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A highly sensitive microRNA (miRNA) biosensor that employs ruthenium oxide nanoparticle (RuO(2) NP)-initiated polymerization of 3,3'-dimethoxybenzidine (DB) and miRNA-templated deposition of an insulating poly(3,3'-dimethoxybenzidine) (PDB) film is described in this work. The biosensor was made of a mixed monolayer of oligonucleotide capture probes (CPs) and 4-mercaptoaniline on a gold electrode. Following hybridization with a RuO(2) NP-tagged target miRNA, a mixture of DB/H(2)O(2) in pH 5.0 0.10 M acetate buffer was applied to the biosensor. The RuO(2) NPs serve as polymerization initiator/catalyst for the polymerization of DB. And the hybridized anionic miRNA strands and free CPs serve as templates, guiding the deposition of PDB. The amount of the deposited PDB and its insulating power directly correlated to the concentration of the target miRNA in solution. Electrochemical impedance spectroscopic tests showed that a linear charge-transfer resistance-concentration relationship from 6.0 fM to 2.0 pM was attained after 60 min of incubation in the DB/H(2)O(2) mixture. There was no cross-hybridization between pre-miRNA and mature miRNA and very little cross-hybridization among closely related miRNA family members even at single-base-mismatched levels. This impedance-based biosensor offers an attractive alternative for miRNA expression profiling and may enable the development of a portable multiplexing miRNA profiling system.

The oxidation of ortho-dianisidine by membrane bound horseradish peroxidase in the presence of sodium dextran sulfate affords a dark-green insoluble product, identified as an unstable meriquinone intermediate previously reported in literature. Cationic and unsubstituted dextrans do not stabilize the intermediate. The highest yield of the intermediate is observed at pH 4.0-5.0 and concentration of dextran sulfate ca. 0.5%. New highly sensitive detection system for peroxidase has been developed on the basis of ortho-dianisidine oxidation to the meriquinone intermediate in the presence of sodium dextran sulfate. Under certain conditions, with lowered sodium dextran sulfate concentrations, a progressive further oxidation of the green intermediate to the yellow-brown final product is observed on passing to higher enzyme concentrations. This finding opens a possibility to develop a detection system in which the color of the mixture of reaction products serves as a measure of the enzyme concentration.

The bromate ion has been identified as an inorganic disinfection by-product (DPB) in water subjected to purification treatment. Its presence arises from the application of oxidation processes to water containing bromide through the action of oxidant agents such as the ozone used in ozonation processes. This ion has been identified as a possible carcinogen by the U.S. Environmental Protection Agency, which recommends a maximum concentration of 10 microgL(-1). The literature reports a broad range of methods for the analysis of bromate in water, among which ion chromatography is the one most widely used, with different detection systems. However, most of the methods described to date require state-of-the-art technology and costly instrumentation, such that they are not readily adaptable to routine analyses. The present work reports a procedure for the spectrophotometric determination of bromate based on the bromination reaction of 3-3' dimethoxybenzidine, o-dianisidine (ODA). The reaction is based on the formation of Br2 in the presence of excess bromide and the later bromination of ODA, generating a product that absorbs at 450 nm. The procedure was set up with Flow Injection Analysis and allows the determination of the analyte in the 8 microg L(-1)-3.3 mg L(-1) range, with a detection limit of 6.0 microg L(-1) and relative standard deviations (n=12, [BrO3-]=8.0 and 30.0 microg L(-1)) of 4.8% and 2.9%, respectively. The determination rate was 10-11 samples/hour.

Laccase production by solid state fermentation (SSF) using an indigenously isolated litter dwelling fungus Fusarium incarnatum LD-3 was optimized. Fourteen medium components were screened by the initial screening method of Plackett-Burman. Each of the components was screened on the basis of 'p' (probability value) which was above 95% confidence level. Ortho-dianisidine, thiamine HCl and CuSO(4) . 5 H(2)O were identified as significant components for laccase production. The Central Composite Design response surface methodology was then applied to further optimize the laccase production. The optimal concentration of these three medium components for higher laccase production were (g/l): CuSO(4) . 5 H(2)O, 0.01; thiamine HCl, 0.0136 and ortho-dianisidine, 0.388 mM served as an inducer. Wheat straw, 5.0 g was used as a solid substrate. Using this statistical optimization method the laccase production was found to increase from 40 U/g to 650 U/g of wheat straw, which was sixteen times higher than non optimized medium. This is the first report on statistical optimization of laccase production from Fusarium incarnatum LD-3.

Summary The peroxidases of winter rye ( Secale cereale L.) seedlings were characterized by means of pH optimum, compartmentation and changes in activity and isoperoxidase pattern during primary leaf development. Peroxidase activity was measured with 9 different hydrogen donors in order to find donor-associated peculiarities. There are a number of parameters that depend on hydrogen donors: i) pH optima; ii) time course; iii) distribution along the leaf; iv) cellular compartmentation and v) isoperoxidase pattern. Gradual changes were found for hydrogen donors according to the character of compartmentation and distribution of peroxidase activity during leaf development, in a sequence dianisidine — guaiacol — catechol — vanillin — ascorbate. In contrast to dianisidine peroxidase activity, which was at a high level in the extracellular fraction of young tissues and sharply increased in older ones, ascorbate peroxidase activity was localized only in protoplasts of juvenile meristematic cells at the basis of the growing leaf. Extraction of tissues with a low ionic strength buffer (0.1 M Na-phosphate, pH 7.8) liberated the main portion (86–91 %) of total peroxidase activity. Both anionic and cationic isoperoxidases were localized intracellularly and extracellularly. The increase in total soluble peroxidase activity during leaf development was associated with the increase in activity of all the presented isoforms, with the exception of A3 and A4, but not with an appearance of new ones.

With an assay that generates free radicals (FR) through photooxidation of dianisidine sensitized by riboflavin, 4 x 10(-5) M captopril, epicaptopril (SQ 14,534, captopril's stereoisomer), zofenopril, and fentiapril [all sulfhydryl (-SH)-containing angiotensin-converting enzyme (ACE) inhibitors] were shown effective scavengers of nonsuperoxide free radicals whereas non-SH ACE inhibitors were not. Captopril was a more effective FR scavenger at pH 5.0 than at pH 7.5. Captopril (2 x 10(-5) M) also scavenged the other toxic oxygen species hydrogen peroxide and singlet oxygen and inhibited microsomal lipid peroxidation. Finally, captopril reduced the amount of superoxide anion-radical detected after neutrophils in whole blood were activated with zymosan, probably by inhibiting leukocyte superoxide production.

A novel method for spectrophotometrical measurement of myeloperoxidase (MPO) activity in plasma with o -dianisidine (DA) as a substrate is proposed. We have determined the optimal conditions, includ- ing the pH and hydrogen peroxide concentration, under which MPO is the main contributor to DA oxidation in plasma. Specific MPO inhibitors, salicylhydroxamic acid or 4-aminobenzoic acid hydrazide, are added to mea- sure the activity of other heme-containing peroxidases (mainly hemoglobin and its derivatives) and subtract their contribution from the total plasma peroxidase activity. Plasma MPO concentrations are quantified by a new enzyme-linked immunosorbent assay (ELISA) developed by us and based on the use of antibodies raised in rats and rabbits. The sensitivity of this ELISA is high: 0.2-250 ng/ml. A direct and significant ( P < 0.0001) correlation was observed between the MPO activities measured spectrophotometrically and the MPO level determined by ELISA in blood samples from 38 healthy donors. The proposed approaches to MPO measure- ment in plasma can be used to evaluate the enzyme activity and concentration, as well as the efficacy of mech- anisms by which MPO is regulated under physiological conditions and against the background of various inflammatory diseases.

The insulin-like growth factor (IGF) family is essential for normal embryonic growth and development and it is highly conserved through vertebrate evolution. However, the roles that the individual members of the IGF family play in embryonic development have not been fully elucidated. This study focuses on the role of IGF-2 in zebrafish embryonic development. Two igf-2 genes, igf-2a and igf-2b, are present in the zebrafish genome. Antisense morpholinos were designed to knock down both igf-2 genes. The neural and cardiovascular defects in IGF-2 morphant embryos were then examined further using wholemount in situ hybridisation, TUNEL analysis and O-dianisidine staining. Knockdown of igf-2a or igf-2b resulted in ventralised embryos with reduced growth, reduced eyes, disrupted brain structures and a disrupted cardiovascular system, with igf-2b playing a more significant role in development. During gastrulation, igf-2a and igf-2b are required for development of anterior neural structures and for regulation of genes critical to dorsal-ventral patterning. As development proceeds, igf-2a and igf-2b play anti-apoptotic roles. Gene expression analysis demonstrates that igf-2a and igf-2b play overlapping roles in angiogenesis and cardiac outflow tract development. Igf-2b is specifically required for cardiac valve development and cardiac looping. Injection of a dominant negative IGF-1 receptor led to similar defects in angiogenesis and cardiac valve development, indicating IGF-2 signals through this receptor to regulate cardiovascular development. This is the first study describing two functional igf-2 genes in zebrafish. This work demonstrates that igf-2a and igf-2b are critical to neural and cardiovascular development in zebrafish embryos. The finding that igf-2a and igf-2b do not act exclusively in a redundant manner may explain why both genes have been stably maintained in the genome.

In this study, for clarifying some possible mechanism of zinc toxicity, the effect of increasing amounts of Zn2+ ion on peroxidase activity was investigated in vitro in serum of cow. The H2O2-mediated oxidation of o-dianisidine was used to assess the peroxidase activity. Results show that after preincubation of serum with 0.2-20 mM Zn2+ concentration for 5 min, peroxidase activity was inhibited compared to the control and decreased rapidly with increasing metal concentrations. The enzyme was completely inhibited after 5 min preincubation in 30 mM Zn2+. When the preincubation of serum and Zn2+ was prolonged to 30 and 60 min, the enzymatic activity decreased more rapidly with increasing metal concentration. Extended exposure of the enzyme to lower concentrations of the metal brought about the same effect as shorter exposure to higher metal concentrations.

Background/Aims A low serum ceruloplasmin concentration is considered diagnostic for Wilson's disease. We aimed to evaluate an enzymatic test for ceruloplasmin oxidase activity and to compare it with the routinely used immunological ceruloplasmin measurement. Methods Serum ceruloplasmin was measured enzymatically with o -dianisidine dihydrochloride as substrate and immunologically. 110 Wilson's disease patients, 52 healthy controls, and 51 patients with impaired liver function not due to Wilson's disease were analyzed. Assay performance was tested by receiver operating characteristic curve analysis, McNemar test, and Spearman's rank correlation. Results The greatest sum of sensitivity and specificity was seen for the enzymatic ceruloplasmin assay at a cut-off point of 55U/L (93.6% and 100%, respectively) and for the immunologic assay at a cut-off point of 0.19g/L (93.6% and 78.8%, respectively). For healthy controls, the differences in specificity between both assays were statistically significant (McNemar, p =0.02). When additionally including patients with impaired liver function into the control group the specificity declined to 84.5% for the enzymatic assay and to 68.9% for the immunologic assay. The correlation between the enzymatic and immunologic assay was high in healthy controls ( r =0.94), but weaker in Wilson's disease patients ( r =0.70) and patients with impaired liver function not due to Wilson's disease ( r =0.65). Conclusions For the enzymatic assay the best cut-off point for predicting Wilson's disease was estimated to be 55U/L. Our data suggest that the enzymatic ceruloplasmin assay is superior to the immunologic assay in diagnosing Wilson's disease and should become the preferred method.

The biochemical characteristics of endogenous macrophage peroxidases (Po), and their relationship to myeloperoxidase (MPO), have heretofore been poorly understood and were examined in the current study. Rat alveolar macrophages (AM) were homogenized and fractionated by differential centrifugation into lysosomal and microsomal fractions. The Po activities in both fractions were separated using HPLC gel-filtration and two main activities were detected. One, in the lysosomal fraction, had a relative molecular mass ( M r ) of 58,000, while the other, associated with the microsomal fraction corresponded to M r 74,000. By comparison MPO from rat polymorphonuclear neutrophils (PMN) had M r 140,000. The 58- and 74-kDa Po activities also differed from MPO with respect to their apparent K m for H 2 O 2 and optimum pH of activity. Using o -dianisidine as a substrate, the K m for H 2 O 2 of the 58- and 74-kDa Po species was 0.4 and 0.19 m m , respectively, compared to 0.011 m m for MPO. Using monochlorodimedon, the corresponding values were 0.22 and 0.195 m m for the 58- and 74-kDa activities and 0.026 m m for MPO. With either substrate, MPO exhibited optimum activity at pH 5.4, compared to 5.2 for the 58-kDa activity and 4.8 for the 74-kDa species. Thus, rat AM contain two endogenous Po activities with biochemical characteristics distinct from those of MPO. Our findings suggest that these activities represent novel peroxidases that may play an important role in the oxidative metabolism of AM.

On the basis of previous observations, we attempted to characterize the effects of various products of phospholipid hydrolysis on neutrophil (PMN) respiratory burst activity. We studied the effects of phos- phorylcholine (PC) and phosphorylethanoline (PE) on superoxide anion production in PMN and in a cell free system. We found that PE but not PC inhibited measured superoxide anion, but that this was not due to inhibition of cellular superoxide generation but to scavenging of generated superoxide anion. Further, utilizing a system based upon the photo-oxidation of O-dianisidine sensitized by riboflavin, we were able to determine that the scavenging effect of PE was not superoxide dismutase (SOD)-like but rather a general scavenging or glutathione (GSH)-like effect. These data underscore the importance of identifying the mechanism of inhibition of superoxide generation by putative inhibitors as being due to a direct cellular effect or to a scavenging property.

To develop artificial hemoproteins that could lead to new selective oxidation biocatalysts, a strategy based on the insertion of various iron-porphyrin cofactors into Xylanase A (Xln10A) was chosen. This protein has a globally positive charge and a wide enough active site to accommodate metalloporphyrins that possess negatively charged substituents such as microperoxidase 8 (MP8), iron(III)-tetra-alpha4-ortho-carboxyphenylporphyrin (Fe(ToCPP)), and iron(III)-tetra-para-carboxyphenylporphyrin (Fe(TpCPP)). Coordination chemistry of the iron atom and molecular modeling studies showed that only Fe(TpCPP) was able to insert deeply into Xln10A, with a KD value of about 0.5 microM. Accordingly, Fe(TpCPP)-Xln10A bound only one imidazole molecule, whereas Fe(TpCPP) free in solution was able to bind two, and the UV-visible spectrum of the Fe(TpCPP)-Xln10A-imidazole complex suggested the binding of an amino acid of the protein on the iron atom, trans to the imidazole. Fe(TpCPP)-Xln10A was found to have peroxidase activity, as it was able to catalyze the oxidation of typical peroxidase cosubstrates such as guaiacol and o-dianisidine by H2O2. With these two cosubstrates, the KM value measured with the Fe(TpCPP)-Xln10A complex was higher than those values observed with free Fe(TpCPP), probably because of the steric hindrance and the increased hydrophobicity caused by the protein around the iron atom of the porphyrin. The peroxidase activity was inhibited by imidazole, and a study of the pH dependence of the oxidation of o-dianisidine suggested that an amino acid with a pKA of around 7.5 was participating in the catalysis. Finally, a very interesting protective effect against oxidative degradation of the porphyrin was provided by the protein.

A method was devised for detecting Lactobacillus mutants that produced amounts of dicarbonyl compounds different from those of their parents. The mutants were recognized by contacting colonies with glass filter paper, which later was sprayed with a solution of o -dianisidine and heated. The amount of dicarbonyl could be estimated by the intensity of the resulting brown spots. The amounts of glyoxal, methylglyoxal, and diacetyl produced by mutants and parents were determined by a high performance chromatography method after growth on two media. Mutants that differed in the production of all three dicarbonyls were noted. The expression of the mutation varied with the medium used.

Control of myeloperoxidase (MPO) may be an important consideration in disorders where excessive PMN elastase activity is a significant factor. There are, however, two mechanisms for the apparent regulation of MPO: 1) inhibit the enzyme directly, and ii) prevent the ensuing HOC1 induced oxidation by using a surrogate reducing agent. Appropriate methodology has been devised to distinguish true MPO inhibitors. With the exception of NaN3, many MPO inhibitors fall into the latter category and do not actually regulate the enzyme. Several potent organic inhibitors have been discovered, which, because of their structural selectivity, appear to associate specifically with a binding site on the enzyme, rather than attaching indiscriminately to a hydrophobic domain. By controlling the enzyme, these compounds protect alpha-1-PI from MPO induced damage, and could serve better than antioxidants to define the role of MPO in elastase induced injury.

3-3'-Dimethoxybenzidine (o-dianisidine, ODA) is oxidised by Br(2), among other oxidants, generating a compound that absorbs at 450 nm, while the non-oxidised reagent absorbs in the UV region. This reaction has been used previously as the basis of a continuous-flow method for the determination of bromate in ozonised water, with a detection limit lower than the maximum permitted for drinking water (10 microg L(-1)). The only interference observed in the method was that due to the chlorite ion (ClO(2)(-)), which generated the same ODA bromation product. Thus, in systems in which O(3) is employed as a disinfectant and disinfection is later enhanced with ClO(-) and ClO(2), there exists the possibility of finding BrO(3)(-) and ClO(2)(-), oxoanions generated as subproducts. The kinetic behaviour of the reaction between bromate and chlorite with bromine in acidic medium is different, allowing the proposal of a continuous-flow method for the simultaneous or sequential determination of both subproducts in water purification systems. None of the other subproducts interfered in the reaction. Kinetic differentiation was achieved by combining the temperature of the reaction and the length of the coils, after which it was possible to determine both analytes sequentially within a concentration range of 6-160 microg L(-1).

4-Aminobyphenyl (4-Ab), benzidine (Bz), and Bz congeners were evaluated for their ability to induce genotoxicity through an oxidative mechanism. The mutagenicity of these compounds was tested in the presence and absence of Aroclor 1254-induced rat S9 mix using Salmonella typhimurium tester strain TA102, which is sensitive to agents producing reactive oxygen species (ROS). In the presence of S9, 4-Ab, Bz, N-acetyl-benzidine, and 3,3 0 -dimethoxybenzidine were strongly mutagenic in TA102, whereas, 3,3 0 ,5,5 0 -tetra-methylbenzidine, 3,3 0 -dimethylbenzidine (O-tolidine), and N,N 0 -diacetylbenzidine were not mutagenic. In addition, 3,3 0 -dichlorobenzidine and 4,4 0 -dinitro-2-biphenylamine were directly mutagenic in TA102. Incorporation of the free radical and metal scavengers, catalase, superoxide dismutase (SOD), butylated hydroxytolune (BHT), and ethylenediamine tetraacetic acid (EDTA) reduced the mutagenic responses of 4-Ab and Bz, whereas heat-inactivated catalase and SOD had no effect. 4Ab and Bz also induced lipid peroxidation in the presence of S9 mix as shown using the thiobarbituric acid reactive substances assay. The results of this study indicate that 4-Ab and Bz induce mutations through the induction of ROS. Environ. Mol. Mutagen. 48:000–000, 2007. V C 2007 Wiley-Liss, Inc.

Pectinesterase (PE), from commercial orange peels or ammonium sulfate fractionation (50-80% saturation) of pea pods, was detected on polyacrylamide gels after native acidic polyacrylamide gel electrophoresis (PAGE) or sodium dodecyl sulfate (SDS)-PAGE by using the synthetic substrate beta-naphthyl acetate (beta-NA). The release of beta-naphthol (at 322 nm) from beta-NA was proportional to PE activity. The PE activity bands on polyacrylamide gels after native acidic PAGE or SDS-PAGE were stained with a combination of tetrazotized o-dianisidine and beta-NA. This fast and sensitive method can be used for enzyme purification and characterization.

Peroxidase oxidation of o-dianisidine, 3,3',5,5'-tetramethylbenzidine, and o-phenylenediamine in the presence of sodium dodecyl sulfate (SDS), an anionic surfactant, was spectrophotometrically studied. It was found that 0.1-100 mM SDS concentrations stabilize intermediates formed in the peroxidase oxidation of these substrates. The cause of the stabilization is an electrostatic interaction between positively charged intermediates and negatively charged surfactant.

We recently published in this journal an article entitled ‘‘Plasma oxidative stress biomarkers, nitric oxide and heat shock protein 70 in trained elite soccer players’’ (Banfi et al. 2005) but our results with the d-ROMs test (Diacron International, Grosseto, Italy) were believed invalid by Harma et al. (2006), who reported the findings of a previous paper of Erel (2005) comparing the above test with the new iron-o-dianisidine/xylenol orange assay. In this article we provide the following evidence strongly indicating that the above authors made some relevant errors in the assessment of the d-ROMs test validity. Firstly, the relationships between d-ROMs test and ferroxidase activity are neither new nor an original finding. Indeed, Alberti et al. (2000), just in the study leading to the definitive validation of the d-ROMs test by electron paramagnetic resonance (EPR) spectrometry, reported that the contribution of ceruloplasmin ferroxidase activity to the typical change of absorbance at 505 nm (DA505/min) in such a test (see below), although not negligible, appeared relatively small. This is because in the d-ROMs test the serum sample is 100-fold diluted. Moreover, Alberti et al. (2000) demonstrated that an amount of sodium azide equivalent to the maximum expected concentration of ceruloplasmin in the serum (i.e. 4.0 lM) causes only an approximately 7% decrease of the DA505/min value. This may explain the reported correlation between d-ROMs test results and ferroxidase activity (Erel 2005). Of course, the above correlation does not mean that d-ROMs test measures only and exactly the serum ferroxidase activity. Indeed, a 70% of the DA505/min value is still detectable even with a five-to-ten fold excess of the azide in the test (Alberti et al. 2000). Furthermore, the found correlation between ferroxidase activity and d-ROMs test (Erel 2005) is not a general rule. For instance, in hemodyalised patients d-ROMs test value was shown to be increased (Gerardi et al. 2002) while the ferroxidase activity of ceruloplasmin was reduced (Roxborough et al. 2000). In this respect, if right that d-ROMs test measures only the ferroxidase activity, Erel (2005) failed to demonstrate and/or explain how an increased ceruloplasmin activity may justify the experimental/clinical findings, which have been reported to be associated to an increased result of d-ROMs test. On the other hand, even granting, for the sake of an argument, that the above relation is the rule, this is not consistent with the recently recognised controversial role of ferroxidase (Shukla et al. 2006). About the lack of response of d-ROMs test during copper-induced lipoprotein autoxidation, apart from the questionable significance of this experimental approach, this finding may be explained by the fact that d-ROMs test is significantly inhibited by the adding of chelants (Alberti et al. 2000) which sequester the iron thus, inhibiting the Fenton’s reaction. Indeed, Erel (2005), in the autoxidation tests used a citrate buffer to obtain lowdensity lipoprotein and ethylendiamine tetra acetate (EDTA) to prevent further oxidation during the eating. This reply refers to the Letter to the Editors, at http://dx.doi.org/ 10.007/s00421-006-0202.

The feasibility and expediency of enzymatic methods application in food analysis is demonstrated by the example of ascorbic acid (AsA) determination in foods. Enzymatic determination of ascorbic acid is based on its action as a second substrate of horseradish (HRP) and peanut (PNP) peroxidases in the reactions of o -dianisidine (OD) and 3,3’,5,5’-tetramethylbenzidine (TMB) oxidation with hydrogen peroxide. The rates of the reactions are monitored spectrophotometrically by measuring the duration of the induction period on kinetic curves plotted in coordinates absorption-time. The proposed procedures are sensitive ( c L = 0.1 μM), simple, and rapid. The procedure using horseradish peroxidase and the reaction of TMB oxidation was used to determine ascorbic acid in fruit juices, milk and sour-milk products for babies’ nutrition.

A multicopper oxidase gene from Staphylococcus aureus was cloned and overexpressed. Purified recombinant multicopper oxidase oxidized the substrate 3,3′-dimethoxybenzidine in the presence of copper. Disruption of mco showed copper sensitivity and H 2 O 2 resistance, suggesting roles for mco in copper homeostasis and oxidative stress response. Northern blot analysis showed copper-induced mco transcription.

Cardiac thrombosis, one of the causes of sudden death throughout the world, plays a principal role in several cardiovascular diseases, such as myocardial infarction and stroke in humans. Data from studies of induction of chemical thrombosis in rodents help to identify substances in our environment that may contribute to cardiac thrombosis. Results for more than 500 chemicals tested in rodents in 2-year bioassays have been published as Technical Reports of the National Toxicology Program (NTP) 〈 http://ntp-server.niehs.nih.gov/index 〉. We evaluated atrial thrombosis induced by these chemical exposures and compared it to similarly induced lesions reported in the literature. Spontaneous rates of cardiac thrombosis were determined for control Fischer 344 rats and B6C3F1 mice: 0% in rats and mice in 90-day studies and, in 2-year studies, 0.7% in both genders of mice, 4% in male rats, and 1% in female rats. Incidences of atrial thrombosis were increased in high-dosed groups involving 13 compounds (incidence rate: 20–100%): 2-butoxyethanol, C.I. Direct Blue 15, bis(2-chloroethoxy)methane, diazoaminobenzene, diethanolamine, 3,3′-dimethoxybenzidine dihydrochloride, hexachloroethane, isobutene, methyleugenol, oxazepam, C.I. Pigment Red 23, C.I. Acid Red 114, and 4,4′-thiobis(6- t-butyl- m-cresol). The main localization of spontaneously occurring and chemically induced thromboses occurred in the left atrium. The literature survey suggested that chemical-induced atrial thrombosis might be closely related to myocardial injury, endothelial injury, circulatory stasis, hypercoagulability, and impaired atrial mechanical activity, such as atrial fibrillation, which could cause stasis of blood within the left atrial appendage, contributing to left atrial thrombosis. Supplementary data referenced in this paper are not printed in this issue of Toxicologic Pathology. They are available as downloadable files at http:taylorandfrancis.metapress.com/openurl.asp?genre=journal&issn=0192-6233 . To access them, click on the issue link for 33(5), then select this article. A download option appears at the bottom of this abstract. In order to access the full article online, you must either have an individual subscription or a member subscription accessed through www.toxpath.org .

An improved method is reported for the determination of catalase activity in honey. We tested different dialysis membranes, dialysis fluid compositions and amounts, dialysis temperatures, sample amounts, and dialysis times. The best results were obtained by dialysis of 7.50 g sample in a cellulose dialysis sack, using two 3 L portions of 0.015 M sodium phosphate buffer (pH 7.0) as the dialysis fluid at 4 degrees C for 22 h. As in previous methods, catalase activity was determined on the basis of the rate of disappearance of the substrate, H202, with the H202 determined spectrophotometrically at 400 nm in an assay system containing o-dianisidine and peroxidase. Trials indicated that the best solvent for the o-dianisidine was 0.2 M sodium phosphate buffer, pH 6.1; the best starting H202 concentration was 3 mM; the best HCl concentration for stopping the reaction was 6 N; and the best sample volume for catalase measurement was 7.0 mL. Precision values (relative standard deviations for analyses of 10 subsamples of each of 3 samples) were high, ranging from 0.48% for samples with high catalase activity to 1.98% for samples with low catalase activity.

Objectives: Oxidative damage of biomolecules occurs as a result of potent free radical reactions. In this study, a novel, colorimetric and fully automated method for measuring total antioxidant response (TAR) against potent free radical reactions is described. Design and methods: Potent free radical reactions were initiated with the production of hydroxyl radical (OH) via Fenton reaction, and the rate of the reactions was monitored by following the absorbance of colored dianisidyl radicals. Ortho-dianisidine (10 mM) and ferrous ammonium sulfate (45 μM) were dissolved in KCl/HCl solution (75 mM, pH 1.8). This mixture was named as Reagent 1 and hydrogen peroxide solution (7.5 mM) as Reagent 2. The OH, produced by mixing of R1 and R2, oxidized o-dianisidine molecules into dianisidyl radicals, leading to a bright yellow-brown color development within seconds. Antioxidants, present in the sample, suppressed the color formation to a degree that is proportional to their concentrations. The method was applied to an automated analyzer and analytical performance characteristics of the assay were determined. Results: Vitamin C and Trolox, reduced glutathione, bilirubin, uric acid and (±)-catechin solutions suppressed the color formation depending on their concentrations. Serum TAR against potent free radical reactions was lower in patients with chronic renal failure (1.13 ± 0.21 mmol Trolox equiv./l) and was higher in the individuals with neonatal icterus (2.82 ± 1.18 mmol Trolox equiv./l) than in healthy subjects (1.54 ± 0.15 mmol Trolox equiv./l). Conclusions: The easy, inexpensive and fully automated method described can be used to measure TAR of samples against potent free radical reactions.

The dynamics of structural and functional changes proceeding in a peroxidase molecule under the effect of temperature was studied. It was shown that peroxidase thermoinactivation proceeds in two consequent stages. Based on the analysis of enzyme peroxidase and oxidase activity and peroxidase spectral and buffer characteristics, it was established that at temperatures from 20 to 55 degrees C reversible conformation there occur changes of the hemoprotein molecule related to consequent unfolding and folding of the protein globule. The influence of temperature of 60 degrees C and above induces the protein globule unfolding and loosing of peroxidase activity.

A method using dianisidine is described to measure promptly the total plasma peroxidase (POX) activity of methemoglobin and other ferrihemes. Methemoglobin (35 mg/dL) is used as POX standard. Three-minute POX activities and total POX concentrations measured by a classic benzidine method were compared in a three-patient trial with citrulline supplementation. High POX values became progressively lower. In two of the patients, 3-minute POX activities were reduced more than total concentrations. Oral citrulline reduced harmful plasma ferriheme levels. Free ferriheme also exhibited POX activity. POX levels may be useful to judge severity in sickle cell anemia and to monitor therapeutic efficacy.

Although benzidine (Bz), 4-aminobiphenyl (ABP), 3,3'-dichlorobenzidine HCl (DCBz), 3,3'-dimethylbenzidine (DMBz), 3,3'-dimethoxybenzidine (DMOBz) and the benzidine congener-based dye trypan blue (TB) produce primarily frameshift mutations in Salmonella typhimurium, the base-substitution strain TA100 also responds to these compounds when S9 is present. Performing DNA sequence analysis, other investigators have shown that ABP induces frameshift, base-pair and complex mutations. Also, it was found that an uninduced hamster liver S9 preparation with glucose-6-phosphate dehydrogenase, FMN, NADH and four times glucose 6-phosphate gave a stronger mutagenic response than the conventional plate incorporation with rat S9 activation mixture for all the compounds tested. Using the base-specific tester strains of S. typhimurium (TA7001-TA7006) with the above reductive metabolic activation system, we surveyed these compounds for the ability to produce specific base-pair substitutions after reductive metabolism. Bz was weakly mutagenic in TA7005 (0.04 revertants/microg). ABP was mutagenic in TA7002 (1.4 revertants/microg), TA7004 (0.6 revertants/microg), TA7005 (2.98 revertants/microg) and TA7006 (0.4 revertants/microg). DCBz was weakly mutagenic in TA7004 (0.01 revertants/microg). It was concluded that benzidine induced some CG-AT transversions in addition to frameshift mutations. ABP induced TA-AT, CG-AT, and CG-GC transversions as well as GC-AT transitions. DCBz induced only GC-AT transitions. Because DMBz, DMOBz and TB were not mutagenic in this base-substitution mutagen detection system, their mutagenic activity was attributed strictly to frameshift mechanisms.

A sensitive and convenient method is described for estimating superoxide dismutase activity using a photochemical augmentation procedure. This method is applicable to both liquid assays and polyacrylamide gel electropherograms. The flux of superoxide is generated by illuminating a reaction mixture containing dianisidine and riboflavin by either a laser source or light from a fluorescent lamp. The oxidation of dianisidine, as sensitized by riboflavin, is enhanced by superoxide dismutase. The increase is linearly dependent on superoxide dismutase concentration. The photochemical reaction is allowed to proceed uninterrupted for a standardized optimum time and intensity of illumination and then terminated by addition of a buffer, 'finibuf', which stabilizes the chromophoric complex formed. This permits the spectrophotometric absorbance measurements of a number of samples collectively and also eliminates the interruption of illumination with the concomitant requirement of a spectrophotometer for constant recording of the absorbance. This method is of utility to both biochemists and clinicians.

Variable availability of the purified dihydrochloride salt of 3,3'-dimethoxybenzidine (DMB; ortho-dianisidine) led us to investigate the effects of reagent purity on the analytical results obtained when this reagent is used in the photometric determination of the disinfection byproduct bromate. After analyte ions are separated by ion chromatography, a solution of DMB (post-column reagent) is added to the eluate and the DMB is oxidized, thereby producing a chromophore detected by its absorbance. Although some commercial products of undefined grade performed well, others did not. Variability was also observed between lots of purified material. Sensitivity at low concentrations (< 5 micrograms L-1 BrO3-) varied by a factor of up to 10. In some cases, the lower limit of detection for photometric detection was greater than that obtained using conductivity detection, as high as 5-7 micrograms L-1 BrO3-. An impurity or several impurities are suspected to be responsible for deviations from linearity at low analyte concentrations. This investigation underscores the need for ensuring reagent purity in environmental analyses. Ideally, chemical manufacturers will meet the needs of analytical chemists who test potable water and begin producing a high grade material in sufficient quantities to meet monitoring requirements. The establishment of third-party standards for a spectrophotometric grade of DMB.2HCl would be helpful in ensuring that a variety of manufacturers could supply products of uniformly high quality that would be suitable for the measurement of bromate in public drinking water supplies.

This method is suitable for the determination of bromate residues in a variety of baked goods. The peer-verified method trial was performed on white bread, multigrain bread, and coffee cake spiked with known levels of potassium bromate. The analytical portion is extracted with deionized water to remove bromate from the bulk of the baked product. The aqueous extract is carried through a series of steps to remove co-extractives that would interfere with the liquid chromatography (LC) in the determinative step or hasten the deterioration of the LC column. The extract is filtered before passing it through a reversed-phase solid-phase extraction (SPE) column and a cation-exchange column in the silver form to remove lipids and chloride, respectively. Ultrafiltration is then used to remove proteins with molecular weights of >30 000 daltons. Finally, a cation-exchange column in the sodium form is used to remove silver ions from the extract. The determinative step uses LC with a reversed-phase column and an ion-pairing agent in the mobile phase. Detection is based on the post-column reaction of bromate with o-dianisidine to form an oxidation product that is quantitated spectrophotometrically at 450 nm. Overall agreement between the submitting and peer laboratories was quite good. For bromate levels of 10–52 ppb, overall mean recoveries were 76.9 and 78.8% for the submitting and peer laboratories, respectively. The standard deviations were higher for the results of the peer laboratory, probably because of the generally higher level of baseline noise present in the chromatograms. The results demonstrate that the method provides adequate accuracy with low-fat as well as high-fat foods. Bromate at levels as low as 5 ppb (ng/g) can be detected with the method.

Free radical (FR) mediated oxidation of low density lipoproteins (LDL) has been implicated in atherogenesis. Probucol is a lipid lowering agent with antioxidant properties which may protect the LDL from FR mediated damage. The specific mechanism by which probucol acts as an antioxidant has not previously been reported. We therefore studied the FR scavenging properties of probucol in vitro and results show that this drug is a powerful superoxide scavenger. This property may be relevant in its use as a lipid lowering drug in the retardation of the atherosclerotic process.

The effect of ofloxacin (CAS 82419-36-1), dapsone (CAS 80-08-0), rifampicin (CAS 13282-46-1) and clofazimine (CAS 2030-63-9) on the generation of superoxide anions was studied in vitro. The drugs were incubated with a superoxide generating system (photochemical reaction between riboflavin and dianisidine). The change in optical density was measured. The optical density was increased with dapsone and ofloxacin and decreased in presence of clofazimine and rifampicin. This result indicates that ofloxacin and dapsone have an antioxidant property, whereas clofazimine and rifampicin behave as pro-oxidants.

Antioxidants (digoxin, quercetin, and ascorbic acid) inhibited the oxidation of o-dianisidine catalyzed by horseradish peroxidase. Digoxin bound with the enzyme-substrate complex which included a stable semioxidized product of o-dianisidine and inhibited horseradish peroxidase by the anticompetitive pattern, while the enzyme inhibition by quercetin followed the mixed pattern. The oxidation of ascorbic acid and o-dianisidine with their combined presence in the reaction mixture was differentiated. o-Dianisidine was oxidized after the oxidation of 90-100% of ascorbic acid was completed. The rate of peroxidase oxidation of ascorbic acid in the presence of o-dianisidine was more than two orders higher than the rate of its individual oxidation and 1.5-2.0 times higher than the rate of o-dianisidine oxidation. Feasible inhibition mechanisms of peroxidase oxidation of o-dianisidine are discussed.

Erythropoietin is known to be an essential hemopoietic growth factor for maturation of erythroid progenitor cells. Like other hemopoietic growth factors, erythropoietin acts as a survival factor that supports maturation of the erythroid progenitor through the suppression of apoptosis. It is unclear whether erythropoietin can also induce differentiation, or if another external regulator is needed to initiate this process. The present study using murine cell lines revealed that maturation of the erythroid lineage requires costimulation by activin A and erythropoietin. Erythropoietin alone dose not induce differentiation and cells stimulated by activin A alone undergo apoptotic death. Costimulation with erythropoietin and activin A, however, rescues the cells from apoptotic death and permits differentiation. Two-step cultivation showed that cells pretreated with activin A no longer need activin A and differentiate in the presence of erythropoietin alone. The action of activin A commits the cell to death or to differentiation, and the presence of erythropoietin enables differentiation through suppression of apoptosis.

Benzidine and its 3,3′-diamino, 3,3′-dimethyl, 3,3′-dimethoxy, 3,3′-difluoro, 3,3′-dichloro, 3,3′-dibromo, 3,3′-dicarbomethoxy and 3,3′-dinitro derivatives together with 2-nitrobenzidine and 3-nitrobenzidine were compared for their in vitro and in vivo genotoxicity. Relative mutagenicity was established with Salmonella strains TA98, TA98/1,8-DNP 6 and TA100 with and without S9 activation. All the derivatives in the presence of S9 were more mutagenic than benzidine with 3,3′-dinitro- and 3-nitro-benzidine having the greatest mutagenicity. Mutagenicity in all 3 strains with S9 activation could be correlated to electron-withdrawing ability of substituent groups, as measured by the basicity of the amines. This correlation was explained on the basis that electron-withdrawing groups could favor the stability of the mutagenic intermediate N -hydroxylamine and also enhance the reactivity of the ultimate mutagenic species, the nitrenium ion. Mutagenicity was also correlated to the energy of the lowest unoccupied molecular orbitals ( E LUMO ). Hydrophobicity was found to have very limited effect on the relative mutagenicity of our benzidine derivatives. The in vivo endpoint was chromosomal aberrations in the bone-marrow cells of mice following intraperitoneal administration of benzidine and its derivatives. In contrast to the in vitro results, while all the amines were genotoxic in vivo, only the 3-nitro derivative had a significant increase in toxicity over benzidine.

The Effects of Prenatal Administration of Azo Dyes on Testicular Development in the Mouse: A Structure Activity Profile of Dyes Derived from Benzidine, Dimethylbenzidine, or Dimethoxybenzidine. Gray, L. E., Jr., and Ostby, J. S. With technical assistance from E. R. Marshall (1993). Fundam. Appl. Toxicol. 20, 177-183. In mice and rats, prenatal exposure to the dye Congo red permanently reduces the number of germ cells in male and female offspring. In the current investigation, nine other dyes structurally related to Congo red were examined for developmental testicular toxicity. In this study, the structural component of the dyes responsible for the prenatal induction of germ cell aplasia was identified. We found that only benzidine-based dyes altered testicular development and caused hypospermatogenesis in mice during adulthood. Dimethyl- and dimethoxybenzidine-based dyes were without effect. Pregnant mice were dosed orally on Days 8-12 of gestation with a benzidine-, dimethlybenzidine-, or a dimethoxybenzidine-based dye and the testes of 45- to 50-day-old male offspring were examined. The testes of postpubertal male offspring exposed to the benzidine-based dyes, Congo red, diamine blue, and Chlorazol Black E, were small and contained some tubules completely devoid of germ cells, but the dimethlybenzidine-based dyes, trypan blue, Evans blue, and benzopurpurin 4B, and the dimethoxybenzidine-based dye, Chicago sky blue, did not alter testicular development in this manner. Azoic diazo component 48, a dimethoxybenzidine congener, and two other diazo dyes, naphthol blue black and Sudan III, were also without effect on the germ cells. Experiments with Chlorazol Black E (CBE) indicate that the period of susceptibility in the male fetus is limited to the period of primordial germ cell migration and division. When CBE was administered on Days 8-10 of gestation it reduced testis weight after puberty by 30%, while treatment after Day 13 did not affect testicular function. It is interesting to note that the structure-activity relationship of the dyes for developmental toxicity following oral administration differs considerably from that produced by maternal ip administration.

Contribution of various hemoproteins to peroxidase oxidation of benzidine and its derivatives as well as effects of these substances on functional state of hepatocytes are discussed. Benzidine and its derivatives were shown to induce those forms of cytochrome P-450 which were involved in accelerated oxidation of the carcinogenic drugs studied as well as affected the glutathione transferase, NADPH-dependent glutathione reductase activities and the activity of antioxidant system enzymes. Increase in content of cytochrome P-450, glutathione-dependent enzymes and other effects specific for adult hepatocytes, which occurred in presence of aminobiphenyls, were accompanied by decrease in content of receptors to epidermal factor of growth regulating the hepatocytes proliferation.

Free radical mechanisms have been implicated in diabetic microangiopathy. Agents that scavenge free radicals may be beneficial. We assessed the scavenging ability of two sulphonylureas, gliclazide and glibenclamide, in vitro. The assay which employs o-dianisidine sensitised by riboflavin can be used to distinguish between superoxide scavengers and general scavengers. The former species lead to an augmentation while the latter has an inhibitory effect. The drugs were added in final concentrations of 0.5, 1.0, 2.5 and 5.0 micrograms/ml. The percentage inhibition (mean +/- S.D.) for each concentration of gliclazide respectively was 11.0 +/- 2.5%, 20.8 +/- 2.9%, 31.4 +/- 2.2% and 47.2 +/- 0.8%. Glibenclamide had no scavenging effects. The results demonstrate that gliclazide is a powerful general free radical scavenger in vitro. We postulate that this scavenging quality of gliclazide may be important in diabetes.

It was found that intoxication of animals with aminobiphenyls leads to the activation of such glutathione-dependent enzymes as glutathione-S-transferase and glutathione reductase. This is accompanied by the induction of activities of individual isoforms of the multifunctional family of glutathione-S-transferases. There was a decrease in the glutathione peroxidase activity after intoxication with benzidine derivatives. It was found that the GSH content in rat liver decreased after benzidine intoxication and sharply increased after effects of 3,3'-dimethylbenzidine and 3,3'-dimethoxybenzidine. In all cases studied there was a diminution in the level of diene conjugates. It was supposed that the specificity of the catalytic glutathione redox system reaction is due to structural peculiarities of the aminobiphenyls being injected. Analysis of functional pairs of glutathione-dependent enzymes revealed a certain imbalance in the antioxidant system function after aminobiphenyl poisoning.

Since various functions of phagocytes can be affected by protease inhibitors, the ability of alpha-1-proteinase inhibitor (alpha 1PI) and alpha-2-macroglobulin (alpha 2M) to modulate the H2O2 production by human polymorphonuclear leukocytes (PMNL) was studied. The preincubation of PMNL for 30 min with these protease inhibitors at concentrations which may occur in human blood diminished in a dose dependent manner their H2O2 generation induced with phorbol myristate acetate. At alpha 1PI 600 mg/dl and alpha 2M 800 mg/dl the H2O2 response decreased to 61 +/- 2 and 58 + 3% (p less than 0.001, n = 4) of the control value obtained with cells preincubated in phosphate buffered saline with glucose, respectively. Autologous serum alone and with addition of pure alpha 1 PI or alpha 2M also suppressed H2O2 release from PMNL. It is suggested that inhibition of H2O2 generation from PMNL may be another additional way by which these serum protease inhibitors may protect tissues (especially lungs) from acute injury related to inflammation.

Dimethoxybenzidine (DMO) and dimethylbenzidine (DM) are used to synthesize dyes such as C.I. Direct Blue 15 and C.I. Acid Red 114, respectively. These commercially used dyes are metabolically degraded to DMO or DM in the intestinal tract of rodents and subsequently DMO and DM are absorbed into the blood stream. Animals were exposed to DMO, DM, or the dyes in the drinking water. Tumors obtained from control and chemical-treated animals were examined for the presence of activated oncogenes by the NIH 3T3 DNA transfection assay. Activated oncogenes were detected in less than 3% (1/38) of the tumors from control animals whereas 68% (34/50) of the tumors from chemical-treated animals contained detectable oncogenes. Activated oncogenes were detected in both malignant (25/36) and benign (9/14) tumors from the chemically treated animals but only in one of 13 malignant tumors from the control animals. The presence of oncogenes in the chemically induced benign tumors suggests that oncogene activation was an early event in those tumors. Southern blot analysis of transfectant DNA showed that the transforming properties of the chemically induced rat tumor DNAs were due to the transfer of an activated H-ras (31/34) or N-ras (3/34) gene. One spontaneous rat tumor DNA was found to contain an activated H-ras gene. Oligonucleotide hybridization analysis indicated that the H-ras oncogenes from chemical-associated tumors contained mutations at codons 12, 13, or 61 whereas the spontaneously activated H-ras gene contained a point mutation at codon 61. These data suggest that activation of cellular ras genes by point mutation is an important step in the induction of tumors, at least in rats, by this class of benzidine-derived dyes. Moreover, in light of common histogenesis of the normal counterparts of many of the chemically induced neoplasms and histological evidence of varied tissue differentiation in some basal cell neoplasms, it is possible that most or all of the chemically induced neoplasms were derived from a common epidermal progenitor stem cell population.

A simple, rapid and inexpensive method (o-DD method) is described for the measurement of hydrogen peroxide released by human polymorphonuclear leukocytes (PMNL) stimulated with phorbol myristate acetate (PMA), n-formyl-methionyl-leucyl-phenylalanine (FMLP) and concanavalin A (Con A). The method is based on the horseradish peroxidase-catalysed oxidation of o-dianisidine by H2O2 which results in the formation of a compound exhibiting an increased absorbance at 470 nm. A linear relationship between the absorbance at 470 nm and the concentration of H2O2 was found in the 1-150 microM range. Using this assay the time course of H2O2 release by PMNL, the dependence of H2O2 release on cell number, the agonist concentration and the presence of cytochalasin B (4.8 micrograms/ml) were studied. The maximal PMNL H2O2 response was found for PMA at 10 ng/ml. Con A at 200 micrograms/ml, FMLP at 300 ng/ml and reached 39 +/- 1.5, 14 +/- 1.2, 2.2 +/- 0.7 nmol for 10(6) cells incubated for 60 min at 37 degrees C. respectively. FMLP under these conditions was a most potent stimulator of myeloperoxidase release (MPO). Cytochalasin B having a weak influence on FMLP- and Con A-mediated H2O2 production enhanced strongly their stimulatory effect on MPO release. It is suggested that measurement of the H2O2 release with the o-DD method could be useful for monitoring PMNL respiratory burst especially in the cases of low MPO release.