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PubMed1996

Comparison of low-volume, Draize and in vitro eye irritation test data. I. Hydroalcoholic formulations.

The first phase in a series of investigations of the relationship between low-volume eye test (LVET) data, Draize eye irritation test data, and comparable data from 25 in vitro assay protocols is presented. These investigations utilize Draize eye test and in vitro assay data generated previously as part of the Cosmetic, Toiletry and Fragrance Association (CTFA) Evaluation of Alternatives Program. LVET data were generated de novo using the same 10 representative hydroalcoholic personal-care formulations. The linear correlation between maximum average score (MAS) as determined by the Draize test and the LVET (LVET-MAS) was 0.93. Comparison of in vitro assay performance with that of the LVET was determined by statistical analysis of the relationship between LVET-MAS and in vitro endpoint. As in the CTFA program, regression modelling is the primary means of enabling such a comparison. The objective is to predict LVET-MAS for a given test material (and to place upper and lower prediction interval bounds in the range in which the LVET-MAS is anticipated to fall with high probability) conditional on observing an in vitro assay score for that material. The degree of confidence in prediction is quantified in terms of the relative widths of prediction intervals constructed about the fitted regression curves. Four assays [EYTEX MPA (membrane partition assay), HET-CAM (hen's egg test-chorioallantoic membrane HET-CAM) I, neutral red release and HET-CAM II] were shown to have the greatest agreement with the LVET. These assays were also among those with low discordance rates relative to the Draize test. Prediction of LVET-MAS values from experimentally determined in vitro scores was more accurate for hydroalcoholic formulations with lower rather than higher irritancy potential.

Animal Testing Alternatives↗
PubMed1992

Toxicity assessment of toxins T-514 and T-544 of buckthorn (Karwinskia humboldtiana) in primary skin and liver cell cultures.

The present study was undertaken to assess and compare the in vitro cytotoxicity of toxins T-514 and T-544 of buckthorn (Karwinskia humboldtiana) using primary cultures of rat hepatocytes and keratinocytes. Cell cultures were exposed to 6, 12, 25 and 50 microM toxins for 2-, 4-, 6- and 24-h periods. Cytotoxicity was determined by release of the cytoplasmic enzyme, lactate dehydrogenase (LDH), in culture media, methylthiazoltetrazolium (MTT) reduction and neutral red (NR) uptake. An increase in LDH leakage was observed in liver cell cultures as early as 2 h with 50 microM T-544 and with 6 microM T-514 and T-544 at 6 h and 24 h, respectively. In the NR assay the toxicity was evident at 2 h with 12 microM T-514 and T-544 and with 6 microM concentrations of both toxins at 6 h. On the other hand, a decrease in MTT reduction was detected at 4 h with 50 microM concentrations of both toxins and with 25 microM T-544 and 12 microM T-514 at 6 h and 6 microM T-514 and T-544 at 24 h. Both toxins were shown to be highly hepatotoxic; T-514 was more toxic than T-544. In the skin cell cultures, the toxicity of the toxins was not as severe and was not expressed until 12 h of exposure.

Animals↗
PubMed1992

A primary culture system of adult rat heart cells for the evaluation of cocaine toxicity.

The complex dose-response relationship by which cocaine (Coc) directly precipitates unfavorable cardiac consequences are not known. There appears to be two diametrically opposed cardiovascular actions of Coc. At low doses, the sympathetic nervous system responses dominate, whereas, at high doses, the local anesthetic actions exert the most powerful effects. The purpose of this study was to describe a dose- and time-dependent Coc cardiotoxicity profile in a model of spontaneously contracting adult primary myocardial cell cultures obtained from 60-90-day-old Sprague-Dawley rats. Indices of toxicity determined included contractility, morphology, lactate dehydrogenase release (LDH), mitochondrial tetrazolium formazan (MTT) production and neutral red (NR) formation. After the cells had been grown in culture for 11 days, they were exposed to 1 x 10(-3), 1 x 10(-5), 1 x 10(-7) and 1 x 10(-9) M Coc for 1-24 h. The two lowest doses of Coc (1 x 10(-7) and 1 x 10(-9) M) had little or no effect on the adult heart cell cultures. However, morphological alterations included vacuolization, granulation and pseudopodia formation as early as 1 h after exposure to the highest doses of Coc (1 x 10(-3) and 1 x 10(-5) M). For all time points observed, the two highest doses of Coc (1 x 10(-3) and 1 x 10(-5) M) significantly depressed contractility and induced significant LDH release. MTT formazan production and NR retention were not significantly different from untreated controls for all treatments. By employing an acute Coc exposure paradigm, these data demonstrate that Coc doses greater than or equal to 1 x 10(-5) M induce direct injurious local anesthetic effects on contractility and morphology of spontaneously contracting adult rat myocardial cells in culture.

Animals↗
PubMed1987

Short-term quantitative in vitro cytotoxicity assay involving an S-9 activating system.

Mouse 3T3 fibroblasts grown in 96-well microtiter plates are used for a test which incorporates an S-9 mixed function oxidase system into a neutral red viability assay for the assessment of the acute cytotoxicity of xenobiotics in vitro. This sensitive, quantitative, semi-automated assay was suitable for the rapid screening of a broad spectrum of substances, including pharmaceuticals, carcinogens, and anti-neoplastic agents. The test was applicable to the analysis of toxic ranges, for the detection of biotransformability of parent compounds and for the evaluation of the cytotoxic effects of chemotherapeutic agents.

Animals↗
PubMed1993

Comparative cytotoxicities of selected minor dietary non-nutrients with chemopreventive properties.

The comparative acute cytotoxicities were determined for a varied spectrum of minor dietary non-nutrients that have been implicated as chemopreventive agents. Cytotoxicity was determined with the neutral red (NR) assay, using BALB/c mouse 3T3 fibroblasts as the bioindicators. Based on midpoint cytotoxicity (NR50) values, the range of cytotoxicity for the different chemicals varied by 1000 times. The sequence of potency was tannic acid, tamoxifen citrate, quercetin, benzyl and phenethyl isothiocyanate > glycyrrhetinic acid > indole-3-carbinol > caffeic acid > phytic acid > vanillin > ellagic acid > D-saccharic acid 1,4-lactone. Vanillin, at slight to moderately toxic concentrations, was the only test agent that induced multinucleation in the 3T3 fibroblasts.

3T3 Cells↗
PubMed1982

Dendritic trees of neurones in the rat supraoptic nucleus.

The cytoarchitecture of the rat supraoptic nucleus was studied using the Golgi-Cox method with a neutral red or Cresyl violet counterstain to allow the limits of the nucleus to be determined accurately. A small number of stained neurones were seen in each of the brains sectioned but they were unevenly distributed in the supraoptic nucleus. There were more cells posteriorly and close to the optic tract. A minority of cells studied (a total of 4) were round (diameter approximately 20 micrograms) and multipolar and were interpreted as interneurones. The majority of stained cells studied (60) had oval cell bodies (mean long diameter 22.7 micrograms in coronal sections) with a single fine axon directed dorsally and medially and 1-3 heavier dendrites passing ventrally. Both cell bodies and dendrites of the major cell type showed spines and both dendrites and axons had some varicosities. The anatomy of the larger cell group (the presumed neurosecretory cells) is thus consistent with their having a substantial synaptic input and the presence of long dendrites (up to 205 micrograms) implies that caution is necessary in the interpretation of experiments where cell activity is monitored while active materials are placed near the cell bodies.

Animals↗
PubMed1984

Vagal and gastric connections to the central nervous system determined by the transport of horseradish peroxidase.

Horseradish peroxidase (HRP, Sigma Type VI) crystals were encased in a parafilm envelope and applied to the transected central ends of the left and right cervical vagus nerves and the anterior and posterior esophageal vagus nerves of adult male hooded rats. Injections of 30% HRP were made into the muscle wall of the fundus and antrum regions of the stomach. After 48 hr survival time, animals were perfused intracardially with a phosphate buffer plus sucrose wash followed by glutaraldehyde and paraformaldehyde fixative. The brain stem, spinal cord and corresponding dorsal root ganglia, superior cervical sympathetic ganglion, and the nodose ganglion were removed and cut into 50 micron sections. All tissue was processed with tetramethylbenzidine (TMB) for the blue reaction according to Mesulum and counterstained with neutral red. Sequential sections were examined under a microscope. Labeled neurons and nerve terminals were identified using bright and dark field condensers and polarized light. In tissue from animals that had HRP applied to the cervical vagus nerves, retrogradely labeled neurons were identified ipsilaterally in the medulla located in the dorsal motor nucleus of the vagus (DMN) and the nucleus ambiguus (NA). Labeled cells extended from the DMN into the spinal cord in ventral-medial and laminae X regions C1 and C2 of cervical segments. Many neurons were labeled in the nodose ganglion. Anterogradely labeled terminals were observed throughout and adjacent to the solitary nucleus (NTS) dorsal to the DMN and intermixed among labeled neurons located in the DMN. In tissue from animals that had HRP applied to the esophageal vagus nerves, similar labeling was observed. However, fewer neurons were identified in the NA, the nodose ganglion, and only in laminae X of the cervical spinal cord segments C1 and C2. Also, very little terminal labeling was observed in and adjacent to the NTS. Labeled neurons in tissue from animals that had HRP injected into the stomach wall were observed bilaterally in the DMN, nodose ganglion, and only in laminae X at the C1 and C2 levels of the spinal cord. Labeled neurons also were observed in the dorsal root ganglia of the thoracic cord. These data indicate that cervical cord and NA neurons are important in the supradiaphragmatic motor innervation by the vagus. Also, many afferents to the NTS originate above the diaphragm. In addition, some afferents from the stomach enter the central nervous system via the thoracic spinal cord.

Animals↗
PubMed1992

A tissue culture assay for direct detection of sodium channel blocking toxins in bacterial culture supernates.

A quantitative assay for sodium channel blocking toxins such as tetrodotoxin and saxitoxin has been developed for use with a microtitre plate reader. Mouse neuroblastoma cells, which die rapidly in the presence of ouabain and veratridine, were protected by tetrodotoxin; surviving cells were detected by their uptake of the vital dye Neutral red which was quantified with a microtitre plate reader at 540 nm. A sigmoidal dose response curve was obtained and tetrodotoxin concentrations were readily measured over the range 10 nM to 500 nM (3.2-160 ng/ml). With this method, sodium channel blocking toxins were detected directly, without processing or concentration, in culture supernates of several marine bacteria, including Shewanella alga, Alteromonas tetraodonis, Listonella (Vibrio) pelagia, V. alginolyticus, V. anguillarum and V. tubiashi. Culture supernates of Shewanella alga contained up to 510 ng/ml of sodium channel blocking toxin (using tetrodotoxin as a standard).

Animals↗
PubMed1989

Arsenic-selenium interactions determined with cultured fish cells.

A fibroblastic cell line derived from gill tissue (designated BG/G) and an epithelioid cell line derived from fin tissue (designed BG/F) of bluegill sunfish (Lepomis macrochirus) were used as the bioindicators in toxicity experiments. The neutral red in vitro cytotoxicity assay served as the endpoint. In both cell lines the sequence of observed cytotoxicity was arsenite greater than arsenate greater than selenite greater than selenate, with each cell type exhibiting comparable ranking of midpoint toxicity (NR50) concentrations. Antagonistic interactions were noted between combinations of arsenics and seleniums. Thus, selenate and selenite (at nontoxic levels) reduced, but did not eliminate, the acute cytotoxicities of arsenate and, to a lesser extent, of arsenite.

Animals↗
PubMed1992

Production of a toxic, novel mammalian metabolite of N-methylcarbazole predicted by a fungal cell model of mammalian metabolism.

The formation of N-hydroxymethylcarbazole (NHMC), carbazole, 1-hydroxy-N-methylcarbazole, 2-hydroxy-N-methylcarbazole, and 3-hydroxy-N-methylcarbazole as products of mammalian liver microsomal metabolism of N-methylcarbazole (NMC) has been documented by several investigators. In previous studies in our laboratory, the fungus Cunninghamella echinulata (ATCC 9244) produced two new metabolites, 3-hydroxy-N-hydroxymethylcarbazole (3-OH-NHMC), and 3-hydroxycarbazole (3-OH-carbazole), in addition to the known mammalian metabolites, NHMC and carbazole. One of the two novel metabolites isolated from the microbial models, 3-OH-NHMC, was also identified and characterized in rat liver microsomes by analytical (HPLC) and spectral (UV and NMR) comparisons with a reference standard. The two metabolites, 3-OH-NHMC and 3-OH-carbazole, were shown to be cytotoxic to cultured rat hepatocytes as assessed by lactate dehydrogenase (LDH) leakage and neutral red (NR) uptake. These studies demonstrate the prospective potential of microbial models for predicting the formation of metabolites from drugs and other xenobiotics in mammalian systems.

Animals↗
PubMed1994

Cytotoxicity of sulphur mustard on a human keratinocyte cell line: direct effects compared to conditioned-medium effects.

Direct toxic effects of sulphur mustard (SM) were compared to SM-conditioned medium effects in a human keratinocyte cell line. Cytotoxicity was evaluated by measurement of cell viability with the Neutral Red uptake assay. Culture directly exposed to SM exhibited cytotoxic dose-response curves after 24, 48 or 72 h. Inhibitory concentration 50 (IC50) appeared to be more than 10 times lower after 72 h than the corresponding value after 24 h. In contrast, no cytotoxic activity was observed in media which were collected 4 h or 24 h after SM exposure. Cell viability was unchanged even if observation was extended to 48 h. Results suggested that SM cytotoxicity was not due to potential mediators secreted by human keratinocytes.

Cell Line↗
PubMed1996

Induction of renin release from isolated glomeruli by inorganic mercury(II).

Mercury(II) ions are known to accumulate in the kidney and their effect upon the renin-angiotensin system has also been described. The question, however, whether mercury(II) also exerts direct effect on the juxtaglomerular cells (JGC) to induce renin release remained to be answered. Suspension of isolated glomeruli was used to measure the mercury(II)-induced renin release in vitro. The glomeruli were isolated from female BALBc mice. HgCl2 was found to be capable of inducing renin release directly from JGC. The effect is concentration-dependent (P < 0.05, r = 0.914 and P < 0.01, r = 0.982, with and without Neutral Red vital staining) and becomes apparent already at a mercury(II) ion concentration as low as 1 microM. The renin-releasing effect of the mercury ion is to be inhibited by dithiothreitol (DTT) (renin activity 20.37 vs. 2.60 ng/ml.h in supernatant) as well as the elevated osmotic concentration of the incubating bath medium (20.37 vs. 6.84 ng/ml.h). This suggests that certain membrane sulfhydryl groups are implicated in the process on the one hand, and it is also in accordance with the known sensitivity of the renin granules to osmotic pressure on the other hand. Light and electron micrographs also demonstrate the direct, effective role of Hg(II) in the renin release process. Therefore, it is assumed that apart from its influence on tubulo-glomerular feedback a direct way of action of mercury(II) on renin release must also be taken into account.

Animals↗
PubMed1991

Serotonin is released from isolated leech ganglia by potassium-induced depolarization.

1. The quantities of serotonin that are released from isolated leech ganglia in vitro were measured with the sensitive neurochemical techniques of HPLC-EC. 2. Segmental ganglia were exposed to elevated concentrations of potassium that depolarize leech serotonin-containing neurons by approximately 35 mV per decade. 3. Each segmental ganglion released on average 0.20 pmol of serotonin during 10 min of incubation in a solution containing 64 mM K+. 4. The rate of serotonin release increased nearly four-fold to 0.74 pmol/10 min when ganglia were incubated in 120 mM K+. 5. The rates of ganglionic serotonin release in 120 mM K+ were quantitatively similar in these three, experimentally important species of leeches: Hirudo medicinalis, Macrobdella decora and Haementeria ghilianii. 6. Ionic substitution experiments with the divalent cations Mg2+ and Co2+ indicated that the release of serotonin from leech ganglia is mediated by a Ca2+ dependent process. 7. The serotonin-uptake blockers, imipramine and chlorimipramine, did not increase the amount of serotonin released in elevated potassium. 8. Vitally staining the identified serotonin-containing neurons with Neutral Red dye did not reduce the quantity of serotonin that was released from the ganglia in elevated potassium. 9. This study demonstrates the capacity of leech ganglia to release the neurochemical serotonin, and the rates of transmitter release increase with the degree of depolarization of serotonin-containing neurons.

Animals↗
PubMed1988

Long-term cytotoxicity of orthodontic direct-bonding adhesives.

The purpose of this study was to examine the potential toxic effects of several orthodontic adhesives immediately after polymerization and at various time intervals up to 2 years postopolymerization by means of an in vitro overlay assay. Adhesive samples were incubated on tissue cultures containing an agar overlay with a vital dye (neutral red). Viable cells were stained red; nonviable cells were unstained. The circular pattern of nonviable cells demonstrated a zone of inhibition that was measured and compared. All materials tested showed cytotoxic effects immediately after polymerization and the toxic effect decreased with time postpolymerization. However, 2 years after initial polymerization, significant zones of inhibition indicating continued in vitro toxicity were still evident in all but one of the adhesives.

Adhesives↗
PubMed1993

Neurodevelopmental toxicity in vitro: primary cell culture models for screening and risk assessment.

Robust models for the evaluation of developmental toxicity are briefly reviewed with emphasis on embryonic brain and retina cells in vitro. Organ slice and aggregate cultures under constant gyratory movement as well as high cell density monolayer ("micromass") cultures are considered as robust models. An in vitro model using high cell density monolayer and re-aggregated cells isolated from embryonic chick brain (ED 6) is presented. Cell development and differentiation of the astrocytes and nerve cells are monitored by marker proteins and cytotoxicity was quantified by neutral red uptake and protein content. Four human teratogens, six possible human teratogens and six unlikely human teratogens were tested in brain and retina cells for their cytotoxic and morphologic effect. All 16 substances were classified correctly except the neurotoxicants MPTP and MPP+, both of which are strong dopaminergic toxicants in vitro as well as in humans and are therefore proposed to be classified as human neuroteratogens. Preliminary data on the lowest effect levels of four potential neurotoxicants (cadmium chloride, Ara-C, Phenytoin, MPTP) in chick brain aggregate cultures correlate surprisingly well with known toxic human plasma levels. Further validation has to be undertaken to confirm these promising results. A battery of such robust in vitro models is proposed that could cover neurodevelopmental toxicity of drugs and chemicals for screening and risk assessment purposes.

Abnormalities, Drug-Induced↗
PubMed1995

Antioxidant and cytotoxic tocopheryl quinones in normal and cancer cells.

We found previously that [d]-alpha-tocopherol (alpha-T) and [d]-gamma-tocopherol (gamma-T) are lipid antioxidants (thiobarbituric acid test) in model systems containing arachidonic acid (AA), cumene hydroperoxide, and Fe3+ and in smooth muscle cell (SMC) cultures challenged with AA. We now show that [d]-alpha-tocopherylquinone (alpha-TQ), [d]-delta-tocopherylquinone (delta-TQ), and [d]-gamma-tocopherylquinone (gamma-TQ) are antioxidants at low concentrations and prooxidants at high concentrations in the model system. Prooxidant activity is greater with gamma-TQ than either alpha-TQ or delta-TQ. Low concentrations of alpha-TQ, delta-TQ, and gamma-TQ are also antioxidants in SMC cultures challenged with AA. Unlike alpha-TQ, partially substituted gamma-TQ and glutathione (GSH) form a Michael adduct which has been purified and characterized. We found previously that alpha-T, gamma-T, and alpha-TQ are mitogenic in SMC. We now report that both delta-TQ and gamma-TQ but not alpha-TQ show concentration-dependent cytotoxicity (changes in morphology, propidium iodide stain) in SMC cultures. Cytotoxicity is greater with gamma-TQ than delta-TQ. An acute lymphoblastic leukemia (ALL) cell line shows greater chemosensitivity (MTT and Neutral Red assays) to gamma-TQ than to either doxorubicin (DOX) or vinblastine (VLB). An ALL cell line resistant to both DOX and VLB retains the same chemosensitivity to gamma-TQ as the drug-sensitive ALL cell line. ALL cell lines are unaffected by either alpha-TQ or the GSH Michael adduct of gamma-TQ. These data show that partially substituted tocopheryl quinones capable of forming Michael adducts are potential chemotherapeutic agents for multidrug-resistant cancer cells.

Animals↗
PubMed1993

Stable expression of human cytochrome P450 1A1 cDNA in V79 Chinese hamster cells and metabolic activation of benzo[a]pyrene.

A V79 Chinese hamster cell line stably expressing human cytochrome P450 1A1 (CYP1A1) was obtained by chromosomal integration of the human CYP1A1 cDNA under the control of the SV40 early promoter. Chromosomal integration was verified by Southern analysis, and effective transcription of the human CYP1A1 cDNA was demonstrated by Northern analysis. The CYP1A1 cDNA-encoded protein was characterized by Western analysis using anti-rat CYP1A1. Intracellular association of CYP1A1 with the endoplasmic reticulum could be visualized by in situ immunofluorescence. Crude cell lysates of the V79 derived cell line was able to catalyze 7-ethoxyresorufin-O-deethylation (EROD) with an activity of about 50 pmol min-1 mg-1 total protein, and an aryl hydrocarbon hydroxylase activity (AHH) of 25 pmol min-1 mg-1. CYP1A1 dependent cytotoxicity, measured by neutral red uptake, and genotoxicity, determined by the frequency of micronucleus formation, of benzo[a]pyrene (B[a]P) and trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (B[a]P-7,8-diol) could be demonstrated at substrate concentrations as low as 10 nM. Thus, this cell line presents a sensitive tool for studying CYP1A1 mediated metabolism of polycyclic aromatic hydrocarbons (PAH). B[a]P and the purified (+)- and (-)-enantiomers of B[a]P-7,8-diol were compared for their mutagenicity. The (-)-enantiomer was found to be 3-5-fold more mutagenic than the (+)-enantiomer.

Animals↗
PubMed1995

A silicon-based biosensor for real-time toxicity testing in normal versus cancer liver cells.

The potentiometric alternating biosensor (PAB) system has been utilized to monitor the effects on normal versus cancer cells of Mitoxantrone, a potent inhibitor of nucleic acid synthesis commonly utilized in the clinical treatment of hepatocarcinomas. In this first series of toxicity tests, we have utilized a primary culture of rat hepatocytes and a stabilized line named HepG2 from a human hepatoma. Primary cultures of hepatocytes represent a unique tool in toxicology/pharmacology as shown in previous works. A suitable microvolume flow chamber has been designed and produced. The chamber is equipped with inlet and outlet circuits and with a fixed transducer (Si/SiO2/Si3N4 chip) facing a cover slip on which cells grow. The PAB system is here utilized only in the pH-sensitive configuration with a single measuring spot, warranting an accurate determination of the change in the extracellular acidifcation rate resulting from drug administration. We have also observed the changes in the acidification rate of 3T6 mouse fibroblasts, induced by a treatment with Ara-C, a well-known DNA antimetabolite. New insights can be obtained from these studies into the drug response of normal versus cancer cells and into the feasibility of real-time PAB monitoring of drug toxicity and efficacy, towards effective human cancer treatment. The effect of increasing drug concentrations on cellular metabolism is here compared with the results obtained from conventional tests (optical microscopy, Neutral Red and Trypan Blue assays).

Animals↗