Reduction Of Tetrazolium Salt Research Articles

Applicability of tetrazolium salts for the measurement of respiratory activity and viability of groundwater bacteria

A study was undertaken to measure aerobic respiration by indigenous bacteria in a sand and gravel aquifer on western Cape Cod, MA using tetrazolium salts and by direct oxygen consumption using gas chromatography (GC). In groundwater and aquifer slurries, the rate of aerobic respiration calculated from the direct GC assay was more than 600 times greater than that using the tetrazolium salt 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl tetrazolium chloride (INT). To explain this discrepancy, the toxicity of INT and two additional tetrazolium salts, sodium 3′-[1-(phenylamino)-carbonyl]-3,4-tetrazolium]-bis(4-methoxy-6-nitro) benzenesulfonic acid hydrate (XTT) and 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), to bacterial isolates from the aquifer was investigated. Each of the three tetrazolium salts was observed to be toxic to some of the groundwater isolates at concentrations normally used in electron transport system (ETS) and viability assays. For example, incubation of cells with XTT (3 mM) caused the density of four of the five groundwater strains tested to decline by more than four orders of magnitude. A reasonable percentage (>57%) of cells killed by CTC and INT contained visible formazan crystals (the insoluble, reduced form of the salts) after 4 h of incubation. Thus, many of the cells reduced enough CTC or INT prior to dying to be considered viable by microscopic evaluation. However, one bacterium ( Pseudomonas fluorescens) that remained viable and culturable in the presence of INT and CTC, did not incorporate formazan crystals into more than a few percent of cells, even after 24 h of incubation. This strain would be considered nonviable based on traditional tetrazolium salt reduction assays. The data show that tetrazolium salt assays are likely to dramatically underestimate total ETS activity in groundwater and, although they may provide a reasonable overall estimate of viable cell numbers in a community of groundwater bacteria, some specific strains may be falsely considered nonviable by this assay due to poor uptake or reduction of the salts.

Rat Neutrophil Phagocytosis Following Feed Restriction

Migration to zymozan and spontaneous migration of neutrophils, ingestion of small hydrophilic particles (HEMA) or large cells (Saccharomyces cerevisiae) and reduction of nitroblue tetrazolium salts (NBT) were investigated in rats with defined food restriction; animals fed ad libitum served as controls. All animals of a specific-pathogen-free grade were housed under constant temperature, humidity, light/dark regime. Stimulated migration to zymozan did not significantly change in animals with slight feed restriction, but it was reduced following both moderate and the greatest restriction. Diet restriction did not significantly influence spontaneous migration of neutrophils. Both chemotactic indices and differentials fall in the first instance but regenerate afterwards. Phagocytic activity of neutrophils diminished during the first two weeks of feed restriction, but a statistically significant decrease was observed only in the group with the greatest restriction (reduction to 20% of normal feed) in the assay using large cells. Phagocytic activity at the end of the experiment, i.e. after 4 weeks of restriction was not significantly different when compared with controls although body weight reduced markedly. The phagocytic index using small particles dropped only after 4 weeks of restriction, whereas in the assay using large cells a decline of phagocytic index was observed after two weeks in the greatest restriction and after four weeks in both restricted groups. Reduction of tetrazolium salts was significantly elevated after both moderate and severe diet restriction, this elevation persisted until the end of the study. Rules for the appropriate prediction of undesirable effects of xenobiotics on phagocytosis from preclinical to clinical studies are discussed.

Genotoxicity of 2-alkylcyclobutanones, markers for an irradiation treatment in fat-containing food—Part I: cyto- and genotoxic potential of 2-tetradecylcyclobutanone

Previous experiments had indicated a slight genotoxic potential both in rat and in human colon cells of a sample of 2-dodecylcyclobutanone, a compound formed by irradiation of food containing palmitic acid in its triglycerides. Up to date, there is no evidence that 2-alkylcyclobutanones occur in non-irradiated foodstuffs, consequently it is prudent to test several members of the class of 2-alkylcyclobutanones which are produced by treatment of fat-containing food with ionising radiation. In this work, 2-tetradecylcyclobutanone (derived from stearic acid) has been tested for its cytotoxic and genotoxic potential. Human colon tumor cell lines, i.e. HT 29 and HT 29 clone 19A, were employed as models for in vitro experiments for cytotoxicity and genotoxicity tests. Cytotoxicity was measured by tetrazolium salt reduction assays (MTT and WST-1) and genotoxicity by determining DNA damage using the Comet Assay. Neither cytotoxic nor genotoxic effects were induced by 2-TCB in HT 29 or HT 29 cl 19A cells at an incubation time of 30 min at 37°C, not even at the highest concentration (400 μM) tested. After prolonged incubation times (1–2 days) at higher concentrations (>50 μM) cytotoxicity did, however, appear. Studies on other 2-alkylcyclobutanones are in progress.

Development of a synchronous enzyme-reaction system for a highly sensitive enzyme immunoassay.

A synchronous enzyme-reaction system using water-soluble formazan and a non-enzymatic electron mediator was developed and applied to an enzyme immunoassay (EIA). The reaction system consists of four steps: (I) dephosphorylation of NADP(+) to produce NAD(+) by alkaline phosphatase (ALP), (II) reduction of NAD(+) to produce NADH with oxidation of ethanol to yield acetaldehyde by alcohol dehydrogenase (ADH), (III) reduction of water-soluble tetrazolium salt (WST-1) to produce formazan by NADH via 1-methoxy-5-methyl-phenazinium methyl sulfate (PMS), and (IV) re-reduction of NAD(+) to produce NADH by ADH. During each cycle, one molecule of tetrazolium is converted to one molecule of formazan. The concentration of formazan during the reaction was given by second-order polynomials of the reaction time. Kinetic studies strongly suggested that the synchronous enzyme-reaction system had the potential to detect an analyte at the attomole level in EIA. On the basis of the kinetic studies, optimal conditions for EIA incorporating the synchronous system were examined. NADP(+) was purified thoroughly to remove minor traces of NAD(+) in the preparation, and an ADH preparation contaminated with the lowest level of ALP activity was used. When the synchronous system was applied to a sandwich-type EIA for human C-reactive protein, the protein was detected with a sensitivity of 50 attomole per well of a micro-titer plate (0.1 ml) in a 1-h reaction. In addition, EIA with water-soluble formazan showed a more quantitative and sensitive result than that with insoluble formazan. These findings indicated that the (WST-1)-PMS system introduced in this study has a great potential for highly sensitive enzyme immunoassay.

Sensitivity of hybrid ovine/rat GH receptors to oGH and rat GH in transfected FDC-P1 mouse myeloid cells in vitro.

We have described previously the properties of two mutant ovine growth hormone receptor extracellular domain (oGHR-ECD) proteins which were created by substituting sequences from the rat GHR at two different locations within the framework of the oGHR-ECD. The first mutation occurred at a region close to the N-terminus of oGHR-ECD between residues Thr 28 and Leu34 and created the protein T28E/N29S/N33K/L34P-oGHR-ECD, where the ovine specific residues T, N, N and L are replaced by their equivalent residues E, S, K and P from the rat protein. This site lies N-terminal to the first element of beta-strand structure in the GHR-ECD and we designated this protein as Site-A mutant. The second mutation was made between residues Serl21 andAsp 124 of oGHR-ECD to produce the protein S121T/E123D/D124E-oGHR-ECD where ovine specific residues are again replaced with the equivalent residues from the rat GHR-ECD. This region lies in a loop structure which joins the two beta-barrel domains of the GHR-ECD. This protein is designated as Site-B mutant. A subsequent analysis confirmed the N-terminal region between residues 28-34 of oGHR-ECD as an important epitope defined by antiserum raised to oGHR-ECD. Also of interest was the finding that mutation at both Sites A and B in oGHR-ECD compromised the affinity of the protein for bovine GH (50-fold for Site-A and 4-fold for Site-B). A comparison of the affinity of wild type oGHR for the highly homologous bovine GH with its affinity for rat GH indicated a 10-fold higher affinity for the ruminant GH than for rat GH. Mutation at Site-A of oGHR-ECD reduced the affinity for rat GH a further 3-fold. However, mutation at Site-B of oGHR-ECD increased the affinity for rat GH 2-3 fold. This indicated that the substitution of rat GHR residues for ovine GHR residues in this part of the protein had a beneficial effect in relation to affinity for rat GH and that this region of the GHR-ECD may contain important specificity determinants. In order to test whether these observed differences in affinity for bovine and rat GH affinities have any biological relevance, we have produced the same ovine --> rat mutations in the context of the full length ovine GHR. Transfection of the cDNAs encoding the wt or mutant GHRs into the mouse myeloid pre-B cell line FDC-P1 to create stably transfected clonal lines, has allowed an examination of the relative activities of ovine and rat GH, using a robust and high throughput bio-assay based on the reduction of a cell permeable tetrazolium salt. In the current manuscript, we report that the decrease in binding affinity previously reported for Site-A and Site-B mutant oGHR extracellular domain proteins is not reflected in compromised biological activities when the same mutations are expressed in the context of the full length oGHR protein. We discuss these findings in the context of the relationships between affinity and activity at the GHR.

Interaction of osmotic and temperature stress on transgenic soybean

The triphenyttetrazotium chloride (TTC) assay is based on the ability of viable cells to metabolically reduce tetrazolium salts to formazan. It was used as a method to screen the viability differences of soybean plants transformed with a P5CR gene in antisense direction controlled by an inducible heat shock promoter (IHSP). Differences were detected between the transgenics and the control plants using this method. The transgenics exhibited a sensitive reaction in that formazan production was reduced following an osmotic stress treatment. The control plants however exhibited a tolerant reaction in that formazan production was highest during stress treatment. The efficiency of protein synthesis in the transgenic plants was lower than in the control plants when the inducible heat shock promoter was activated. This indicated that underexpression of the P5CR gene resulted in declined protein synthesis due to proline shortage. The transgenics had a lower seed production than the control plants indicating that the antisense P5CR gene also influenced seed production negatively. The screening methods showed that the control plants were the most viable of the plants tested. The decrease in the proline level due to the activated antisense P5CR gene yielded plants that are directly or indirectly more sensitive to osmotic stress. It appears that the antisense transgenics were not able to withstand the osmotic stress due to the decline in proline synthesis. These findings indicate that proline plays an adaptive role during osmotic stress in soybean plants

Triphenyltin acetate-induced cytotoxicity and CD4 + and CD8 + depletion in mouse thymocyte primary cultures

Organotin compounds (OTs) find application worldwide as catalysts, stabilizers and biocides. Triphenyltin derivatives (TPs), including the fungicide triphenyltin acetate (TPTA), are OTs mostly used in our country. Some OTs were proved to be immunotoxic and in this paper the cytotoxicity, the possible selective activity upon definite lymphocyte subsets as well as the antiproliferative effect of TPTA was investigated in vitro by using primary cultures of mouse thymocytes. TPTA (5, 10 and 25 μM) was cytotoxic to these cells, as demonstrated by the significant ( P<0.05) reduction of the cell viability percentage (trypan blue dye exclusion test), the neutral red uptake and the reduction of tetrazolium salts to formazan products (MTT assay). These overt effects were already noticed after 4 h of exposure to TPTA. The fungicide otherwise significantly reduced, after 24 h of incubation, the percentage of mature single positive thymocytes, particularly the CD4 +/CD8 − one. Finally, a significative dose-dependent inhibition of the T-cell mitogen-induced cell proliferation was observed in thymocytes exposed to 1 and 8 μM TPTA. These results are indicative of the TPTA immunotoxic properties, according to previous published reports concerning the in vitro and in vivo toxicity of some di- and triorganotin compounds.

β-amyloid induces caspase-dependent early neurotoxic change in PC12 cells: correlation with H 2O 2 neurotoxicity

We examined neurotoxic effects of Aβ(25–35), an active fragment of β-amyloid (Aβ), and compared the effect with H 2O 2 neurotoxicity in PC12 cells. Aβ(25–35) induced the loss of mitochondria function as detected using a tetrazolium salt (WST-1) reduction assay and decreased the number of cells adhering to collagen type 1-coated plates. Aβ(25–35) did not induce cell death, as detected by Hoechst 33342/propidium iodide staining. The caspase tetrapeptide inhibitor z-IETD-fluoromethylketone (FMK) and z-LEHD-FMK inhibited the attenuation of WST-1 reduction induced by Aβ(25–35) and H 2O 2, while the caspase-3 inhibitor z-DEVD-FMK afforded protection only against H 2O 2 neurotoxicity. Caspase-3 protease activity was increased by treatment of H 2O 2 but not Aβ(25–35). Thus, Aβ(25–35) induces early neurotoxic events by activating caspases other than caspase-3. H 2O 2-induced oxidative stress may not be implicated in Aβ-induced neurotoxic pathways.

Pterin interactions with distinct reductase activities of NO synthase

Besides oxidizing l-arginine, neuronal NO synthase (NOS) NADPH-dependently reduces various electron acceptors, including cytochrome c and tetrazolium salts. The latter NADPH diaphorase reaction is used as a NOS-specific histochemical stain. Both reductase activities have been utilized to analyse electron transfer mechanisms within NOS. Basal l-arginine turnover by homodimeric NOS is enhanced by exogenous tetrahydrobiopterin, and the intra-subunit electron flow may include intermediate trihydrobiopterin. In the present work we have investigated the possible role of the tetrahydrobiopterin binding site of NOS in its reductase activities by examining the effects of anti-pterin type (PHS) NOS inhibitors. Although the type I anti-pterin, PHS-32, which does not affect basal dimeric NOS activity, also had no effect on either reductase activity, the type II anti-pterin, PHS-72, which inhibits basal NOS activity, inhibited both reductase activities and the NADPH diaphorase histochemical stain. Pterin-free NOS monomers catalysed both cytochrome c and tetrazolium salt reduction. Our data suggest that both NOS reductase activities are independent of tetrahydrobiopterin. However, occupation of an exosite near the pterin site in NOS by type II anti-pterins may interfere with the electron flow within the active centre, suggesting that steric perturbation of the pterin binding pocket or reductase interaction contribute to the mechanism of inhibition by this class of NOS inhibitors.

Pterin interactions with distinct reductase activities of NO synthase.

Besides oxidizing L-arginine, neuronal NO synthase (NOS) NADPH-dependently reduces various electron acceptors, including cytochrome c and tetrazolium salts. The latter NADPH diaphorase reaction is used as a NOS-specific histochemical stain. Both reductase activities have been utilized to analyse electron transfer mechanisms within NOS. Basal L-arginine turnover by homodimeric NOS is enhanced by exogenous tetrahydrobiopterin, and the intra-subunit electron flow may include intermediate trihydrobiopterin. In the present work we have investigated the possible role of the tetrahydrobiopterin binding site of NOS in its reductase activities by examining the effects of anti-pterin type (PHS) NOS inhibitors. Although the type I anti-pterin, PHS-32, which does not affect basal dimeric NOS activity, also had no effect on either reductase activity, the type II anti-pterin, PHS-72, which inhibits basal NOS activity, inhibited both reductase activities and the NADPH diaphorase histochemical stain. Pterin-free NOS monomers catalysed both cytochrome c and tetrazolium salt reduction. Our data suggest that both NOS reductase activities are independent of tetrahydrobiopterin. However, occupation of an exosite near the pterin site in NOS by type II anti-pterins may interfere with the electron flow within the active centre, suggesting that steric perturbation of the pterin binding pocket or reductase interaction contribute to the mechanism of inhibition by this class of NOS inhibitors.

The Role of Plasma Membrane in Bioreduction of Two Tetrazolium Salts, MTT, and CTC

Despite widespread use of various tetrazolium assays, the mechanisms of bioreduction of these compounds have not been fully elucidated. We investigated the capacity of tetrazolium salts to penetrate through intact cell plasma membranes. 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) and 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) tetrazolium salts appear to represent examples of species that are reduced by different mechanisms. We provide evidence suggesting that MTT readily crosses intact plasma membranes and is reduced intracellularly. MTT appears to be reduced by both plasma membrane and intracellular reductases; reducing cells are not damaged and remain metabolically active for at least 45 min. In contrast, CTC remains extracellular with respect to viable cells and thus requires plasma membrane permeable electron carrier to be reduced efficiently. However, reduction of CTC in the presence of an electron carrier inflicts damage on plasma membranes. The intracellular vs extracellular sites of reduction of tetrazolium salts were established on the basis of deposition of formazans. Crystals of formazan were detected using fluorescence or backscattered light confocal laser microscopy. We postulate that the capacity of a tetrazolium salt to cross intact plasma membranes constitutes an important experimental variable which needs to be controlled in order to correctly interpret the outcome of tetrazolium assays designed to measure cellular production of oxygen radicals, activity of mitochondrial, cytosolic, or outer membrane reductases, etc.

Iron-induced Lipid Peroxidation in Human Skin-derived Cell Lines: Protection by a Red Orange Extract.

Although photodamage and photoprotection have already been extensively studied in cultured cells, few data have been reported in the literature regarding the in vitro behaviour of skin cells toward a chemical stress, such as iron-induced lipid peroxidation. We investigated the susceptibilities of two human skin-derived cell lines (NCTC 2544 keratinocytes and HFFF2 fibroblasts) to lipid peroxidation induced by FeSO4/histidine, FeSO4/ascorbate and Fe2(SO4)3/ADP. NCTC 2544 cells were more susceptible than HFFF2 cells to lipid peroxidation (assessed by measuring the content of malondialdehyde [MDA]) with iron/ascorbate and iron/ADP as pro-oxidants whereas, with iron/histidine, the same level of MDA production was achieved (about 10nmol/mg protein) in the two cell populations. On the basis of these results, one experimental model ( (iron/histidine) was selected to assess the protective effect of a mixture of two classical antioxidants, Trolox C™ (50μM) and Vitamin C (1mM), added to the cell cultures according to various protocols. The maximal decrease of MDA production in both cell lines was obtained when the antioxidant mixture and the pro-oxidant were added simultaneously to the cultures. By using the same experimental design, NCTC 2544 and HFFF2 cells were exposed to a standardised extract of red oranges (ROE; 0.025-0.5mg/ml), the main active principles of which (anthocyanins 3.1%, hydroxycinnamic acid 2.07%, and flavanone glycosides 8.1%) possess antioxidant activity. Cells treated with ROE, that were still over 90% viable, as evaluated by means of neutral red uptake and tetrazolium salt reduction tests, showed a significant and dose-dependent inhibition of MDA production. This study provides new information about the behaviour of cultured skin cells exposed to chemically induced oxidative stress, and provides further support to the possibility of using skin-derived human cell lines in the evaluation of the effectiveness of antioxidant ingredients for new drugs and/or cosmetics.

Endothelial and Smooth Muscle Changes of the Thoracic and Abdominal Aorta with Various Types of Cryopreservation

Background. There is a need for cryopreserved arterial allografts in vascular surgery. Vitrification was examined as an alternative to slow cooling. Function of endothelial and adjacent cells was evaluated in the thoracic and abdominal regions of the porcine aorta.Material and Methods. Aortas from young pigs were harvested and cryopreserved with several treatments: slow cooling at 1°C/min with 10% dimethyl sulfoxide (DMSO) and vitrification with a vitrification solution (VS), performed either in the presence (medium) or in the absence (air) of the cryoprotective medium surrounding the sample. Tetrazolium salt reduction and oxygen consumption were used to assess the mitochondrial activity of the endothelial cells and smooth muscle cells from the aorta.Results. Fresh aorta showed an increased function from below the aortic arch to the iliac branch. Exposure to cryoprotective solutions reduced significantly the oxygen consumption of the abdominal sections. Samples from the thoracic region had a better response to cryopreservation than those from the abdominal region. There was a significant reduction in tetrazolium salt reduction and oxygen consumption when the samples were cryopreserved immersed in the medium. Significant flaking of the endothelium was observed after vitrification and contributes generally to lower the function.Conclusion. The thoracic region of the aortic wall was more tolerant to the preservation injury, and freezing with DMSO in air offered a better choice of preservation.

Cytochemical and immunocytochemical methods for electron microscopic detection of glucose-6-phosphate dehydrogenase in brain areas

This paper reports on protocols for the cytochemical and immunocytochemical determination of the glucose-6-phosphate dehydrogenase (G6PD) in brain areas by electron microscopy (EM). The cytochemical assay consists of a pre-embedding staining of small and flat tissue blocks, which were first mildly fixed and then floated in a staining mixture based on the reduction of tetrazolium salts by NADPH. Tissue blocks were then washed, post-fixed in OsO 4, dehydrated through graded ethanol concentrations and embedded in resin. Ultrathin sections were then obtained and observed at the EM. The immunocytochemical technique was performed on completely fixed tissues of perfused animals. After the tissue embedding in resin, ultrathin sections were obtained and treated with a primary anti-erythrocyte G6PD antibody, produced and purified in our laboratory. The immunostaining was performed with secondary gold-conjugated antibody. Gold grains were well evident by EM analysis thus revealing the G6PD protein in the subcellular compartments. These protocols are useful to detect peculiar populations of neurons which express high levels of G6PD to sustain processes of neural plasticity in some brain areas. Themes: Development and regeneration Topics: Cell division and differentiation

Selenium causes growth inhibition and apoptosis in human brain tumor cell lines.

We examined the effect of the trace element selenium on human glioma cell lines: T98G, U373MG, and U87MG, in addition to dermal fibroblast cells. Cultures were incubated with sodium selenite, and the following parameters were studied: cell growth, mitochondrial function, and ultrastructure. Cell growth was assayed by counting the number of viable cells after treatment with selenium. Mitochondrial function was analyzed using the MTT (tetrazolium salt reduction) assay. Apoptosis was determined by evaluating nuclear chromatin condensation by electron microscopy. The results indicated that selenium had a significant inhibitory effect on the growth of the tumor cells but had little effect upon dermal fibroblasts which had been passaged numerous times. Selenium also induced mitochondrial damage as shown by MTT assay in two brain tumor cell lines and in minimally passaged fibroblasts, but it had little effect upon the high-passage fibroblasts. Ultrastructurally, mitochondria had electron-dense inclusions resulting from selenium treatment. High rates of apoptosis were induced by selenium in the tumor cell lines and in the minimally passaged fibroblasts, whereas the fibroblasts with a high number of passages had some resistance to selenium treatment. This study correlates the adverse effects of selenium on mitochondrial function, inhibition of cell growth, and apoptosis and shows that selenium similarly affects three different brain tumor cell lines and minimally passaged fibroblasts. Further, the results with fibroblasts show that some types of cells after repeated passages can develop resistance to selenium damage.

Density gradient separation of active and non-active cells from natural environments.

We present a method for the selective, physical separation of active and non-active bacterial cells from natural communities. The method exploits the reduction of tetrazolium salts to form insoluble formazan crystals intracellularly in response to the addition of different oxidisable substrates. The intracellular deposition of formazan alters the bouyant density of active cells enabling them to be separated by density gradient centrifugation. The method has been successfully applied to the fractionation and collection of large whole cell sub-populations of active and non-active cells from sea-water samples. Removal of the bands from the density gradient, followed by PCR amplification and DGGE analyses showed distinct differences in the PCR amplicon diversity associated with the active and non-active cell fractions; an indication of changes in bacterial community structure in response to the addition of oxidisable substrate. Thus, based on their in situ respiration potential, the approach enables the cytochemical enrichment and molecular characterisation of mixed bacterial populations in natural environments.

Energy sources of non-starter lactic acid bacteria isolated from Cheddar cheese

Abstract The range of carbon sources available in cheese curd during maturation that could be used as energy and growth substrates by 60 cultures of non-starter lactic acid bacteria isolated from Cheddar cheese was determined by the detection of tetrazolium salt reduction in Biolog MT1 microplates ™ . There were marked inter-species and strain differences in the range of carbon substrates catabolized by the 11 Lactobacillus spp. and 2 Weissella spp. examined. Sugars were used widely among the NSLAB with 90, 100 and 85% of the isolates metabolizing lactose, glucose and galactose, respectively. In addition, ribose, N -acetyl-galactosamine and sialic acid, potentially derived from nucleic acid and casein deglycosylation, were catabolized by 58, 48 and 22% of the isolates, respectively. Lactic acid was also a potential substrate for 15% of the isolates but Tween 80 was not an effective substrate. Although 50% of the NSLAB removed citric acid from the growth medium it was not an independent energy source. Peptides and amino acids were also catabolized by up to 27% of the NSLAB provided that an exogenous source of α -ketoglutaric acid was present to facilitate the aminotransferase-mediated transamination degradative pathway. The MT1 microplate method facilitates the rapid screening for isolates able to establish in the cheese curd and for the detection of specific metabolic activities in isolates undergoing evaluation for use as adjunct cultures in cheesemaking trials.

Concordance between tumour cell activation by epidermal growth factor and alteration of cell sensitivity to cisplatin and lymphokine-activated killer cell activity.

In this study the impact of epidermal growth factor (EGF) on chemosensitivity and susceptibility to lymphokine-activated killer (LAK) cytolysis on six cell lines (one super expressing EGFr i.e. HN5, three high expressing i.e. Hep2, KB and MCF-7 and two low expressing i.e. Fen and HN15) was investigated using the tetrazolium salt reduction assay (MTT) as measured by optical density (OD). Hep2, KB, MCF-7 and Fen lines showed a dose-related inhibition to cisplatin (from 19% to 80%). Treatment of EGFr positive cell lines, Hep2, KB and MCF-7 but not EGFr negative Fen by EGF prior to exposure to cisplatin inhibited the cells by between 10-15% (p<0.05). Exposure of HN5 line to EGF (0.1 ng/ml) prior to LAK assay, led to a decrease in tumour killing (13%, p<0.05). However, at 0.01 ng/ml the pre-treatment enhanced tumour sensitivity. These data indicated that pre-exposure of tumour cells to EGF altered their response to cisplatin and LAK killing and this depended on the degree of EGFr expression. These data may prove helpful for pre-selection of patients for an appropriate therapy.

Estradiol 17β inhibition of LDL oxidation and endothelial cell cytotoxicity is opposed by progestins to different degrees

Progestins oppose the effects of estrogens in many biological systems, but it is not known if progestins oppose the antioxidant effects of estrogen and to differing degrees. To test these questions, the effects of various sex steroids on LDL oxidation and cytotoxicity were studied in the absence or presence of endothelial cells. Freshly isolated LDL was incubated in the presence of Cu ++ in the absence or presence of cultured bovine aortic endothelial cells in phenol red-free medium and without or with hormones in 0.5% ethanol. The hormones included 17β-estradiol (E 2), progesterone (Pg), norgestimate (NGM), levonorgestrel (LNG), and medroxyprogesterone acetate (MPA). LDL oxidation was measured as formation of conjugated dienes, lipid peroxides, and TBARS, and cyotoxicity by tetrazolium salt reduction (MTT reduction). Progestins diminished conjugated diene lag phase, accelerated lipid peroxide and TBARS production in the absence and presence of cells and accelerated cytotoxicity. When E 2 and progestin were incubated with cells at a molar ratio of 1:5, lipid peroxides were reduced from baseline by E 2 alone 31%, E 2/Pg 29%, E 2/NGM 16%, E 2/LNG 9% (all P<0.05 or more) and E 2/MPA 8% (ns) (E 2 or E 2/Pg>E 2/NGM, E 2/LNG and E 2/MPA [ P<0.001]; E 2/NGM>E 2/LNG or E 2/MPA [ P<0.05]). MTT reduction followed a similar gradient, greatest with E 2 alone, least with E 2/MPA. Conclusions: Progestins promote LDL oxidation and, conjointly, endothelial cell cytotoxicity. Progestins oppose the antioxidant and cytoprotective effects of estrogen when given in combination. MPA and LNG have the strongest prooxidant and cytotoxic effects, which may limit the cardiovascular benefit of estrogen during combined administration in vivo.

Reduction of a tetrazolium salt, CTC, by intact HepG2 human hepatoma cells: subcellular localisation of reducing systems

Cell-mediated reduction of tetrazolium salts, including MTT, XTT, MTS, NBT, NTV, INT, in the presence or absence of intermediate electron carriers is used as a convenient test for animal or bacterial cell viability. Bioreduction of tetrazolium is considered an alternative to a clonogenic assay and a thymidine incorporation assay. However, correlation between clonogenic potential and capacity to reduce tetrazolium has not been demonstrated convincingly. Moreover, despite a wide use of tetrazolium viability assays, the mechanism and subcellular localisation of reducing systems or species in viable intact cells have not been fully elucidated. We report evidence indicating that a tetrazolium salt CTC can be reduced in the presence as well as in the absence of an electron carrier by viable HepG2 human hepatoma cells. CTC-formazan is formed within or at the outer surface of plasma membranes. We hypothesise that in the presence of an electron carrier the electron donors active in the reduction of CTC are located in the intracellular compartment, as well as in plasma membranes. However, in the absence of an electron carrier, the reduction occurs primarily via a plasma membrane-associated enzymatic system or species.