Effects of mineral dust on generation of superoxide radicals and hydrogen peroxide by alveolar macrophages, granulocytes and monocytes

Phagocytosis of quartz dust by alveolar macrophages and monocytes of rabbits and human monocytes and granulocytes is accompanied by stimulation of substrateless recovery of nitroblue tetrazolium to formazan. It reflects activation of oxygen-dependent bactericidal phagocyte system and generation of active oxygen forms. Less fibrogenic and cytotoxic dust of aluminium oxide increased formazan formation insignificantly. Extracellular generation of superoxide radicals and hydrogen peroxide was not discovered during phagocytosis of quartz by alveolar macrophages and monocytes. Incubation of human granulocytes with silica caused, on contrary, considerable increase in exogenous generation of superoxide radicals and hydrogen peroxide. Less fibrogenic dust of aluminium oxide under the conditions had no effect on generation of hydrogen peroxide and induced acute decrease in generation of superoxide radicals by granulocytes. The obtained results testify both to the essential part of active oxygen form during pathologic processes with pneumoconiosis, and also to a great similarity among biochemical processes, characterizing interaction of alveolar macrophages and monocytes with mineral dust.     

Adhering lung macrophages produce superoxide demonstrated with desferal-Mn(IV)

It has been shown that H2O2, the dismutation product of O2., is produced at cell-surface interfaces. Nevertheless, the relationships between the degree of attachment itself, type of surface, and O2. production are not clear. Superoxide production can be measured by the O2.-dependent reduction of nitroblue tetrazolium to an insoluble formazan. Superoxide dismutase (SOD) may be unable to scavenge O2. produced between alveolar macrophages (AM) and a surface. Desferal-Mn(IV) (Des-Mn), a low molecular weight mimic of SOD, is protective against paraquat toxicity in vivo, presumably because of specificity for O2-. Using that assumption, Des-Mn was used to measure O2. production that occurred during adherence of AM. AM suspensions were placed on fibronectin-coated glass coverslips or uncoated glass coverslips or non-stick tissue culture plates. Adherence to the surfaces varied with fibronectin greater than glass greater than non-stick and the percent formazan positive cells was 60, 24, and 4, respectively. With SOD present, the percentage of formazan positive cells were 40, 17, and 2; however, in the presence of Des-Mn the percent stained cells was 4, 4, and 0. When phorbol myristate acetate (PMA) was added during adherence, the percent of formazan positive cells was 82, 57, and 44, respectively. With PMA, Des-Mn was able to inhibit 88-100% of formazan staining whereas SOD inhibition decreased more markedly with increasing adherence. These results indicated that the degree of attachment correlated with both the degree of NBT reduction and the relative effectiveness of Des-Mn versus SOD to scavenge O2..     

Cell-based quantitative evaluation of the MTT assay

OBJECTIVE: To analyze the bioreduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) on a per cell basis and evaluate its modulation as a function of different stages of cell metabolism. STUDY DESIGN: Following MTT bioreduction, total optical density (TOD), cell area and specific activity (TOD/area) of V79 cells and cultured macrophages were recorded for individual cells by means of digital image analysis. The effect of different serum (0-10% vol/vol) or genistein (0-100 microM) concentrations was used to modulate the MTT-specific activity response. RESULTS: As cells in culture are heterogeneous in cell size, the contribution of each cell to the total amount of formazan formed per dish is variable. The production of formazan per cell as a result of MTT bioreduction was found to be proportional to cell size. CONCLUSION: Specific MTT-reducing activity was analyzed in phagocytes and nonphagocyte cells, revealing the utility of this variable in evaluating the MTT assay at the single-cell level.     

Role of distinct subpopulations of peritoneal macrophages in the regulation of reactive oxygen species release.

It has been reported in vitro that during the respiratory burst of phagocytic cells the superoxide anion production per cell shows a negative relation with the cell density. This process has been described as autoregulation. The aim of this work was to analyze the superoxide anion production in thioglycollate-elicited peritoneal macrophage exudates to evaluate the importance of the peritoneal cavity environment in the autoregulation process. 12-O-tetradecanoylphorbol-13-acetate (PMA) was used to stimulate the respiratory burst and superoxide anion production was measured evaluating the intracellular formazan deposits that precipitate as a result of nitro blue tetrazolium (NBT) reduction. We have demonstrated a negative correlation between superoxide anion production and cell density in the peritoneal cavity in macrophages challenged with PMA. The response of individual cells was analyzed by means of an image analyzer, measuring the amount of formazan per cell and cell-size changes during the process of activation. The results revealed that the decrease in individual cell response as a function of higher cell densities were due to a significant increase in the amount of basal reaction macrophages. Concomitantly, the number of reactive cells remained unchanged irrespective of the cell density of the population. A direct correlation between cell size and superoxide anion production was observed. This phenomenon was demonstrated in SENCAR and Balb/c strains. However, macrophages from SENCAR mice showed greater superoxide anion production than those from Balb/c.The differences between strains could be associated to the increased sensitivity to PMA tumor promotion of SENCAR mice. Based on this property, macrophages from SENCAR mice were stimulated with opsonized zymosan, a particulate stimulus that reflects the interaction macrophage-microorganism during the phagocytic process. This data will contribute to the knowledge of infection control. We conclude that variations in basal reaction cells modulates the macrophage activation response when excess macrophages are recruited to the peritoneum. This is demonstrated using different stimuli, thus suggesting that this response may be applied to a wide variety of stimuli-macrophage interactions. The differences between strains may be associated to the increased sensitivity to PMA tumor promotion of SENCAR mice.     

Cytotoxic Amyloid Peptides Inhibit Cellular 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) Reduction by Enhancing MTT Formazan Exocytosis

Abstract: Amyloid β peptide (Aβ) neurotoxicity is believed to play a central role in the pathogenesis of Alzheimer's disease. An early indicator of Aβ toxicity is the inhibition of cellular 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction to MTT formazan, a widely used assay for measuring cell viability. In this report we show that Aβ and other cytotoxic amyloid peptides such as human amylin dramatically enhance MTT formazan exocytosis, resulting in the inhibition of cellular MTT reduction. Only the amyloid peptides that are known to be cytotoxic enhanced MTT formazan exocytosis. Basal MTT formazan exocytosis and amyloid peptide-enhanced MTT formazan exocytosis are blocked by several drugs with diverse known effects. These and other data suggest that MTT formazan exocytosis is a multistep process and that cytotoxic amyloid peptides enhance MTT formazan exocytosis through an intracellular signal transduction pathway.     

Localization of MTT formazan in lipid droplets. An alternative hypothesis about the nature of formazan granules and aggregates

MTT (3-(4, 5-dimethyl-2-thiazolyl)-2, 5-dihphenyltetrazolium bromide) assay is a widely used method to assess cell viability and proliferation. MTT is readily taken up by cells and enzymatically reduced to formazan, a dark compound which accumulates in cytoplasmic granules. Formazan is later eliminated by the cell by a mechanisms often indicated as exocytosis, that produces characteristic needle-like aggregates on the cell surface. The shape of formazan aggregates and the rate of exocytosis change in the presence of bioactive amyloid beta peptides (Abeta) and cholesterol. Though the cellular mechanisms involved in MTT reduction have been extensively investigated, the exact nature of formazan granules and the process of exocytosis are still obscure. Using Nile Red, which stains differentially neutral and polar lipids, and a fluorescent analog of cholesterol (NBD-cholesterol), we found that formazan localized in lipid droplets, consistent with the lipophilic nature of formazan. However, formazan granules and aggregates were also found to form after killing cells with paraformaldehyde fixation. Moreover, formazan aggregates were also obtained in cell-free media, using ascorbic acid to reduce MTT. The density and shape of formazan aggregates obtained in cell-free media was sensitive to cholesterol and Abeta. In cells, electron microscopy failed to detect the presence of secretory vesicles, but revealed unusual fibers of 50 nm of diameter extending throughout the cytoplasm. Taken together, these findings suggest that formazan efflux is driven by physico-chemical interactions at molecular level without involving higher cytological mechanisms.     

Steroid Hormones Block Amyloid Fibril-Induced 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) Formazan Exocytosis: Relationship to Neurotoxicity

Abstract: Perhaps the most reproducible early event induced by the interaction of amyloid β peptide (Aβ) with the cell is the inhibition of cellular 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction. We recently demonstrated that cytotoxic amyloid peptides such as Aβ and human amylin inhibit cellular MTT reduction by dramatically enhancing MTT formazan exocytosis. We now show the following: (a) Insulin and glucagon, when converted to fibrils with β-pleated sheet structure, induce MTT formazan exocytosis that is indistinguishable from that induced by Aβ. NAC35, an amyloidogenic fragment of α-synuclein (or NACP), also induces MTT formazan exocytosis. (b) All protein fibrils with the β-pleated sheet structure examined are toxic to rat hippocampal neurons. (c) Many sterol sex hormones (e.g., estradiol and progesterone) block amyloid fibril-enhanced MTT formazan exocytosis as well as MTT formazan exocytosis in control cells by acting at a common late step in the exocytic pathway. Steroids fail, however, to protect hippocampal neurons from acute amyloid fibril toxicity. These findings suggest that the ability to enhance MTT formazan exocytosis and to induce neurotoxicity are common biological activities of protein fibrils with β-pleated sheet structure but that enhanced MTT formazan exocytosis is not sufficient for acute Aβ neurotoxicity.     

Osmotic induced stimulation of the reduction of the viability dye 2,3,5-triphenyltetrazolium chloride by maize roots and callus cultures

Live cells can reduce colorless 2,3,5-triphenyltetrazolium chloride (TTC) to a red insoluble compound, formazan. Maize (Zea mays) callus, when osmotically stressed by 0.53 mol/L mannitol, produced 7-times or more formazan than untreated control callus. This result was seen with all osmotica tested and could not be attributed to differences in TTC uptake rate or accumulation, increased respiration rate as measured by O2 uptake, or to de novo protein synthesis. Increased formazan production could be detected after 2.5 h of exposure to osmotic stress and leveled off after 48 h of exposure. The increased formazan production was only detected when callus was moved from high osmotic medium to low osmotic, TTC-containing medium. Abscisic acid increased TTC reduction only when added in combination with 0.53 mol/L mannitol. Incubation of maize seedling roots with 0.53 mol/L mannitol also increased formazan production as seen visually. Further studies are needed to determine the cause of the increased formazan production. These results show that TTC viability measurements must be carefully evaluated with appropriate controls to confirm their validity.     

Relationship of MTT reduction to stimulants of muscle metabolism

MTT, a positively charged tetrazolium salt, is widely used as an indicator of cell viability and metabolism and has potential for histochemical identification of tissue regions of hypermetabolism. In the present study, MTT was infused in the constant-flow perfused rat hindlimb to assess the effect of various agents and particularly vasoconstrictors that increase muscle metabolism. Reduction of MTT to the insoluble formazan in muscles assessed at the end of experiments was linear over a 30 min period and production rates were greater in red fibre types than white fibre types. The vasoconstrictors, norepinephrine (100 nM) and angiotensin (10 nM) decreased MTT formazan production in all muscles but increased hindlimb oxygen uptake and lactate efflux. Veratridine, a Na(+) channel opener that increases hindlimb oxygen uptake and lactate efflux without increases in perfusion pressure, also decreased MTT formazan production. Membrane stabilizing doses (100 microM) of (+/-)-propranolol reversed the inhibitory effects of angiotensin and veratridine on MTT formazan production. Muscle contractions elicited by stimulation of the sciatic nerve, reversed the norepinephrine-mediated inhibitory effects on MTT formazan production, even though oxygen consumption and lactate efflux were further stimulated. Stimulation of hindlimb muscle oxygen uptake by pentachlorophenol, a mitochondrial uncoupler, was not associated with alterations in MTT formazan production. It is concluded that apart from muscle contractions MTT formazan production does not increase with increased muscle metabolism. Since the vasoconstrictors angiotensin and norepinephrine as well as veratridine activate Na(+) channels and the Na(+)/K(+) pump, energy required for Na(+) pumping may be required for MTT reduction. It is unlikely that vasoconstrictors that stimulate oxygen uptake do so by uncoupling respiration.     

A cytochemical staining procedure for succinate dehydrogenase activity in pre-ovulatory mouse oocytes embedded in low gelling temperature agarose.

We describe a cytochemical staining procedure for succinate dehydrogenase (SDH) activity in pre-ovulatory mouse oocytes. The oocytes were embedded in low gelling temperature agarose and treated with caffeine before cytochemical staining in the presence of nitro blue tetrazolium (NBT), phenazinemethosulfate (PMS), and succinate. This resulted in intense staining of the oocytes by formazan precipitate. The level of aspecific formazan production in the absence of succinate was very low. We applied the procedure to oocytes matured in vitro and found that the location of the formazan precipitate as a result of SDH activity correlated well with the location of mitochondria. The chromatin of the cytochemically stained oocytes could subsequently be analyzed by means of the DNA-specific fluorochrome DAPI. In pre-ovulatory oocytes, we found a correlation between chromatin organization and the location of mitochondria: in oocytes with an intact germinal vesicle the mitochondria were uniformly distributed in the cytoplasm, as shown by fine grains of formazan precipitate. In oocytes with condensed chromatin the mitochondria apparently had clustered, because the formazan precipitate was more coarse in these cells.     

Inhibition of xanthine oxidase by pterins

The effect of a panel of pterins on xanthine oxidase was investigated by measuring formation of urate from xanthine as well as formazan production from nitroblue tetrazolium. The pterin derivatives, depending on their chemical structure, decreased urate as well as formazan generation: 200 μM neopterin and biopterin suppressed urate formation (90% from baseline) and formazan production (80% from baseline) as well. Their reduced forms, 7,8-dihydroneopterin and 5,6,7,8-tetrahydrobiopterin, showed a lesser but still strongly diminishing influence (40% from baseline). Another oxidized pterin namely leukopterin showed only a weak inhibitory effect. Xanthopterin, a known substrate of xanthine oxidase, had a strong effect on urate formation (80% inhibition), but a lesser effect on formazan production (30% reduction). When iron-(III)-EDTA complex was added to the reaction mixture all the effects were more pronounced. Superoxide dismutase, which removes superoxide anion by dismutation intooxygen, decreased formazan production in addition to pterin derivatives and had a small but enhancing effect on urate formation. Also the reductant N-acetylcysteine had an additive effect to pterins to diminish formazan production in a dose-dependent way. The results of our study suggest that depending on their chemical structure pterins reduce superoxide anion generation by xanthine oxidase.     

Variables Affecting Two Electron Transport System Assays

Several methodological variables were critical in two commonly used electron transport activity assays. The dehydrogenase assay based on triphenyl formazan production exhibited a nonlinear relationship between formazan production (dehydrogenase activity) and sediment dilution, and linear formazan production occurred for 1 h in sediment slurries. Activity decreased with increased time of sediment storage at 4°C. Extraction efficiencies of formazan from sediment varied with alcohol type; methanol was unsatisfactory. Phosphate buffer (0.06 M) produced higher activity than did either U.S. Environmental Protection Agency reconstituted hard water or Tris buffer sediment diluents. Intracellular formazan crystals were dissolved within minutes when in contact with immersion oil. Greater crystal production (respiration) detected by a tetrazolium salt assay occurred at increased substrate concentrations. Test diluents containing macrophyte exudates produced greater activity than did phosphate buffer, U.S. Environmental Protection Agency water, or ultrapure water diluents. Both assays showed decreases in sediment or bacterial activity through time.     

Control of oxidative stress in microcirculation of spontaneously hypertensive rats

One mechanism for organ damage in individuals with arterial hypertension may be due to oxygen free radical production. This study was designed to localize free radicals in a microvascular network of mature spontaneously hypertensive rats (SHRs) and normotensive Wistar-Kyoto (WKY) rats. Because glucocorticoids play a role in pressure elevation of SHRs, we investigated their role in microvascular free radical formation. Oxygen radical production in mesentery was detected by tetranitroblue tetrazolium reduction to formazan aided by digital light-absorption measurements. Formazan deposits were observed in the endothelial cells and lumens of all microvessels and in lymphatic endothelia but were fewer in tissue parenchyma. The formazan distribution in younger (14-16 wk old) WKY rats and SHRs was heterogeneous with low values in capillaries and small arterioles/venules (<30 microm) but enhanced deposits in larger venules. Adrenalectomy served to reduce the formazan density in SHRs to the level of WKY rats, whereas dexamethasone supplementation of the adrenalectomized rats caused elevation in the larger venules of SHRs. In older (40 wk old) SHRs, formazan levels were elevated in all hierarchies of microvessels. After pressure reduction was employed with chronic hydralazine treatment, the formazan deposits were reduced in all locations of the microcirculation in both WKY rats and SHRs. Elevated formazan deposits were also found in lymphatic endothelium. These results suggest that oxygen free radical production is elevated in both high- and low-pressure regions of SHR microcirculation via a process that is controlled by glucocorticoids. Older SHRs have higher formazan levels than younger SHRs in all microvessels. Chronic hydralazine treatment, which serves to reduce arterial blood pressure, attenuates tetranitroblue tetrazolium reduction in WKY rats and SHRs even in venules of the microcirculation, which has no micropressure elevation. Free radical production may be a more global condition in SHRs and may not be limited to arteries and arterioles.     

A quantitative colorimetric method to evaluate the functional state of human polymorphonuclear leukocytes

The colorimetric assay previously described by Mosmann for the measurement of cell viability and proliferation has been modified for the assessment of the functional state of human polymorphnuclear cells (PMNs). The ability of PMNs to reduce the tetrazolium salt MTT to formazan reflects directly the degree of stimulation induced by various agents. The underlying mechanism of MTT-reduction to formazan seems to be similar to that of nitroblue tetrazolium (NBT)-reduction. In contrast to the NBT-reduction assay, the formazan produced from MTT can easily be measured by an ELISA reader. Parallel experiments revealed a qualitative correlation between the concentration of formazan produced from MTT and the concentration of cytochrome C reduced by PMNs. Although oxidative burst may not be the actual lytic mechanism in cellular cytotoxicity of PMN, we also observed an association between MTT-reduction capacity and the cytotoxic activity of PMNs from normal donors in antibody dependent cellular cytotoxicity. Our results indicate that the MTT-reduction assay can be employed to estimate the functional state of polymorphnuclear granulocytes.     

Disruption of functional activity of mitochondria during MTT assay of viability of cultured neurons

The MTT assay based on the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium in the cell cytoplasm to a strongly light absorbing formazan is among the most commonly used methods for determination of cell viability and activity of NAD-dependent oxidoreductases. In the present study, the effects of MTT (0.1 mg/ml) on mitochondrial potential (ΔΨ_m), intracellular NADH, and respiration of cultured rat cerebellum neurons and isolated rat liver mitochondria were investigated. MTT caused rapid quenching of NADH autofluorescence, fluorescence of MitoTracker Green (MTG) and ΔΨ_m-sensitive probes Rh123 (rhodamine 123) and TMRM (tetramethylrhodamine methyl ester). The Rh123 signal, unlike that of NADH, MTG, and TMRM, increased in the nucleoplasm after 5-10 min, and this was accompanied by the formation of opaque aggregates of formazan in the cytoplasm and neurites. Increase in the Rh123 signal indicated diffusion of the probe from mitochondria to cytosol and nucleus due to ΔΨ_m decrease. Inhibition of complex I of the respiratory chain decreased the rate of formazan formation, while inhibition of complex IV increased it. Inhibition of complex III and ATP-synthase affected only insignificantly the rate of formazan formation. Inhibition of glycolysis by 2-deoxy-D-glucose blocked the MTT reduction, whereas pyruvate increased the rate of formazan formation in a concentration-dependent manner. MTT reduced the rate of oxygen consumption by cultured neurons to the value observed when respiratory chain complexes I and III were simultaneously blocked, and it suppressed respiration of isolated mitochondria if substrates oxidized by NAD-dependent dehydrogenases were used. These results demonstrate that formazan formation in cultured rat cerebellum neurons occurs primarily in mitochondria. The initial rate of formazan formation may serve as an indicator of complex I activity and pyruvate transport rate.     

Progressive loss of the macrophage respiratory burst in oxygen toxicity

The respiratory burst of rat alveolar macrophages stimulated by a variety of agents declines as a function of time of exposure to hyperoxia. Previous studies have evaluated this effect in terms of the stimulated O₂⨪ production of a population of cells. The present study was designed to determine whether this decline is due to a “turning off” of the respiratory burst activity of some cells within the alveolar macrophage population or a general suppression of the activity of all cells. The phorbol myristate acetate (PMA) initiated respiratory burst of individual rat alveolar macrophages was monitored using the reaction of nitroblue tetrazolium (NBT), which results in the formation of a precipitate on active cells. The formazan staining was evaluated using black and white photographs of the cells and comparison to a scale constructed from photographed cells of four differing intensities of staining. Frequency distributions indicated that when the respiratory burst capability in the population of alveolar macrophages is impaired approximately 50% by oxygen exposure and/or culture in plastic vessels with artificial media, there is a gradual shift in NBT reduction rather than an “all or nothing” mechanism, in which the distribution would have reflected a shift from darkly stained cells to the very lessened or negligible staining observed at the end stage of oxygen toxicity.     

Precise cytochemical measurement of neotetrazolium formazan by scanning and integrating microdensitometry

Summary The extinction values of the formazan of neotetrazolium, deposited in tissue sections, have been measured using a Vickers M 85 scanning and integrating microdensitometer with a scanning spot of 0.25 diameter. These values have been correlated with the amount of formazan in the same sections as determined by elution and spectrophotometry. The extinction at 520 nm did not increase linearly with increasing amounts of formazan in sections incubated for different times. This has been shown to be due to the presence of two formazan components which have different absorption characteristics; a red diffuse colour with a molar extinction coefficient of 20580 and a more granular purple product with a coefficient of 7370. However, at 585 nm, the isosbestic wavelength, the extinction was directly proportional to the amount of formazan in the section, irrespective of the nature of the end product; at this wavelength the molar extinction coefficient was 7200.These results have shown that scanning and integrating microdensitometry can be used for the precise measurement of the amount of neotetrazolium formazan, in picograms, in a single cell of group or cells in a tissue section.     

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.     

Quantitative determination of superoxide in plant leaves using a modified NBT staining method

Introduction – In plants, the ROS (reactive oxygen species) level is tightly regulated because their accumulation produces irreversible damage leading to cell death. However, ROS accumulation plays a key role in plant signaling under biotic or abiotic stress. Although various methods were reported to evaluate ROS accumulation, they are restricted to model plants or provide only qualitative information. Objective – Develop a simple method to quantify superoxide radicals produced in plant tissues, based on the selective extraction of the formazan produced after nitroblue tetrazolium (NBT) reduction in histochemical staining. Methodology – Plant leaves were stained with a standard NBT method and the formazan precipitated in tissues was selectively extracted using chloroform. The organic phase was dried and formazan residue dissolved in dimethylsulfoxide–potassium hydroxide and quantified by spectrophotometry. The method was tested in strawberry plant leaves under different stressing conditions. Results – Formazan extracted from leaves subjected to stress conditions showed similar absorption spectra to those obtained from standard solutions using pure formazan. Calibration curves showed a linear relationship between absorbance and formazan amounts, within the range 0.5–8 µg. Outcomes suggested that formazan was retained in the solid residue of leaf tissues. This protocol allowed us to quantify superoxide radicals produced under different stress conditions. Conclusions – Chloroform allowed a selective formazan extraction and removal of potential endogenous, exogenous or procedural artefacts that may interfere with the quantitative determination. This protocol can be used to quantify the superoxide produced in plant tissues using any traditional qualitative NBT histochemical staining method.     

p-benzoquinone and p-hydroquinone as histochemical reagents

Summary Studies on the rate controlling factors of the MTT-Hydroquinone lipoprotein reaction show that the formazan production is quantitatively linear on time of incubation, and dependent on temperature and hydroquinone concentration. Experiments indicate that the reproducibility is sufficient for the reaction to be used as a reference parameter in the numerical expression of dehydrogenase activities in tissue sections. A ratio system is described in which the formazan produced by the dehydrogenase is expressed in terms of the formazan from the non-enzymatic lipoprotein reaction, and a nomenclature for a unit measure is proposed. The method has been tested on guinea pig liver and heart, and the range of variation investigated.