Reduction Of Tetrazolium Salt Research Articles

The Effect of Detergents on the Reduction of Tetrazolium Salts

Detergents, such as Triton X-100, markedly increase the reduction of tetrazolium salts by xanthine oxidase plus xanthine, or by NADH. This effect of detergent, in the case of the xanthine oxidase catalyzed process, is seen aerobically but not anaerobically. Increasing the rate of accumulation of formazan, whether by increasing the concentration of the tetrazolium salt or by adding detergent, decreased susceptibility to inhibition by superoxide dismutase or by O2. These results are accommodated by a scheme of reactions the essence of which is the univalent reduction of the tetrazolium to an uncharged tetrazoinyl radical which can reduce O2to[formula]or which can partition into the detergent micelles and there dismute to generate the stable formazan.

Interleukin-1α and -β modulation of luteinized human granulosa cell oestrogen and progesterone biosynthesis

This study was designed to test the hypothesis that interleukin-1 alpha (IL-1 alpha) and beta directly affect progesterone, and oestradiol production in cultures of purified human granulosa cells. Luteinized granulosa cells were obtained from women during in-vitro fertilization cycles. Granulosa cells with and without associated white blood cells were cultured in the presence of IL-1 alpha and IL-1 beta (0.5-50 ng/ml) for 48 h. Media were changed at 24 h intervals and assayed for progesterone and oestradiol. In separate experiments, granulosa cell viability was assessed with the tetrazolium salt reduction assay, haemocytometer cell counts, and Trypan blue dye exclusion. Our results indicate that progesterone synthesis by basal and human chorionic gonadotrophin (HCG)-stimulated granulosa cells co-cultured with white blood cells was inhibited by 5.0 ng/ml of IL-1 alpha and IL-1 beta at 48 h of culture. In the presence of white blood cells, granulosa cell oestradiol synthesis was inhibited by IL-1 beta but not IL-1 alpha. Oestradiol was inhibited after both 24 and 48 h of culture and was maximally affected by 5.0 ng/ml of IL-1 beta. In contrast, basal and HCG-stimulated oestradiol production by granulosa cells cultured free of white blood cells was inhibited only by IL-1 alpha. IL-1 alpha at 5.0 ng/ml produced maximal inhibition of basal oestradiol (57%) and HCG-stimulated oestradiol (41%) production at 48 h of culture. Gonadal steroid inhibition by IL-1 alpha and IL-1 beta was not mediated through cytotoxic or antiproliferative effects on granulosa cells. Specificity of the granulosa cell response to IL-1 alpha and IL-1 beta was demonstrated by abrogation of steroid inhibition with anti-IL-1 alpha and IL-1 beta neutralizing antibodies. In conclusion, IL-1 alpha directly inhibited the production of oestradiol by human ovarian granulosa cells. IL-1 alpha and IL-1 beta also exerted indirect effects on steroid production via white blood cells that are usually present in granulosa cell cultures if steps are not taken to remove them. These data support the hypothesis that cytokines play an important role in intra-ovarian regulation of steroid biosynthesis.

Photoelectrochemical Reduction of Tetrazolium Salts to Formazans on Surfaces of Semiconductor Powders in Alcohol Solutions

Abstract The formation of colored formazans from the reduction of colorless tetrazolium salts was found to occur on the surfaces of photoexcited TiO2 and ZnO powders in ethanol solutions. The yields of the conversions of 2,3,5-triphenyltetrazolium chloride to 1,3,5-triphenylformazan and of 2,5-diphenyl-3-(l-naphthyl)tetrazolium chloride to 1,3-diphenyl-5-(l-naphthyl)formazan were 41.6% and 40.0%, respectively, after a 25-min UV irradiation in the TiO2 suspension, whereas they were 55.8% and 35.2%, respectively, after a 60-min UV irradiation in the ZnO suspension. These reactions proceeded via photoelectrochemical reduction by the electrons from the conduction bands of the semiconductors, but were quenched in the presence of oxygen or Ag+ ions.

Alcohol solutions of triphenyl-tetrazolium chloride as high-dose radiochromic dosimeters

The radiolytic reduction of colorless tetrazolium salts in aqueous solution to the highly colored formazan dye is a well-known acid-forming radiation chemical reaction. Radiochromic thin films and three-dimensional hydrocolloid gels have been used for imaging and mapping absorbed dose distributions. The high solubility of 2,3,5-triphenyl-tetrazolium chloride (TTC) in alcohols provides a useful liquid dosimeter (45 mM TTC in aerated ethanol) and shows a linear response of absorbance increase (λmax = 480 nm) with dose over the range 1–16 kGy. The linear molar absorption coefficient (ɛm) for the formazan at the absorption peak is 1.5 × 103 m2 mol−1, and the radiation chemical yield for the above solution is G (formazan) = 0.014 μmol J−1. The irradiation temperature coefficient is about 0.8 percent per degree Celsius rise in temperature over the temperature range 0–30 °C but is much larger between 30° and 60 °C. The unirradiated and irradiated solutions are stable over at least five days storage at normal laboratory temperature in the dark, but when stored in daylight at elevated temperature, the unirradiated solution in sealed amber glass ampoules undergoes slow photolytic dye formation, and the irradiated solution experiences initial fading and subsequent reversal (photochromism) when exposed to direct sunlight.

Oxidative metabolism in nonculturable Helicobacter pylori and Vibrio vulnificus cells studied by substrate-enhanced tetrazolium reduction and digital image processing

Growing and nonculturable cells of Helicobacter pylori and Vibrio vulnificus were studied for the capacity to reduce tetrazolium salts in order to elucidate the possible physiological basis for the proposed "viable but nonculturable" (VNC) state. Initial difficulties in obtaining consistent reduction of rho-iodonitrotetrazolium violet (INT) by H. pylori led us to develop a method for studying the effect of adding exogenous substrates on these reactions. The established procedure provided a profile of substrate enhancement of oxidative activity revealed by INT reduction which was related to both the identity and physiological state of the organism studied. Representation and interpretation of these enhancement profiles were facilitated by digital image processing. Nonculturable cells of H. pylori produced by carbon and nitrogen starvation in air lost all INT-reducing capacity in 24 h when stored at 37 degrees C, while 99% of those produced at 4 degrees C retained oxidative activity for at least 250 days when tested in the presence but not in the absence of succinate, alpha-ketoglutarate, or aspartate. Activity was detected at similar levels in cells with coccoid and spiral shapes. In contrast, only 1% of nonculturable cells of V. vulnificus, produced under conditions previously reported to induce the VNC state in this organism, retained intrinsic INT-reducing capacity; no substrate-enhanced activity occurred in the remainder of the population. Thus, there was no common pattern of oxidative activity indicative of a VNC state in both test organisms. Nonculturable cells of H. pylori can retain several different oxidative enzyme activities; whether these indicate viability or the persistence of cells as "bags of enzymes" remains to be established.

Cytotoxicity in vitro of organotin compounds to fish hepatoma cells PLHC-1 ( Poeciliopsis lucida)

Cytotoxicity in vitro to fish hepatoma cells PLHC-1 has been analyzed for a series of 21 organotin compounds consisting of all degrees of alkylation and arylation. The sensitivity of the neutral red (NR) assay and the tetrazolium salt reduction (MTT) assay was similar for most of the organotin compounds. Cytotoxic effects were found at concentrations between 10 −8 M and 10 −2 M. For various trisubstituted organotin compounds, including tributyltin and triphenyltin, which are used in antifouling paints and as pesticides, respectively, cytotoxic concentrations in the range of 10 −8 M to 10 −6 M were observed. Based on the concentration reducing NR uptake by 50% (NR 50), the sequence of Cytotoxicity amongst butyltins was tributyltin ⪢ bis(tributyl)-tin ⪢ dibutyltin ⪢ tetrabutyltin ⪢ butyltin ⪢ tin(IV). Tributyltin induced effects on cell functions quickly, as a reduction in NR uptake by 30% was recorded after 30 min exposure to 4 · 10 −7 M. The ranking order in Cytotoxicity in the MTT assay of phenylated organotins was triphenyltin ⪢ diphenyltin ⪢ phenyltin ⪢ tin(IV). Cytotoxic concentrations of tributyltin and triphenyltin measured in the bromodeoxyuridine (BrdU) assay and with the crystal violet (CV) staining method do not differ significantly from those determined in the NR and MTT assay. Tri-and disubstituted organotin compounds exhibit a significant correlation between the noctanol/water partition coefficient (logK ow) and the NR 50 ( n = 10, r = 0.86, P = 0.001) and the MTT 50 values ( n = 11, r = 0.85, P = 0.001), respectively. In addition, good qualitative correlations between in vitro Cytotoxicity data and in vivo fish toxicity data were found (for NR assay n = 8, r = 0.86, P = 0.001; for MTT assay n = 9, r = 0.80, P = 0.001). he results indicate that the in vitro Cytotoxicity assays using PLHC-1 cells are useful tools for the estimation of the acute toxicity to fish of organotins and possibly other compounds.

Interferon gamma inhibits luteinized human granulosa cell steroid production in vitro

OBJECTIVE: The purpose of this study was to determine whether interferon gamma affects luteinized human granulosa cell progesterone, estrone, and estradiol production in the presence and absence of associated white blood cells by either cytotoxic or antiproliferative mechanisms. STUDY DESIGN: Luteinized granulosa cells were isolated by Percoll centrifugation from women during in vitro fertilization cycles. Some cell suspensions were further treated with anti-CD45 magnetic immunobeads to remove associated white blood cells. Granulosa cells with and without white blood cells were cultured in the presence of interferon gamma (0.5 to 50 ng/ml) for 48 hours. Medium was changed at 24-hour intervals, and spent medium was assayed for progesterone, estrone, and estradiol. In separate experiments granulosa cell viability was assessed with the tetrazolium salt reduction assay. RESULTS: Interferon gamma significantly inhibited granulosa cell progesterone production in both basal and human chorionic gonadotropin stimulated cells cocultured with white blood cells in a concentration-dependent manner, whereas cells cultured free of white blood cells demonstrated less inhibition. In the absence of interferon gamma a more profound increase in granulosa cell progesterone synthesis was found in human chorionic gonadotropin-stimulated cultures without associated white blood cells. Interferon gamma inhibited granulosacell estrone and estradiol production in basal cultures containing white blood cells in both a time- and concentration-dependent manner. Estrone production was not affected by interferon gamma in human chorionic gonadotropin-stimulated granulosa cell cultures containing white blood cells, whereas estradiol secretion was decreased at 48 hours with 50 ng/ml interferon gamma. Both estrone and estradiol synthesis were inhibited by 50 ng/ml interferon gamma in granulosa cel cultures free of white blood cells. In cultures free of interferon gamma, granulosa cell estrone and estradiol secretion was not affected by human chorionic gonadotropin stimulation compared with basal controls regardless of the presence or absence of white blood cells. All concentrations of interferon gamma used had no effect on granulosa cell viability at any time point tested. CONCLUSION: Our data suggest that interferon gamma affects granulosa cell steroid production both independently and in synergy with associated white blood cells and further supports the hypothesis that interferon gamma may be an important intraovarian regulator of ovarian steroid production during the luteal phase.

Neutrophil NADPH oxidase activity in chronic myeloproliferative and myelodysplastic diseases by microscopic and photometric assays.

Neutrophil NADPH oxidase is a membrane-bound enzyme complex responsible for the reduction of oxygen to superoxide anion in the respiratory burst. Impaired neutrophil function has often been reported in myeloproliferative diseases and myelodysplastic syndromes (MDSs), but its laboratory features have not been well characterized. Traditionally, NADPH oxidase activity has been evaluated with a microscopic method by nitroblue tetrazolium salt reduction to blue-black insoluble formazan granules identified in positive neutrophils by microscopy. We investigated neutrophil NADPH oxidase in 22 patients with chronic myeloproliferative disorders (cMPDs) and 15 patients with MDSs, using the microscopic method as well as a photometric microplate assay, which monitored the cytochemical reaction for 30 min. The relationship between cMPD and patient susceptibility to infections was also investigated. In the photometric assay, the mean enzyme activity in MDSs and cMPDs was lower than in normal subjects. NADPH oxidase activity was greater in cMPDs (except myelofibrosis) than in MDSs (except chronic myelomonocytic leukemia), and these differences were less evident with microscopy. Moreover, for cMPDs, patients with susceptibility to infections showed a lower NADPH oxidase activity than patients without infections.

Multiple Use of Blotted Polyvinylidene Difluoride Membranes Immunostained with Nitro Blue Tetrazolium

A simple procedure for reusing polyvinylidene difluoride membranes with bound proteins reacted with antibodies and developed by the nitro blue tetrazolium method (M. S. Blake, K. H. Johnston, G. J. Russell-Jones, and E. C. Gotschlich (1984) Anal. Biochem. 136, 175-179) is described. The blue diformazan precipitate produced by the reduction of the nitro blue tetrazolium salt was dissolved by soaking the membrane in dichloro- or trichloromethane for 10-60 min at room temperature. After equilibration in methanol and then in distilled water the first and second antibodies were removed by incubating the membrane in 0.2 M glycine-HCl buffer, pH 2.2, for 90 min at room temperature. These cleaned membranes could be successively reused with different monoclonal antibodies or preimmune serum up to 21 times. The intensity of the spots or electroblotted bands and their specific reaction pattern with different monoclonal antibodies remained unchanged, and no background was observed when the membranes were reacted with preimmune serum. This procedure makes it possible to reuse membranes freshly developed or stored for long periods without loss of resolution.

Survival and Recovery of Methanotrophic Bacteria Starved under Oxic and Anoxic Conditions

The effects of carbon deprivation on survival of methanotrophic bacteria were compared in cultures incubated in the presence and absence of oxygen in the starvation medium. Survival and recovery of the examined methanotrophs were generally highest for cultures starved under anoxic conditions as indicated by poststarvation measurements of methane oxidation, tetrazolium salt reduction, plate counts, and protein synthesis. Methylosinus trichosporium OB3b survived up to 6 weeks of carbon deprivation under anoxic conditions while maintaining a physiological state that allowed relatively rapid (hours) methane oxidation after substrate addition. A small fraction of cells starved under oxic and anoxic conditions (4 and 10%, respectively) survived more than 10 weeks but required several days for recovery on plates and in liquid medium. A non-spore-forming methanotroph, strain WP 12, displayed 36 to 118% of its initial methane oxidation capacity after 5 days of carbon deprivation. Oxidation rates varied with growth history prior to the experiments as well as with starvation conditions. Strain WP 12 starved under anoxic conditions showed up to 90% higher methane oxidation activity and 46% higher protein production after starvation than did cultures starved under oxic conditions. Only minor changes in biomass and morphology were seen for methanotrophic bacteria starved under anoxic conditions. In contrast, starvation under oxic conditions resulted in morphology changes and an initial 28 to 35% loss of cell protein. These data suggest that methanotrophic bacteria can survive carbon deprivation under anoxic conditions by using maintenance energy derived solely from an anaerobic endogenous metabolism. This capability could partly explain a significant potential for methane oxidation in environments not continuously supporting aerobic methanotrophic growth.

Effect of High Glucose on Cellular Proliferation and Lipid Peroxidation in Cultured Vero Cells

Nephropathy is common among the diabetic population. However, the molecular mechanisms by which hyperglycemia can cause alteration in kidney structure and function is not known. In this study, we examined the effect of high glucose levels on cellular growth and membrane lipid peroxidation in Vero cells, an African green monkey kidney cell line. Cell growth was assessed by a tetrazolium salt reduction test (MTT); and lipid peroxidation was measured by thiobarbituric acid reactivity. Our results show that elevated levels of glucose (25, 50 and 100 mM) can cause up to 50% reduction in cell growth in cultured Vero cells as compared with controls (8 mM). Also, we observed a significant increase in the amount of oxidative damage in Vero cells cultured with elevated glucose concentrations. This study demonstrates that hyperglycemia can cause increased lipid peroxidation and affect the cellular proliferation in a monkey kidney cell line.

On the mechanism of the multistep reduction of tetrazolium salts with special reference to the involvement of tetrazolium radicals

Thin layer chromatography (TLC) was performed to analyze which products are formed when unstable tetrazolinyl radicals are generated during the reduction of tetrazolium salts under physiological conditions of biochemical and histochemical assays. It was found in aqueous media that irrespective the assay conditions, reduction of 2,3-di(4-nitrophenyl)-5-phenyl-2H-tetrazolium chloride (p-DNTTC) or 2,3-di(4-nitrophenyl)-5-t-butyl-2H-tetrazolium chloride (t-butyl-DNTC) always lead to production of formazan and never to generation of the corresponding 2(4-nitrophenyl)-5-phenyl- (respectively t-butyl-) tetrazoles by splitting off a nitrophenyl side chain from the tetrazolium ring as a cation. This reaction is known to occur in non-aqueous media (Neugebauer, 1973). Because p-DNTTC is analogous to tetrazolium salts that are used in biochemical and histochemical assays, it is concluded that reduction of tetrazolium salts leads to formation of formazans only under these conditions.

Enhancement of growth hormone bioactivity by zinc in the eluted stain assay system

The effects of ionic zinc (Zn2+) on human (h) GH bioactivity have been examined using a lactogenic bioassay. The potencies of pituitary-derived hGH (IRP 80/505), recombinant 22K hGH (IRP 88/624), pituitary-derived human PRL (IRP 84/500), and a recombinant methionyl 20-kilodalton variant of hGH in the presence of selected concentrations of ZnCl2 were investigated with an eluted stain assay that uses Nb2 rat lymphoma cells. This precise colorimetric bioassay is based upon the reduction of a yellow tetrazolium salt, 3-[4,5-dimethyl-thiazol-2-yl]2,5-di-phenyl-tetrazolium bromide, to its purple formazan by lactogen-activated Nb2 cells. Zinc (6-100 microM) enhanced the bioactivity of low doses (< 0.045 nM) of both pituitary-derived and recombinant 22K hGH, although the magnitude of enhancement was considerably less than might have been anticipated from previous binding studies (13). Higher concentrations of pituitary-derived hGH (> 0.045 nM) were inhibited by Zn2+. The bioactivity of recombinant methionyl 20K hGH was greatly enhanced by zinc (3-100 microM). In contrast to hGH, the bioactivity of hPRL was not potentiated by Zn2+. These discriminatory effects of Zn2+ when stimulating via the lactogenic receptor are in concordance with the results of previous radioligand binding studies (13). The striking enhancement of 20K hGH lactogenic bioactivity was observed at Zn2+ concentrations within the physiological range for normal human serum (5-20 microM).

Enhancement of growth hormone bioactivity by zinc in the eluted stain assay system.

The effects of ionic zinc (Zn2+) on human (h) GH bioactivity have been examined using a lactogenic bioassay. The potencies of pituitary-derived hGH (IRP 80/505), recombinant 22K hGH (IRP 88/624), pituitary-derived human PRL (IRP 84/500), and a recombinant methionyl 20-kilodalton variant of hGH in the presence of selected concentrations of ZnCl2 were investigated with an eluted stain assay that uses Nb2 rat lymphoma cells. This precise colorimetric bioassay is based upon the reduction of a yellow tetrazolium salt, 3-[4,5-dimethyl-thiazol-2-yl]2,5-di-phenyl-tetrazolium bromide, to its purple formazan by lactogen-activated Nb2 cells. Zinc (6-100 microM) enhanced the bioactivity of low doses (< 0.045 nM) of both pituitary-derived and recombinant 22K hGH, although the magnitude of enhancement was considerably less than might have been anticipated from previous binding studies (13). Higher concentrations of pituitary-derived hGH (> 0.045 nM) were inhibited by Zn2+. The bioactivity of recombinant methionyl 20K hGH was greatly enhanced by zinc (3-100 microM). In contrast to hGH, the bioactivity of hPRL was not potentiated by Zn2+. These discriminatory effects of Zn2+ when stimulating via the lactogenic receptor are in concordance with the results of previous radioligand binding studies (13). The striking enhancement of 20K hGH lactogenic bioactivity was observed at Zn2+ concentrations within the physiological range for normal human serum (5-20 microM).

Assay of Succinate Dehydrogenase Activity by a Colorimetric-Continuous Method Using Iodonitrotetrazolium Chloride as Electron Acceptor

A spectrophotometric assay method for determining succinate dehydrogenase activity is described in which iodonitrotetrazolium chloride is used as a final electron acceptor. The enzyme activity is determined by measuring the formation of formazan due to the tetrazolium salt reduction. The assay is continuous, rapid, simple, and sensitive, and may be used in the determination of enzyme activity either in tissue homogenates or as a marker of the mitochondrial fraction in cell fractionation procedures.

THE EFFECTS OF ZINC ON LACTOGEN STIMULATION OF Nb2 CELLS USING AN ELUTED STAIN ASSAY (ESTA)

Ionic zinc (Zn) has been reported to enhance binding of human growth hormone (hGH), but not prolactin (PRL), to the human PRL receptor at a concentration of ~50uM (1). We have investigated the effect of zinc on hGH bioactivity using a lactogenic bioassay. The potencies of selected doses of both hGH and PRL in the presence of increasing concentrations of ZnCl2 were investigated with an Eluted Stain Assay (ESTA) which used Nb2 cells. This colorimetric bioassay is based upon the reduction of a yellow tetrazolium salt, MTT, to its purple formazan by lactogen-activated Nb2 cells. Zinc enhanced the bioactivity of hGH but not PRL. Progressive enhancement of a low dose (0.5mU/L; 9pM) of hGH (IRP 80/505) was observed over the Zn concentration range of 6-100uM; higher Zn concentrations were inhibitory. Potentiation of bioactivity was observed only with low doses of hGH; with >2.5mU hGH/L, 50uM Zn inhibited the response. The bioactivity of PRL was consistently inhibited by 50uM Zn (PRL range 0.2-50mU/L; 0.4-105pM), inhibition being greater with higher PRL doses. We conclude that in this precise and sensitive bioassay, zinc has a differential effect on hGH and PRL bioactivities which concords with the radioligand studies but potentiation of bioactivity was only observed with low hGH doses. It was optimal at ~50uM Zn but was far less than might have been anticipated from the binding studies, when the affinity increased by 8000-fold. This discrepancy was not explained by significant endogenous Zn in the bioassay medium (only 2uM by Atomic Absorption Spectroscopy). Our findings therefore do not support the earlier suggestion (1) that Zn is crucial for the lactogenic bioactivity or hGH. (1) Cunningham B.C, Bass S, Fuh G, Wells JA 1990 Science 250:1709-1712.

Cytotoxicity of 28 MEIC Chemicals to Rat Hepatocytes using Two Viability Endpoints: Correlation with Acute Toxicity Data in Rat and Man

28 of the chemicals on the MEIC list were tested on rat hepatocytes in culture. Firstly, the metabolic capacities of the cell model were characterised, i.e. cytochrome P450 content and conjugation activities. Two independent endpoints, MTT (tetrazolium salt) reduction (for mitochondrial integrity) and neutral red uptake (NRU, for lysosomal integrity), were then used to measure the cytotoxicities of the test compounds after incubation with the cells for 24 hours. The relative toxicities of the test chemicals were measured by the determination of IC50 values for each parameter. Statistical analysis revealed a correlation between the results obtained in the two assays. The NRU assay, however, proved to be more sensitive than the MTT reduction assay. When the in vitro results were compared with those obtained from an in vivo data bank, good agreement was found with the acute toxicities of the test products in rats, with the exception of thioridazine, malathion, copper sulphate (all overestimated) and potassium cyanide (underestimated). In addition, a good correlation between basal cytotoxicity values for rat hepatocytes and LD50 for rats or lethal doses for humans was observed. These results suggest that the MTT reduction and/or NRU assays could be useful indicators of the cytotoxic potential of chemicals in rat hepatocyte cultures and thus provide information on the intrinsic lethal toxicity of compounds and their metabolites to rats and humans.

The role of oxygen in the reduction of tetrazolium salts with nadh mediated by 5-methyl phenazonium methyl sulfate. An EPR and voltammetric study

The reduction of tetrazolium salts (namely INT, MTT and BNT) with NADH in the presence of catalytic amounts of PMS (5-methylphenazonium methylsulfate) was studied, and the influence of oxygen on the system was considered. The redox potentials of all the investigated compounds, the kinetic measurements, as well as the application of the Marcus theory confirmed that PMS and tetrazolium ions are reduced by NADH through an inner-sphere mechanism. The EPR investigations led to the detection of all possible radical species coming from PMS and tetrazolium ions and are in agreement with a mechanism which excludes any role of oxygen in the system NADH/PMS/tetrazolium for the formation of formazan.

底泥中のマイクロサイトの分布構造に関する実験的研究

Micro-structure in sediments of a tidal river, which is composed of microsites of various ranges of Eh level, is experimentally investigated. At first, the cytochrome oxidase activity in the sediments is measured to study the distribution of aerobic microorganisms. Sediments with a high ignition loss are high in the activity and dense in aerobic microsites down to 5mm from the water-sediment interface. On the other hand, those with a low ignition loss are low in the activity and rather disperse in aerobic microsites down to 4cm from the interface. Secondly, the reduction of tetrazolium salts in the sediments is examined to obtain distributions of reduced microsites. The results indicate that they exist patchy in the oxic and anoxic parts of the sediments and their spacial ratio increases with depth. Finally, Eh in the sediments is measured with handmade micro-electrodes with 17μm in tip diameter and 500μm in length, whose dimension is the same size or smaller than the average size of the microsites. The values of Eh measured in vertical and horizontal planes of the sediments range from positive to negative values.

Cutaneous toxicity testing in organ culture: Neutral red uptake and reduction of tetrazolium salt (MTT)

Rabbit full thickness skin can be maintained viable for 7 days using a two-compartment organ culture model. We report that this model is useful for assessing cutaneous toxicity. 11 chemicals were each applied topically and then cytotoxicity was determined using two different assays. Mitochondrial activity was assessed by measuring reduction of the tetrazolium salt MTT, while uptake of the vital dye neutral red was used as a parameter of membrane damage. Conversion of MTT was inhibited in a dose-dependent way by all of the chemicals tested except dimethyl sulphoxide. Furthermore, when the skin explants were kept in culture after exposure to the test agents, both repair of initial damage and delayed toxicity could be observed to some extent. Uptake of neutral red was affected dose-dependently by six out of the 11 chemicals tested. The data indicate that a strong correlation exists between the cytotoxicity of chemicals and their effect on the conversion of MTT in skin organ culture. The MTT assay reported here offers the possibility of studying both acute and delayed cytotoxicity, and to some extent, recovery from initial damage.