HRP/[Zn-Cr-ABTS] redox clay-based biosensor: design and optimization for cyanide detection

A novel inexpensive and simple amperometric biosensor, based on the immobilization of HRP into redox active [Zn-Cr-ABTS] layered double hydroxide, is applied to the determination of cyanide. The electrochemical transduction step corresponds to the reduction at 0.0 V of ABTS+* enzymatically formed in the presence of H2O2. The biosensor has a fast response to H2O2 (8s) with a linear range of 1.7 x 10(-9) to 2.1 x 10(-6) M and a sensitivity of 875 mA M(-1) cm(-2). The apparent Michaelis-Menten constant (KMapp) is 12 microM. The detection of cyanide is performed via its non competitive inhibiting action on the HRP/[Zn-Cr-ABTS] electrode. The concentration range of the linear response and the apparent inhibition constant (ki) are 5 x 10(-9) to 4 x 10(-8) and 1.4 x 10 (-7) M, respectively.     

Hybridization signal enhancement via long-stranded probe for detection of DNA using aquadichloro (benzimidazole)-copper(II) as hybridization indicator

A simple and sensitive method for electrochemical detection of DNA was designed. This DNA sensor was based on a "sandwich" detection strategy, which involved a long capture probe DNA immobilized on glassy carbon electrodes that flanked both the reference DNA and target DNA. Electrochemical signals were measured by cyclic voltammetry (CV) and differential pulse voltammetry (DPV) using aquadichloro(benzimidazole)-copper(II), Cu(bzim)(H(2)O)Cl(2), as an electroactive indicator. An improving amount of Cu(bzim)(H(2)O)Cl(2) was interacted with the hybrid DNA via the incorporation of a long-probe DNA and a reference DNA in this sensor. As a result of this effect, this sensor design significantly enhanced the sensitivity. With 48-mer probe DNA and 27-mer reference DNA, the proposed method could be used for detection of 21-mer ssDNA ranging from 1.32 x 10(-7) to 2.52 x 10(-6) M with a detection limit of 2.94 x 10(-8) M. Electrochemical DNA biosensors were also developed using the same long-probe sequence as the target sequence with the novel hybridization indicator, Cu(bzim) (H(2)O)Cl(2). The detection limits for the complementary 21-mer target and 27-mer target were 9.52 x 10(-8) M and 5.81 x 10(-8) M, respectively. The results showed that the sensor with long-probe DNA and reference DNA is far more sensitive than that with nonswitch assay.     

Detection and quantification of Escherichia coli O157:H7 in environmental samples by real-time PCR

AIMS: To apply the real-time Polymerase chain reaction (PCR) method to detect and quantify Escherichia coli O157:H7 in soil, manure, faeces and dairy waste washwater. METHODS AND RESULTS: Soil samples were spiked with E. coli O157:H7 and subjected to a single enrichment step prior to multiplex PCR. Other environmental samples suspected of harbouring E.coli O157:H7 were also analysed. The sensitivity of the primers was confirmed with DNA from E.coli O157:H7 strain 3081 spiked into soil by multiplex PCR assay. A linear relationship was measured between the fluorescence threshold cycle (C T ) value and colony counts (CFU ml(-1)) in spiked soil and other environmental samples. The detection limit for E.coli O157:H7 in the real-time PCR assay was 3.5 x 10(3) CFU ml(-1) in pure culture and 2.6 x 10(4) CFU g(-1) in the environmental samples. Use of a 16-h enrichment step for spiked samples enabled detection of <10 CFU g(-1) soil. E. coli colony counts as determined by the real-time PCR assay, were in the range of 2.0 x 10(2) to 6.0 x 10(5) CFU PCR (-1) in manure, faeces and waste washwater. CONCLUSIONS: The real-time PCR-based assay enabled sensitive and rapid quantification of E. coli O157:H7 in soil and other environmental samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The ability to quantitatively determine cell counts of E.coli O157:H7 in large numbers of environmental samples, represents considerable advancement in the area of pathogen quantification for risk assessment and transport studies.     

Biosensor based on polyaniline-Prussian Blue/multi-walled carbon nanotubes hybrid composites

In this work, a novel route for fabrication polyaniline (PANI)-Prussian Blue (PB) hybrid composites is proposed by the spontaneous redox reaction in the FeCl(3)-K(3)[Fe(CN)(6)] and the aniline solution. With the introduction of multi-walled carbon nanotubes (MWNTs), the PANI-PB/MWNTs system shows synergy between the PANI-PB and MWNTs which amplified the H(2)O(2) sensitivity greatly. A linear range from 8 x1 0(-8) to 1 x 10(-5)M and a high sensitivity 508.1 8 microA microM cm(2) for H(2)O(2) detection are obtained. The composites also show good stability in neutral solution. A glucose biosensor was further constructed by immobilizing glucose oxidase (GOD) with Nafion and glutaraldehyde on the electrode surface. The performance factors influencing the resulted biosensor were studied in detail. The biosensor exhibits excellent response performance to glucose with the linear range from 1 to 11 mM and a detection limit of 0.01 mM. Furthermore, the biosensor shows rapid response, high sensitivity, good reproducibility, long-term stability and freedom of interference from other co-existing electroactive species.     

Chromium(III)-mediated structural modification of glycoprotein: impact of the ligand and the oxidants

The interaction of three types of chromium(III) complexes, [Cr(salen) (H2O2]+, [Cr(en)3]3+, and [Cr(EDTA) (H2O)]- with AGP has been investigated. [Cr(salen) (H2O2]+, [Cr(en)3]3+ and [Cr(EDTA) (H2O]- bind to Human alpha1-acid glycoprotein with a protein:metal ratio of 1:8, 1:6, and 1:4, respectively. The binding constant, K(b) was estimated to be 1.37 +/- 0.12 x 10(5) M(-1), 1.089 +/- 0.05 x 10(5) M(-1) and 5.3 +/- 0.05 x 10(4) M(-1) for [Cr(salen) (H2O2]+, [Cr(en)3]3+, and [Cr(EDTA) (H2O)]-, respectively. [Cr(en)3]3+ has been found to induce structural transition of AGP from the native twisted beta sheet to a more compact alpha-helix. The complexes, [Cr(salen) (H2O2]+ and [Cr(EDTA) (H2O]-, in the presence of H2O2, have been found to bring about nonspecific cleavage of AGP, whereas [Cr(en)3]3+ does not bring about any protein damage. Treatment of [Cr(salen) (H2O)2]+-protein adduct with iodosyl benzene on the other hand led to site specific cleavage of the protein. These results clearly demonstrate that protein damage brought about by chromium(III) complexes depends on the nature of the coordinated ligand, nature of the metal complex, and the nature of the oxidant.     

Inhibitory effects of hot water extract of the Stevia stem on the contractile response of the smooth muscle of the guinea pig ileum

The effects of a hot water extract of the stem of Stevia rebaudiana on the smooth muscle of isolated guinea pig ileum were investigated. The butyl alcohol layer of the extract antagonized the contractions of the isolated guinea pig ileum induced by histamine (1 x 10(-5) M) and acetylcholine (1 x 10(-5) M) in a concentration-dependent manner. The butyl alcohol layer of the extract also showed inhibition of CaCl(2) (1 x 10(-3)-3.8 x 10(-1) M)-induced contractions. The antagonism of the extract was considered to be non-specific, but this action might be related to an influx of extracellular Ca(2+).With column chromatography preparation, the active component was assumed to be as stevioside. The antagonistic effects exerted by the stem extract of Stevia rebaudiana contributed to the gastroprotective activity of the extract in animals fed dietary histamine.     

Inhibition of immunoglobulin G-catalyzed hydrogen peroxide generation by dexamethasone and piroxicam

In the present study, we established a simple and physiologically acceptable in vitro assay system to measure H2O2 generated by human immunoglobulin G (IgG) and other proteins. In addition, the effects of various drugs were also tested in this method. We found that UV irradiation (280 nm) of the test solutions for 1 h at 37 degrees C produced suitable conditions to test the effects of these drugs. The test solution contained 100 microg/ml IgG in 50 mM phosphate buffer (pH 7.4), and 1% dimethylformamide (DMF), a solvent used to dissolve each drug. Phosphate anions were preferable for H2O2 generation. H2O2 concentration in the irradiated sample was determined by continuous photometric measurement of absorption (O.D.) at 340 nm for 600 sec. The decrease in O.D. was due to the oxidation of NADPH by H2O2 mediated by the glutathione redox cycle. H2O2 generation was expressed as O.D.(340 nm decrease/400 sec). IgG (100 microg/ml) generated 6-7 microM H2O2/h. With irradiation, most cytokines, proteins and enzymes failed to generate significant amounts of H2O2. The formation of H2O2 from H2O and UV light-induced singlet oxygen (1O2) was demonstrated by the inhibitory effects of 1O2 quenchers. Dexamethasone (IC50: 6 ng/ml = 1.4x10(-8) M) blocked H2O2 generation catalyzed by IgG. This action was not mediated by binding to the glucocorticoid receptor. Piroxicam (IC50: 20 ng/ml = 6.0 x 10(-6) M) and diclofenac.Na (IC50: 500 ng/ml = 1.6 x 10(-5) M), but not indomethacin, also blocked H2O2 generation. The mechanism underlying the inhibition of IgG-catalyzed H2O2 generation is not clear; however, the possibility exists that these drugs intercept, or interfere with, the approach of water molecules at the catalytic interface(s) of the IgG.     

Electrochemical characterization and application of azurin-modified gold electrodes for detection of superoxide

A novel biosensor for superoxide radical (O(2)(*-)) detection based on Pseudomonas aeruginosa azurin immobilized on gold electrode was designed. The rate constant of azurin reduction by O(2)(*-) was found to be 10(5)M(-1)s(-1) in solution and five times lower, i.e., 0.2 x 10(5)M(-1)s(-1), for azurin coupled to gold by 3,3'-dithiobis(sulfosuccinimidylpropionate) (DTSSP). The electron transfer rate between the protein and the electrode ranged from 2 to 6s(-1). The sensitivity of this biosensor to O(2)(*-) was 6.8 x 10(2)Am(-2)M(-1). The response to the interference substances, such as uric acid, H(2)O(2), and dimethylsulfoxide was negligible below 10 microM. The electrode was applied in three O(2)(*-) generating systems: (i) xanthine oxidase (XOD), (ii) potassium superoxide (KO(2)), and (iii) stimulated neutrophil granulocytes. The latter was compared with luminol-amplified chemiluminescence. The biosensor responded to O(2)(*-) in all three environments, and the signals were antagonized by superoxide dismutase.     

Amplification effect and mechanism of action of ET-1 in U-46619-induced vasoconstriction in pig skin

The aim of this study was to investigate if a low concentration of endothelin-1 (ET-1; 8 x 10(-10) M) may amplify the skin vasoconstrictor effect of other vasoactive substances in the pathogenesis of skin vasospasm. Pig skin flaps (6 x 16 cm) were perfused with Krebs buffer equilibrated with 95% O(2) and 5% CO(2) at 37 degrees C and pH 7.4. Skin perfusion pressure measured by a pressure transducer and skin perfusion assessed by the dermofluorometry technique were used for assessment of skin vasoconstriction. We observed that ET-1 (8 x 10(-10) M) significantly amplified the concentration-dependent (10(-7)-10(-5) M) skin vasoconstrictor effect of norepinephrine. More importantly, we observed for the first time that this low concentration of ET-1 also amplified the concentration-dependent (10(-8)-10(-6) M) skin vasoconstrictor effect of the thromboxane A(2) mimetic U-46619, and this amplification effect of ET-1 was completely blocked by the protein kinase C (PKC) inhibitor chelerythrine (5 x 10(-6) M). Conversely, the PKC activator phorbol 12,13-dibutyrate (10(-7) M) amplified the vasoconstrictor effect of U-46619. Furthermore, the sensitivity of the skin vasculature to the vasoconstrictor effect of extracellular Ca(2+) in U-46619-induced skin vasoconstriction was significantly enhanced in the presence of 8 x 10(-10) M ET-1. Finally, the cyclooxygenase inhibitor indomethacin (5 x 10(-6) M) did not affect the amplification effect of ET-1 on U-46619-induced skin vasoconstriction. We conclude that a low concentration of ET-1 can amplify the skin vasoconstrictor effect of U-46619 independent of endogenous cyclooxygenase products, and the mechanism may involve activation of PKC and increase in sensitivity of the contractile apparatus to Ca(2+) in smooth muscle cells.     

Kinetic study of the reaction between vitamin E radical and alkyl hydroperoxides in solution

Kinetic study of the reaction between vitamin E radical and alkyl hydroperoxides has been performed, as a model for the reactions of lipid hydroperoxides with vitamin E radical in biological systems. The rates of reaction of hydroperoxides (n-butyl hydroperoxide 1, sec-butyl hydroperoxide 2, and tert-butyl hydroperoxide 3) with vitamin E radical (5,7-diisopropyl-tocopheroxyl 4) in benzene solution have been determined spectrophotometrically. The second-order rate constants, k-1, obtained are 1.34 x 10(-1) M-1s-1 for 1, 2.42 x 10(-1) M-1s-1 for 2, and 3.65 x 10(-1) M-1s-1 for 3 at 25.0 degrees C. The result indicates that the rate constants increase as the total electron donating capacity of the alkyl substituents at alpha-carbon atom of hydroperoxides increases. The above rates, k-1, are about seven order of magnitude lower than those, k1, for the reaction of vitamin E with peroxyl radical.     

Study on the polarographic catalytic wave of vitamin P in the presence of persulfate and its application

The polarographic catalytic wave of vitamin P in the presence of persulfate was studied by linear potential scan polarography and cyclic voltammetry. Vitamin P yielded a single reduction wave in acidic aqueous solution, which was ascribed to a 2e(-), 2H(+) reduction of the carbonyl group in the C-4 position. Actually, the carbonyl group C=O first underwent a 1e(-), 1H(+) reduction to form a neutral free radical, and the further 1e(-), 1H(+) reduction of the free radical was simultaneous with its following chemical reactions. When S(2)O(2-)(8) was present, the free radical of vitamin P was oxidized by both S(2)O(2-)(8) and its reduction intermediate, the sulfate radical anion SO(*-)(4), to regenerate the original, which resulted in the production of a polarographic catalytic wave of vitamin P. Based on this catalytic wave, a novel method for the determination of vitamin P was proposed. In 0.02 M tartaric acid-sodium tartrate (pH 3.3) buffer containing 5.0 x 10(-3) M K(2)S(2)O(8), the peak potential of the catalytic wave was -1.42 V (vs SCE) and the peak current was rectilinear to the vitamin P concentration in the range of 8.0 x 10(-9)-1.0 x 10(-6) M (r = 0.9994, n = 13). The catalytic wave of 2.0 x 10(-7) M vitamin P enhanced the polarographic current 70 times compared with the corresponding reduction wave. The detection limit was 2.0 x 10(-9) M, and the relative standard deviation at the 2.0 x 10(-7) M level was 0.7% (n = 15). The proposed method was used for the determination of vitamin P content in the pharmaceutical preparation of tablets and the medicinal plant Sophora japonica L. without previous separation.     

Pulsed high-field gradient in vivo NMR spectroscopy to measure diffusional water permeability in Corynebacterium glutamicum

Pulsed high-field gradient in vivo NMR spectroscopy was used to measure diffusional water permeability in cell suspensions of the Gram-positive bacterium Corynebacterium glutamicum. Two different regions of H2O mobility were detected. One was characterized by the apparent coefficient of self-diffusion, D(1 app) = (4.6-12.7)x10(-8) cm(2) s(-1), depending on the observation time t. The other region was characterized by D(2) = 1.4x10(-5) cm(2) s(-1). The value of D(2) was similar to the diffusion coefficient of H2O in free water and in extracellular biological fluids. Restricted diffusion could be demonstrated for the slower process (D(1)). It was attributed to the cytoplasm of the cells. The membrane permeability, P(d H2O), for C. glutamicum was (4.8+/-0.4)x10(-3) cm s(-1). It compared favorably with values reported for human erythrocytes and was higher by a factor of about 100 compared to the diffusional permeability for ethanol, P(d ethanol), in Zymomonas mobilis. Addition of HgCl2, a water channel inhibitor in eukaryotes, decreased P(d H2O) in C. glutamicum by a factor of approximately 8. To our knowledge, these are the first functional studies of water transport in prokaryotes that yielded quantitative data, viz., transmembrane water permeability expressed through D(H2O) and P(d H2O).     

Detection of Escherichia coli O157:H7 using immunomagnetic separation and absorbance measurement

An assay system for detection of Escherichia coli O157:H7 was developed based on immunomagnetic separation of the target pathogen from samples and absorbance measurement of p-nitrophenol at 400 nm from p-nitrophenyl phosphate hydrolysis by alkaline phosphatase (EC 3.1.3.1) on the "sandwich" structure complexes (antibodies coated onto micromagnetic beads--E. coli O157:H7-antibodies conjugated with the enzyme) formed on the microbead surface. The effects of immunoreaction time, phosphate buffer concentration, pH and temperature on the immunomagnetic separation of E. coli O157:H7 from samples were determined and the conditions used for the separation were 1-h reaction time, 1.0 x 10(-2) M PBS, pH 8.0 and 33 degrees C in this system. The effects of MgCl(2) concentration, Tris buffer concentration, pH and temperature on the activity of alkaline phosphatase conjugated on the immuno-"sandwich" structure complexes were investigated after immunomagnetic separation of the target pathogen and the conditions used for the enzymatic amplification were 1.0 x 10(-4) M MgCl(2), 1.0 M Tris buffer, pH 8.0, 28 degrees C and 30-min reaction time during the assay. The selectivity of the system was examined and no interference from the other pathogens including Salmonella typhimurium, Campylobacter jejuni and Listeria monocytogenes was observed. Its working range was from 3.2 x 10(2) to 3.2 x 10(4) CFU/ml, and the relative standard deviation was 2.5-9.9%. The total detection time was less than 2 h.     

Studies of the reactivity of HO2/O2- with unsaturated hydroperoxides in ethanolic solutions

A study of the reactivity of HO2/O2- with unsaturated hydroperoxides/peroxides was carried out in a stopped-flow spectrophotometer equipped with an O2--generating plasma lamp. The results show that, in 80% aqueous ethanol solution containing either 0.05 M H2SO4 (for HO2 studies) or 0.005 M KOH (for O2- studies), these oxy-radicals do not react with oleic acid hydroperoxide, linoleic acid hydroperoxide, 1-hydroperoxy-2-cyclooctene, and tert-butyl allyl peroxide. These findings are discussed in the light of conflicting evidence concerning the reaction of HO2/O2- with organic hydroperoxides/peroxides.     

Effects of selective hypoglossal nerve stimulation on canine upper airway mechanics.

Electrical stimulation of the hypoglossal (XII) nerve has been demonstrated as an effective approach to treating obstructive sleep apnea. The physiological effects of conventional modes of stimulation (i.e., genioglossus activation or whole XII nerve stimulation), however, have yielded inconsistent and only partial alleviations of hypopneic or apneic events. Although selective stimulation of the multifasciculated XII nerve offers many stimulus options, it is not clear how these will functionally affect the upper airway (UAW). To study these effects, animal experiments in eight beagles were performed to investigate changes in the UAW resistance and critical pressure during simulated expiration (n = 4) and inspiration (n = 4). During expiration, nonselective XII nerve stimulation yielded the greatest improvement in UAW resistance (-0.66 +/- 0.11 cm H2O x l(-1) x min(-1)), compared with that for selective activation of the geniohyoid (-0.29 +/- 0.09 cm H2O x l(-1) x min(-1)), genioglossus (-0.31 +/- 0.12 cm H2O x l(-1) x min(-1)), and hyoglossus/styloglossus (0.37 +/- 0.06 cm H2O x l(-1) x min(-1)) muscles. For simulated inspiration, on the other hand, only whole XII nerve stimulation (-0.9 +/- 0.4 cm H2O) and coactivation of the genioglossus + hyoglossus/styloglossus muscles (-1.18 +/- 0.6 cm H2O) produced significant (P < 0.05) improvements in UAW stability (i.e., lowered critical pressure), compared with baseline (-0.52 +/- 0.32 cm H2O). The results of this study suggest that a multicontact nerve electrode can be used to achieve both UAW dilation and patency, comparable to that obtained with nonselective stimulation, by selectively activating the various branches of the XII nerve.     

An osmometer for aqueous biopolymer solutions

A sensitive osmometer for the study of the osmotic pressure of aqueous protein solutions is presented. Equilibrium is reached within 1 to 2 h and is reproducible to about 1 N.m-2 (= 0.01 cm H2O). The method allows the determination of dissociation constants of proteins down to 1 x 10(-8) M.     

Properties of a novel periplasmic catalase-peroxidase from Escherichia coli O157:H7

A subset of catalase-peroxidases are distinguished by their periplasmic location and their expression by pathogens. Kinetic and spectral properties have not been reported for any of these enzymes. We report the cloning, expression, isolation, and characterization of KatP, a periplasmic catalase-peroxidase from Escherichia coli O157:H7. Absorption spectra indicated a mixture of heme states dominated by the pentacoordinate and hexacoordinate high-spin forms. Apparent k(cat) values for catalase (1.8x10(4) s(-1)) and peroxidase (77 s(-1)) activities were greater than those of other catalase-peroxidases. However, apparent K(M) values for H2O2 were also higher (27 mM for catalase and 3 mM for peroxidase). Ferric KatP reacted with peracetic acid to form compound I (8.8x10(3) M(-1) s(-1)) and with CN(-) to form a ferri-cyano complex (3.9x10(5) M(-1) s(-1)) consistent with other catalase-peroxidases. The isolation and characterization of KatP opens new avenues to explore mechanisms by which the periplasmic catalase-peroxidases may contribute to bacterial virulence.     

Biphasic effect of hydrogen peroxide on skeletal muscle arteriolar tone via activation of endothelial and smooth muscle signaling pathways.

We hypothesized that hydrogen peroxide (H2O2) has a role in the local regulation of skeletal muscle blood flow, thus significantly affecting the myogenic tone of arterioles. In our study, we investigated the effects of exogenous H2O2 on the diameter of isolated, pressurized (at 80 mmHg) rat gracilis skeletal muscle arterioles (diameter of approximately 150 microm). Lower concentrations of H2O2 (10(-6)-3 x 10(-5) M) elicited constrictions, whereas higher concentrations of H2O2 (6 x 10(-5)-3 x 10(-4) M), after initial constrictions, caused dilations of arterioles (at 10(-4) M H2O2, -19 +/- 1% constriction and 66 +/- 4% dilation). Endothelium removal reduced both constrictions (to -10 +/- 1%) and dilations (to 33 +/- 3%) due to H2O2. Constrictions due to H2O2 were completely abolished by indomethacin and the prostaglandin H2/thromboxane A2 (PGH2/TxA2) receptor antagonist SQ-29548. Dilations due to H2O2 were significantly reduced by inhibition of nitric oxide synthase (to 38 +/- 7%) but were unaffected by clotrimazole or sulfaphenazole (inhibitors of cytochrome P-450 enzymes), indomethacin, or SQ-29548. In endothelium-denuded arterioles, clotrimazole had no effect, whereas H2O2-induced dilations were significantly reduced by charybdotoxin plus apamin, inhibitors of Ca(2+)-activated K+ channels (to 24 +/- 3%), the selective blocker of ATP-sensitive K+ channels glybenclamide (to 14 +/- 2%), and the nonselective K(+)-channel inhibitor tetrabutylammonium (to -1 +/- 1%). Thus exogenous administration of H2O2 elicits 1) release of PGH2/TxA2 from both endothelium and smooth muscle, 2) release of nitric oxide from the endothelium, and 3) activation of K+ channels, such as Ca(2+)-activated and ATP-sensitive K+ channels in the smooth muscle resulting in biphasic changes of arteriolar diameter. Because H2O2 at low micromolar concentrations activates several intrinsic mechanisms, we suggest that H2O2 contributes to the local regulation of skeletal muscle blood flow in various physiological and pathophysiological conditions.     

Application of the chemiluminescent assay to cytotoxicity test: detection of menadione-catalyzed H2O2 production by viable cells.

Menadione-catalyzed H2O2 production by viable cells is proportional to viable cell number. The correlations between the viable cell number and the concentration of H2O2 produced are determined with the rapid chemiluminescent assay (S. Yamashoji, T. Ikeda, and K. Yamashoji, 1989, Anal. Biochem. 181, 149-152). This chemiluminescent assay of viable cells requires only 10 min and is much faster than NR (neutral red) inclusion and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reduction assays, which require 3-5 h. When viable cells are incubated with antitumor drugs, detergents, mycotoxins, and glycoalkaloids for 24-48 h, a decrease in menadione-catalyzed H2O2 production in a dose- or incubation time-dependent manner is observed. In general, the 50% inhibition concentration determined by the chemiluminescent assay is lower than that determined by NR inclusion and MTT reduction assays, and the order of relative cytotoxic effects of agents is the same among these assays. Furthermore, clear cytotoxic effects are observed by the chemiluminescent assay after 1 h exposure of trypsinized cells to toxic compounds. Therefore, the chemiluminescent assay is expected to be more useful for the rapid detection of cytotoxic compounds than NR inclusion and MTT reduction assays.