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PubMed1985

Increased lipoperoxide value and glutathione peroxidase activity in blood plasma of type 2 (non-insulin-dependent) diabetic women.

The lipoperoxide values and glutathione peroxidase activity in blood plasma, along with the glutathione peroxidase, catalase and cupro-zinc superoxide dismutase activities in erythrocytes were investigated in 60 women with Type 2 (non-insulin-dependent) diabetes mellitus and in 71 healthy women. The mean lipoperoxide value and the mean plasma glutathione peroxidase activity in the diabetic patients were significantly higher than those in the control subjects (lipoperoxide p less than 0.001, plasma glutathione peroxidase activity p less than 0.01). The plasma glutathione peroxidase activities did not, however, correlate with the plasma lipoperoxide values. The erythrocyte glutathione peroxidase activity was approximately ten times higher than that of the plasma glutathione peroxidase activity, nor did they correlate with each other. In contrast to the findings of other authors on the activities of the protective enzymes in erythrocytes against oxidative damage, there were no significant differences of erythrocytes glutathione peroxidase, catalase and superoxide dismutase activities between diabetic and control women.

Adult↗
PubMed2003

Peroxidase and superoxide dismutase activities in fig leaves in response to ambient air pollution in a subtropical city.

Urban air pollution is a serious problem in both developing and developed countries, and antioxidant enzyme activities in plants have been suggested as a useful bioindicator of air pollution. In this study, the seasonal and spatial variability of peroxidase and superoxide dismutase activities were measured in leaves of Ficus microcarpa at eight sampling sites in the Taipei metropolitan area and one background site in rural area at each month for a year. The spatial pattern of peroxidase activity in figs collected from the Taipei metropolitan area was similar to the spatial pattern of O3 concentration in the Taipei metropolitan area. The peroxidase activities of Ficus microcarpa were significantly higher at sampling sites from the outer zone of the metropolitan area than those from the inner zone of the metropolitan area in spring and summer. On the other hand, the spatial pattern of superoxide dismutase activity in fig leaves did not show significant differences between the inner and outer zones of the Taipei metropolitan area. In addition, peroxidase activities, but not superoxide dismutase activities, of Ficus microcarpa were significantly higher in sites with high traffic density than those in low traffic density sites. Even though peroxidase activities in Ficus microcarpa tended to be higher in high traffic density sites or some sites with high ozone concentration, site-specific changes of peroxidase activity in Ficus microcarpa due to O3 pollution were not clearly observed in this study. Based on these results, neither peroxidase nor superoxide dismutase in Ficus microcarpa is a sensitive bioindicator for O3 pollution, although peroxidase shows some potential to be used as a general bioindicator of air quality.

Air Pollutants↗
PubMed2006

A quantitative evaluation of peroxidase inhibitors for tyramide signal amplification mediated cytochemistry and histochemistry.

Many peroxidase inhibitors have been used in horseradish peroxidase (HRP) mediated immunostaining and in situ hybridization to quench background peroxidase activity. However, the efficacy of these inhibitors has been controversial, partially due to the lack of a quantitative study. Tyramide signal amplification (TSA) is much more sensitive than other HRP-mediated methods but its super-sensitivity also demands effective inhibition of background peroxidase activity. In searching for an effective peroxidase inhibitor, we have systematically evaluated the efficacy of several peroxidase inhibitors by quantifying the fluorescence intensity in cultured fibroblasts and tissue sections treated with the inhibitors. For cultured cells, 0.05 mM of phenylhydrazine and 1 unit/ml of glucose oxidase gave only moderate inhibition of HRP activity while 1 mM of sodium azide (NaN(3)), 3% of hydrogen peroxide (H(2)O(2)), NaN(3)/H(2)O(2) combined and 0.02 N hydrochloric acid (HCl) provided more complete inhibition. However, the inhibitory effect of NaN(3)/H(2)O(2) is reversible upon removal of the inhibitors and followed by incubation and wash to mimic antibody interactions. Similar results were obtained from rat skin wound tissues that have strong endogenous peroxidase activity. Our results recommend the use of HCl and caution the use of phenylhydrazine, glucose oxidase, NaN(3) and H(2)O(2) as potent peroxidase inhibitors.

Animals↗
PubMed1998

Infiltration of peroxidase-producing eosinophils into the lamina propria of patients with ulcerative colitis.

Little information is available to explain the pathogenesis of ulcerative colitis (UC). In this study, we focused on eosinophils in the lamina propria of the mucosa of patients with UC in the active phase. Biopsy specimens were taken from 17 patients with UC in the active phase, 17 in the inactive phase, and 20 control patients, and submitted for histochemical staining for peroxidase and chloroacetate esterase for microscopic examination. Both peroxidase-producing and chloroacetate esterase-producing cells in the lamina propria increased markedly in the active phase (8.3 +/- 3.1/0.01 mm2 and 6.6 +/- 2.7/0.01 mm2, respectively), compared with values in the inactive phase (0.8 +/- 0.6/0.01 mm2 and 1.3 +/- 0.6/0.01 mm2) or in the controls (1.3 +/- 0.8/0.01 mm2 and 1.3 +/- 0.4/0.01 mm2). Triple staining for peroxidase, chloroacetate esterase, and nonspecific esterase in the specimens revealed that the peroxidase-producing cells constituted a different population from that of neutrophils, macrophages/monocytes, or basophils. A monoclonal antibody specific for eosinophil peroxidase stained almost all infiltrated peroxidase-producing cells. These results indicated that eosinophils with strong peroxidase activity had infiltrated the lamina propria in UC, suggesting an allergic background and the involvement of released peroxidase in the mucosal damage characteristic of UC.

Carboxylic Ester Hydrolases↗
PubMed2003

Peroxidase from Catharanthus roseus (L.) G. Don and the biosynthesis of alpha-3',4'-anhydrovinblastine: a specific role for a multifunctional enzyme.

We have characterized a basic peroxidase with alpha-3',4'-anhydrovinblastine (AVLB) synthase activity, which was purified from Catharanthus roseus leaves. This enzyme was the single peroxidase isoenzyme detected in C. roseus leaves, and the single AVLB synthase activity detected in C. roseus extracts. It was observed that the monomeric substrates of AVLB, vindoline and catharanthine, are both suitable electron donors for the oxidizing intermediates of the basic peroxidase, compounds I and II. Results also showed that the reaction proceeds by a radical-propagated mechanism. Substrate specificity studies of the enzyme revealed that it was also able to oxidize several common peroxidase substrates, indicating a broad range of substrate specificity that is characteristic of class III plant peroxidases. Cytochemical studies showed that the enzyme is localized in C. roseus mesophyll vacuoles, in individual spots at the inner surface of the tonoplast. This particular location suggests a meaningful spatial organization that led to the proposal of a metabolic channeling model for the peroxidase-mediated synthesis of AVLB. The importance of this type of mechanism in the regulation of peroxidase isoenzyme functions in vivo is discussed. In view of the results obtained it is concluded that the basic peroxidase present in C. roseus leaves fulfills all the requirements to be considered as an AVLB synthase, and it is proposed that this specific function of this multifunctional enzyme is determined by metabolic channeling resulting from specific protein-protein interactions.

Catharanthus↗
PubMed2001

Characterization of a catalase-peroxidase from the hyperthermophilic archaeon Archaeoglobus fulgidus.

A putative perA gene from Archaeoglobus fulgidus was cloned and expressed in Escherichia coli BL21(DE3), and the recombinant catalase-peroxidase was purified to homogeneity. The enzyme is a homodimer with a subunit molecular mass of 85 kDa. UV-visible spectroscopic analysis indicated the presence of protoheme IX as a prosthetic group (ferric heme), in a stoichiometry of 0.25 heme per subunit. Electron paramagnetic resonance analysis confirmed the presence of ferric heme and identified the proximal axial ligand as a histidine. The enzyme showed both catalase and peroxidase activity with pH optima of 6.0 and 4.5, respectively. Optimal temperatures of 70 degrees C and 80 degrees C were found for the catalase and peroxidase activity, respectively. The catalase activity strongly exceeded the peroxidase activity, with Vmax values of 9600 and 36 U mg(-1), respectively. Km values for H2O2 of 8.6 and 0.85 mM were found for catalase and peroxidase, respectively. Common heme inhibitors such as cyanide, azide, and hydroxylamine inhibited peroxidase activity. However, unlike all other catalase-peroxidases, the enzyme was also inhibited by 3-amino-1,2,4-triazole. Although the enzyme exhibited a high thermostability, rapid inactivation occurred in the presence of H2O2, with half-life values of less than 1 min. This is the first catalase-peroxidase characterized from a hyperthermophilic microorganism.

Archaeoglobus fulgidus↗
PubMed2005

Isolation and characterization of a polymorphic stigma-specific class III peroxidase gene from Senecio squalidus L. (Asteraceae).

A novel stigma-specific class III peroxidase gene, SSP (Stigma-Specific Peroxidase), has been isolated from the self-incompatible daisy Senecio squalidus L. (Asteraceae). Expression of SSP in flower buds is developmentally regulated, with maximal levels of expression coinciding with anthesis, when stigmas are most receptive to pollen and when self-incompatibility is fully developed. In situ hybridization revealed SSP expression to be localized exclusively to the specialized secretory epidermal cells (papillae) of the stigma, which receive and discriminate pollen. SSP is therefore the first tissue-specific and cell-specific peroxidase gene identified in a plant. SSP belongs to a distinct clade of class III plant peroxidases that possess two introns, instead of the more normal situation of three conserved introns. The deduced amino acid sequence of SSP revealed a 27 amino acid signal peptide, suggesting that the SSP protein is secreted to the cell wall of the stigmatic papillae. In-gel peroxidase activity assays showed that SSP has relatively low peroxidase activity compared to other, as yet uncharacterized, peroxidases present in stigmatic extracts. Six SSP alleles have been cloned from different lines of S. squalidus carrying a range of self-incompatibility (S)-alleles but there was no consistent association between the presence of a particular SSP allele and S-genotype indicating that SSP is not the female determinant of SSI in S. squalidus. Nevertheless, the precise expression of SSP in stigmatic papillae suggests that it may have a more general function in pollen-stigma interactions, or alternatively in protection of stigmas from pathogen attack. Extensive database screens have identified homologues of SSP in other plant species, but available expression data for these genes indicates that none are flower-specific, suggesting that SSP represents a new functional type of class III peroxidase specific to the stigma. We discuss the possible function(s) of S. squalidus SSP in pollen-stigma interactions and in protection of stigmas from pathogen attack.

Alleles↗
PubMed1991

Characterization and partial amino acid sequence of human plasma glutathione peroxidase.

Human plasma glutathione peroxidase was purified to homogeneity and partially sequenced. Overlapping peptide fragments from three endopeptidase digests permitted the determination of one sequence of 32 contiguous amino acids and one sequence of 23 contiguous amino acids. Five additional unique peptide sequences without obvious overlaps were obtained. The sequence of 32 amino acid residues aligns with positions 82-113 of human cytosolic glutathione peroxidase with nine mismatches without gaps or insertions. The sequence of 23 amino acid residues aligns with positions 157-178 with six mismatches and an insertion of one residue. Three additional peptide sequences with no obvious sequence homology to glutathione peroxidase can be aligned based on the sequence of a cDNA clone encoding plasma glutathione peroxidase that was isolated from a human placental library. The plasma enzyme is a homotetramer composed of 21-kDa subunits which cannot reduce phospholipid hydroperoxides. These results indicate that the plasma glutathione peroxidase is distinct from both the classical cytosolic enzyme and the monomeric phospholipid hydroperoxide glutathione peroxidase. Only a negligible amount of glutathione peroxidase activity was detected in bile, indicating that the liver exports plasma glutathione peroxidase exclusively to the circulation.

Amino Acid Sequence↗
PubMed1989

Stable high level expression of human thyroid peroxidase in cultured Chinese hamster ovary cells.

An expression plasmid containing both human thyroid peroxidase and mouse dihydrofolate reductase cDNAs was transfected into chinese hamster ovary cells. The stably transformed cells constitutively expressed immunoreactive thyroid peroxidase on the cell surface. These cells were further used to establish a subline producing a large amount of thyroid peroxidase by selecting clones resistant to methotrexate. The molecular weight of the expressed thyroid peroxidase was the same as purified human thyroid peroxidase. This expressed protein had peroxidase activity when determined by guaiacol oxidation. Furthermore, the expressed thyroid peroxidase was immunoreactive to sera of patients with autoimmune thyroid disease in which autoantibodies to thyroid peroxidase appeared.

Animals↗
PubMed1986

Prevention of microsomal production of hydroxyl radicals, but not lipid peroxidation, by the glutathione-glutathione peroxidase system.

The glutathione-glutathione peroxidase system is an important defense against oxidative stress. The ability of this system to protect against iron-catalyzed microsomal production of hydroxyl radicals [oxidation of 4-methylmercapto-2-oxo-butyrate (KMBA)] and lipid peroxidation was evaluated. When rat liver cytosol was added to microsomes, strong inhibition against KMBA oxidation was observed. No protection was found when the cytosol was boiled or dialyzed. In the latter case, the addition of 0.5 mM glutathione restored almost complete protection, whereas in the former case protection could be restored by the addition of both glutathione and glutathione peroxidase. Cysteine could not replace glutathione, nor could glutathione S-transferase replace glutathione peroxidase. The glutathione-glutathione peroxidase system was also very effective in decreasing production of hydroxyl radicals stimulated by the addition of menadione or paraquat to microsomes. In the absence of cytosol, the addition of glutathione plus glutathione peroxidase was also effective; however, 5 mM glutathione was necessary to protect against KMBA oxidation. The effective concentration of glutathione required for protection was lowered when glutathione reductase was added to the system, to regenerate reduced glutathione. These results indicate that low concentrations of glutathione in conjunction with glutathione peroxidase plus reductase can be very effective in preventing microsomal formation of hydroxyl radicals catalyzed by iron and other toxic compounds. Microsomal lipid peroxidation was decreased 40% by glutathione alone, and this decrease was potentiated in the presence of glutathione reductase. In contrast to KMBA oxidation, the combination of glutathione plus glutathione peroxidase was not any more effective than glutathione alone in preventing lipid peroxidation. The differences in sensitivities of microsomal lipid peroxidation and KMBA oxidation to glutathione peroxidase suggest that these two processes can be distinguished from each other, and that free H2O2 and hydroxyl radicals are involved in KMBA oxidation, but not lipid peroxidation.

Animals↗
PubMed1987

Differential effect of cadmium on GSH-peroxidase activity in the Leydig and the Sertoli cells of rat testis. Suppression by selenium and the possible relationship to heme concentration.

In the testes of rats treated with cadmium acetate (7 or 20 mumoles/kg, 24 hr, s.c.), the activity of glutathione (GSH)-peroxidase was increased. At the same time, the activity of glutathione disulfide (GSSG)-reductase and the cellular GSH concentration were decreased significantly. The basal activity of peroxidase in the Leydig and the Sertoli cell populations was comparable. However, the magnitude of increases in the activities markedly differed in the two cell populations, with that of the Sertoli cells increasing to nearly 450% of the control value in response to treatment with 20 mumoles/kg Cd2+. In the Leydig cells, the enzyme activity in response to the same treatment increased to only about 170% of the control value. Cd2+ treatment increased the concentration of heme in the microsomal and the smooth and rough endoplasmic reticulum fractions of the whole testis, as well as in the microsomal fractions of the Leydig and the Sertoli cells. As with the peroxidase activity, the two cell populations vastly differed in their susceptibilities to Cd2+ treatment, with the Sertoli cells being more severely affected by the metal. In the Sertoli cells the microsomal heme concentration was increased by approximately 11-fold, whereas only a 2-fold increase in the Leydig cells was noted. The increase in GSH-peroxidase activity was not due to the peroxidase activity of GSH-S-transferases, insofar as an increase in transferase activity was not observed in the Leydig and the Sertoli cells. Treatment of rats with sodium selenite (10 mumoles/kg, s.c.) 30 min before Cd2+ treatment (20 mumoles/kg) fully suppressed the above-described spectrum of effects of Cd2+ in the testis. Also, sodium selenite at a lower dose of 5 mumoles/kg prevented an increase in GSH-peroxidase activity. It is hypothesized that increased GSH-peroxidase activity in the Leydig and the Sertoli cells constitutes an adaptive response to increased cellular levels of heme and to the free radicals generated by the heme molecule. Selenium prevents the increase in GSH-peroxidase activity by circumventing the increase in cellular heme concentration. The protection is believed to be related, at least in part, to increased production of cellular GSH.

Animals↗
PubMed1995

Horseradish peroxidase inhibition by thiouracils.

In this paper, the activity of horseradish peroxidase was further determined in the presence of several uracil derivatives. The rate of guaiacol peroxidation decreases in presence of 2-thiouracil and of 6-n-propyl-2-thiouracil, but is not changed by 6-n-propyluracil nor uracil. Thus, thiouracils inhibit horseradish peroxidase in a noncompetitive form. The binding of 6-n-propyl-2-thiouracil, 2-thiouracil, 6-n-propyluracil and uracil with horseradish peroxidase shows difference spectra due to changes in the environment of heme group in peroxidase. Then, the binding sites for these uracil derivatives are in an hydrophobic pocket at the heme periphery of peroxidase. The lesser binding rates were for uracil and propyluracil, which did not inhibit the peroxidase activity. These results point to the thiol group in uracils as responsible for the inhibition of peroxidase activity through interaction with an allosteric binding site, in peroxidase heme environment.

Antithyroid Agents↗
PubMed1990

Cloning and expression of a lignin peroxidase gene from Streptomyces viridosporus in Streptomyces lividans.

A lignin peroxidase gene was cloned from Streptomyces viridosporus T7A into Streptomyces lividans TK64 in plasmid pIJ702. BglII-digested genomic DNA (4-10 kb) of S. viridosporus was shotgun-cloned into S. lividans after insertion into the melanin (mel+) gene of pIJ702. Transformants expressing pIJ702 with insert DNA were selected based upon the appearance of thiostrepton resistant (tsrr)/mel-colonies on regeneration medium. Lignin peroxidase-expressing clones were isolated from this population by screening of transformants on a tsr-poly B-411 dye agar medium. In the presence of H2O2 excreted by S. lividans, colonies of lignin peroxidase-expressing clones decolorized the dye. Among 1000 transformants screened, 2 dye-decolorizing clones were found. One, pIJ702/TK64.1 (TK64.1), was further characterized. TK64.1 expressed significant extracellular 2,4-dichlorophenol (2.4-DCP) peroxidase activity (= assay for S. viridosporus lignin peroxidase). Under the cultural conditions employed, plasmidless S. lividans TK64 had a low background level of 2.4-DCP oxidizing activity. TK64.1 excreted an extracellular peroxidase not observed in S. lividans TK64, but similar to S. viridosporus lignin peroxidase ALip-P3, as shown by activity stain assays on nondenaturing polyacrylamide gels. The gene was located on a 4 kb fragment of S. viridosporus genomic DNA. When peroxidase-encoding plasmid, pIJ702.LP, was purified and used to transform three different S. lividans strains (TK64, TK23, TK24), all transformants tested decolorized poly B-411. When grown on lignocellulose in solid state processes, genetically engineered S. lividans TK64.1 degraded the lignocellulose slightly better than did S. lividans TK64. This is the first report of the cloning of a bacterial gene coding for a lignin-degrading enzyme.

Cloning, Molecular↗
PubMed1985

A comparison between wheat germ agglutinin-and choleragenoid-horseradish peroxidase as anterogradely transported markers in central branches of primary sensory neurones in the rat with some observations in the cat.

Horseradish peroxidase conjugates of either the lectin wheat germ agglutinin or choleragenoid, the binding subunit of cholera toxin, were injected into the L5 spinal ganglion of adult rats. This enabled comparison of these two conjugates as anterograde tracers in the primary sensory system. After a postoperative survival of 4 h to 30 days, the rats were perfused and frozen sections from spinal and medullary regions receiving primary afferents were processed for horseradish peroxidase histochemistry with tetramethylbenzidine as the chromogen. Additional observations were made in two adult cats. Following injection of wheat germ agglutinin-horseradish peroxidase the labelling appeared mostly as small-sized granules. The concentration of labelled primary afferents in the grey matter of the spinal cord was greatest in the marginal zone and the substantia gelatinosa and less pronounced in the deep parts of the dorsal horn. Labelling was also found in a region lateral to the central canal and in the ventral horn. Following injection of choleragenoid-horseradish peroxidase the labelling appeared mainly as larger-sized granular profiles. The concentration of labelled primary afferents was greatest in the deep part of the dorsal horn and pronounced in a region lateral to the central canal and in the ventral horn. All these regions are known to receive large calibre fibres. The marginal zone and the substantia gelatinosa, known to receive fine calibre fibres, showed almost no labelling in the rat. In the cat, however, there was somewhat more labelling in the substantia gelatinosa. Labelling of neuronal cell bodies indicating transneuronal transport was seen after injection of wheat germ agglutinin-horseradish peroxidase. Transneuronal labelling did not seem to occur after injection of choleragenoid-horseradish peroxidase. The present findings show that wheat germ agglutinin-and choleragenoid-horseradish peroxidase give rise to markedly different labelling patterns. A possible explanation for the different labelling in the marginal zone and substantia gelatinosa could be that certain primary sensory neurones lack either receptors for choleragenoid on their neuronal plasma membrane or the ability to transport the choleragenoid-horseradish peroxidase complex.

Animals↗
PubMed2005

Purification, characterization and evaluation of extracellular peroxidase from two Coprinus species for aqueous phenol treatment.

Non-ligninolytic fungal peroxidases produced by Coprinus cinereus UAMH 4103 and Coprinus sp. UAMH 10067 were purified, characterized and evaluated as cost-effective alternatives to horseradish peroxidase for aqueous phenol treatment. Purified Coprinus peroxidases exhibited a molecular weight of 36 kDa on matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Although the catalytic properties of the two Coprinus peroxidases were nearly identical in both crude and purified forms, the stabilities were substantially different. The peroxidase from Coprinus sp. UAMH 10067 was more stable at 50 degrees C and under basic conditions (up to pH 10) than the enzyme from C. cinereus UAMH 4103. The former enzyme also performed better at pH 9 than the latter one in aqueous phenol treatment. The phenol removal efficiency of the Coprinus peroxidase was comparable to those of previously studied plant peroxidases. The broader working pH and higher thermal and alkaline stability of the peroxidase from Coprinus sp. UAMH 10067 may be advantageous for its application to industrial wastewater treatment.

Biodegradation, Environmental↗
PubMed2005

Role of peroxidases in Parkinson disease: a hypothesis.

Extensive research has been done to elucidate the underlying molecular events causing neurodegenerative diseases such as Parkinson disease, yet the cause and the individual steps in the progression of such diseases are still unknown. Here we advance the hypothesis that, rather than or in addition to inorganic radical molecules, heme-containing peroxidase enzymes may play a major role in the etiology of Parkinson disease. This hypothesis is based on the following considerations: (1) several heme-containing enzymes with peroxidase activity are present in the substantia nigra pars compacta; (2) these peroxidases have the ability to catalyze the oxidation of proteins and lipids; (3) certain heme peroxidases are known to destroy cells in vivo; (4) heme peroxidases have the stability and specificity that could account for the fact that specific molecules and cells are subject to damage in Parkinson disease, rather than a random destruction; (5) heme peroxidase activity could account for certain reactions in connection with parkinsonism that thus far have not been adequately explained; and (6) the participation of a heme peroxidase could explain some recent observations that are inconsistent with the oxyradical theory. The peroxidase-catalyzed oxidative pathway proposed here does not preclude the participation of apoptosis as an additional mechanism for cell destruction.

Animals↗
PubMed2006

PeroxiBase: a class III plant peroxidase database.

Class III plant peroxidases (EC 1.11.1.7), which are encoded by multigenic families in land plants, are involved in several important physiological and developmental processes. Their varied functions are not yet clearly determined, but their characterization will certainly lead to a better understanding of plant growth, differentiation and interaction with the environment, and hence to many exciting applications. Since there is currently no central database for plant peroxidase sequences and many plant sequences are not deposited in the EMBL/GenBank/DDBJ repository or the UniProt KnowledgeBase, this prevents researchers from easily accessing all peroxidase sequences. Furthermore, gene expression data are poorly covered and annotations are inconsistent. In this rapidly moving field, there is a need for continual updating and correction of the peroxidase superfamily in plants. Moreover, consolidating information about peroxidases will allow for comparison of peroxidases between species and thus significantly help making correlations of function, structure or phylogeny. We report a new database (PeroxiBase) accessible through a web server with specific tools dedicated to facilitate query, classification and submission of peroxidase sequences. Recent developments in the field of plant peroxidase are also mentioned.

Databases, Genetic↗
PubMed2002

Mechanism of peroxidase-mediated oxidation of carcinogenic o-anisidine and its binding to DNA.

2-Methoxyaniline (o-anisidine) is a urinary bladder carcinogen in both mice and rats. Since the urinary bladder contains substantial peroxidase activity, we investigated the metabolism of this carcinogen by prostaglandin H synthase (PHS), a prominent enzyme in the urinary bladder, and lactoperoxidase as model mammalian peroxidases. Horseradish peroxidase (HRP)-mediated oxidation of o-anisidine was also determined and compared with the reactions catalyzed by mammalian peroxidases. All three peroxidases oxidized o-anisidine via a radical mechanism. Using HPLC combined with electrospray tandem mass spectrometry, we determined that peroxidases oxidized o-anisidine to a diimine metabolite, which subsequently hydrolyzed to form a quinone imine. Two additional metabolites were identified as a dimer linked by an azo bond and another metabolite consisting of three methoxybenzene rings, which exact structure has not been identified as yet. Using [14C]-labeled o-anisidine, we observed substantial peroxidase-dependent covalent binding of o-anisidine to DNA, tRNA and polydeoxynucleotides [poly(dX)]. The 32P-postlabeling assay (a standard procedure and enrichment of adducts by digestion with nuclease P1 or by extraction into 1-butanol prior to 32P-labeling) was employed as the second method to detect and quantitate binding of o-anisidine to DNA. Using these versions of the 32P-postlabeling technique we did not observe any DNA adducts derived from o-anisidine. The o-anisidine-DNA adducts became detectable only when DNA modified by o-anisidine was digested using three times higher concentrations of micrococcal nuclease and spleen phosphodiesterase (MN/SPD). We found deoxyguanosine to be the target for o-anisidine binding in DNA using poly(dX) and deoxyguanosine 3'-monophosphate (dGp). A diimine metabolite of o-anisidine is the reactive species forming adducts in dGp. The results strongly indicate that peroxidases play an important role in o-anisidine metabolism to reactive species, which might be responsible for its genotoxicity, and its carcinogenicity to the urinary bladder in rodents. The limitation of the 32P-postlabeling technique to analyze DNA adducts derived from o-anisidine as a means to estimate its genotoxicity is discussed.

Aniline Compounds↗