds.

Animals↗
PubMed2004

Morphological characterization of renal cell lines (BGM and VERO) exposed to low doses of lead nitrate.

The response to lead nitrate has been assessed in two cell lines of renal origin. The range of toxic concentrations was determined by Neutral Red assay after 24-h of exposure. Morphological changes in the Buffalo Green Monkey (BGM) and VERO cell lines after exposure to subcytotoxic doses (1.38 mM and 1.04 mM, respectively) equivalent to EC10 (effective concentrations 10%) of lead nitrate were evaluated at the ultrastructural level by transmission microscopy. The most notable finding in treated cells was the presence of inclusion bodies in the form of irregular granules of varying size in both cytoplasm and lysosomes. Cell membrane integrity was not affected. The number of phagolysosomes and myeline figures associated to the inclusion bodies was higher than in the control cultures. We conclude that the phagolysosomic mechanism fails to digest this metal ion and the BGM and VERO renal cell lines can be considered as useful tools for toxicological studies involving lead nitrate.

Animals↗
PubMedDate not supplied

Early cytotoxic effects induced by bis-chloroethyl sulphide (sulphur mustard): [Ca2+]i rise and time-dependent inhibition of B77 fibroblast serum response.

Early cytotoxic events were studied on B77 fibroblasts. Cells were treated with sulphur mustard (SM) in short-term experiments in which cell viability was unchanged, as evaluated by the neutral red cytotoxicity test. This treatment was correlated to two early signs of cytotoxicity. The intracellular Ca2+ concentration [Ca2+]i level in SM-treated Fura-2-loaded fibroblasts showed a significant dose-dependent increase. This observed rise was sustained, in contrast to the Ca2+ signal induced by serum, and was already visible 5-10 min after the addition of SM to cell suspensions in vitro. Modification of the extracellular Ca2+ concentration in the medium had no effect on the cytosolic calcium rise caused by SM, suggesting release from intracellular Ca2+ pools. Furthermore, a time-dependent inhibition of the [Ca2+]i transient increase induced by growth-factors (as evaluated by the fetal calf serum (FCS) response) was observed within the first hour of exposure. These latter results suggest that early alterations of calcium distribution induced by SM could be one of the earliest markers of SM intoxication.

Animals↗