In-Situ Imaging of Azoreductase Activity in the Acute and Chronic Ulcerative Colitis Mice by a Near-Infrared Fluorescent Probe.

Azoreductase (AzoR) is an essential reductive enzyme which is closely associated with the intestinal disease such as ulcerative colitis (UC). To date, only a few fluorescent probes for detecting AzoR activity in bacteria or cells have been constructed successfully. It is still challenging to design fluorescent probes for in situ monitoring AzoR in vivo. In this paper, a near-infrared (NIR) fluorescent probe (Cy-Azo) based on hemicyanine is designed and synthesized. The emission of the probe is located at 735 nm in the NIR region, which is favorable for its application in vivo. In addition, Cy-Azo shows high sensitivity to AzoR activity with 17-fold fluorescence enhancement and is particularly selective to AzoR over other enzymes, ions, and amino acids. Meanwhile, a possible response mechanism (the azo group in Cy-Azo is reduced by AzoR and cleaved resulting in the production of Cy-NH2) was proposed and verified by HPLC, MS, and theory calculation. In addition, based on low cell cytotoxicity, Cy-Azo is successfully applied in visualizing the activity of AzoR in two cell lines (HCT116 and HepG2 cells) and three types of bacteria (E. coli, S. aureus, and P. aeruginosa). In particular, due to its NIR emission, the probe can monitor AzoR activity in acute and chronic UC mice models. To our knowledge this is the first fluorescent probe for detecting AzoR activity in vivo, which can provide much important information for the diagnosis and treatment of UC.

Activation of protein phosphatase 2A is responsible for increased content and inactivation of respiratory chain complex i induced by all-trans retinoic acid in human keratinocytes.

This study presents the effect of all-trans retinoic acid (ATRA) on cell growth and respiratory chain complex I in human keratinocyte cultures. Keratinocyte treatment results in increased level of GRIM-19 and other subunits of complex I, in particular of their carbonylated forms, associated with inhibition of its enzymatic activity. The results show that in keratinocytes ATRA-promoted phosphatase activity controls the proteostasis and activity of complex I.

NOX1 mediates chemoresistance via HIF1α/MDR1 pathway in gallbladder cancer.

NADPH oxidase 1 (NOX1) plays a key role in tumorigenesis and metastasis through generating reactive oxygen species (ROS), an important intracellular signaling molecule. However, how it is expressed in gallbladder cancer (GBC) tissue sample and whether it associates with GBC chemoresistance have never been investigated. Our study analyzed the relationship between NOX1 expression and cisplatin-sensitivity both in vivo and in vitro. We found that reduced NOX1 expression promoted cisplatin efficiency in GBC-SD cells, whereas overexpression of which potentially inhibited the sensitivity of cisplatin in SGC-996 cells. Further study into the mechanism we found that increased NOX1 expression elevated intracellular ROS levels, which then activated HIF-1α/MDR1 pathway. These findings established NOX1 a novel accelerant of chemoresistance in GBC, and NOX1-targeted therapeutics might be exploited as a strategy for increasing the efficacy of cisplatin treatment.

Expression of GW112 and GRIM-19 in colorectal cancer tissues.

PURPOSE: To investigate the expression of GW112 and GRIM-19 in colorectal cancer tissues. METHODS: Immunohistochemistry and semi-quantitative PCR were used to simultaneously detect the levels of expression of GW112 and GRIM-19 in colorectal cancer tissues and normal colorectal tissues in 39 cases. RESULTS: Expression of GW112 protein and mRNA were significantly higher in colorectal cancer tissues than in normal tissues (p<0.05). Expression of GRIM-19 protein and mRNA were significantly lower in colorectal cancer tissues than in normal tissues (p<0.05). GW112 gene mRNA copy number(GAPDH gene mRNA copy number were 0.53 ± 0.21 and 1.81 ± 0.65 in normal colorectal tissues and colorectal cancer tissues respectively, and GRIM-19 gene mRNA copy number/GAPDH gene mRNA copy number were 1.15 ± 0.29 and 1.74 ± 0.0.44 in colorectal cancer tissues and normal colorectal tissues, respectively. Expression of GW112 gene mRNA was significantly higher in colorectal cancer tissues than in normal tissues (p<0.05), and expression of GRIM- 19 gene mRNA was significantly lower in colorectal cancer tissues than in normal tissues (p<0.05). CONCLUSION: High expression of GW112 in colorectal cancer tissues and reduced expression of GRIM-19 in colorectal cancer tissues may be associated with abnormal proliferation of cancer cells and are possibly one of the reasons for development of colorectal cancer, which can provide effective targets for clinical treatment of this disease.

Toward high-throughput screening of NAD(P)-dependent oxidoreductases using boron-doped diamond microelectrodes and microfluidic devices.

Although oxidoreductases are widely used in many applications, such as biosensors and biofuel cells, improvements in the function of existing oxidoreductases or the discovery of novel oxidoreductases with greater activities is desired. To increase the activity of oxidoreductases by directed evolution, a powerful screening technique for oxidoreductases is required. In this study, we demonstrate the utility of boron-doped diamond (BDD) microelectrodes for quantitative and potentially high-throughput measurement of the activity of NAD(P)-dependent oxidoreductases. We first confirmed that BDD microelectrodes can quantify the activity of low concentrations (10-100 pM) of glucose-6-phosphate dehydrogenase and alcohol dehydrogenase with a measuring time of 1 ms per sample. In addition, we found that poisoning of BDD microelectrodes can be repressed by optimizing the pH and by adding l-arginine to the enzyme solution as an antiaggregation agent. Finally, we fabricated a microfluidic device containing a BDD electrode for the first time and observed the elevation of the oxidation current of NADH with increasing flow rate. These results imply that the combination of a BDD microelectrode and microfluidics can be used for high-throughput screening of an oxidoreductase library containing a large number (>10(6)) of samples, each with a small (nanoliter) sample volume.

Highly enhanced electrochemiluminescence based on pseudo triple-enzyme cascade catalysis and in situ generation of co-reactant for thrombin detection.

In this work, a novel pseudo triple-enzyme cascade catalysis amplification strategy was employed to fabricate a highly sensitive electrochemiluminescence (ECL) aptasensor for thrombin (TB) detection. The signal amplification of the proposed aptasensor was based on the synergistic catalysis of glucose dehydrogenase (GDH) and hemin/G-quadruplex to generate a co-reactant in situ for the ECL of peroxydisulfate. Gold nanorods (AuNRs) conjugated with GDH and hemin/G-quadruplex were used as the secondary aptamer bioconjugate (TBA II) in this aptasensor. TB was sandwiched between TBA II and a thiol-terminated TB aptamer which self-assembled on the AuNRs-modified electrode. The pseudo triple-enzyme cascade catalysis was completed as follows: firstly, GDH could effectively catalyze the oxidation of glucose to gluconolactone, coupling with the reduction of ß-nicotinamide adenine dinucleotide hydrate (NAD(+)) into ß-nicotinamide adenine dinucleotide hydrogen (NADH). Then, the hemin/G-quadruplex acted as NADH oxidase, could rapidly oxidize NADH into NAD(+) accompanied with the generation of H2O2. Simultaneously, the hemin/G-quadruplex served as the horseradish peroxidase (HRP)-mimicking DNAzyme that further catalyzed the reduction of H2O2 to generate O2in situ. Then the O2 produced acted as the co-reactant of peroxydisulfate, resulting in significant ECL signal amplification and highly sensitive ECL detection. The proposed aptasensor showed a wide linear range of 0.0001-50 nM with a low detection limit of 33 fM (S/N = 3) for TB determination. The present work demonstrated that the novel strategy has great advantages of sensitivity, selectivity and reproducibility, which hold new promise for highly sensitive bioassays applied in clinical detection.

Substrains of inbred mice differ in their physical activity as a behavior.

Recent studies strengthen the belief that physical activity as a behavior has a genetic basis. Screening wheel-running behavior in inbred mouse strains highlighted differences among strains, showing that even very limited genetic differences deeply affect mouse behavior. We extended this observation to substrains of the same inbred mouse strain, that is, BALB/c mice. We found that only a minority of the population of one of these substrains, the BALB/c J, performs spontaneous physical activity. In addition, the runners of this substrain cover a significantly smaller distance than the average runners of two other substrains, namely, the BALB/c ByJ and the BALB/c AnNCrl. The latter shows a striking level of voluntary activity, with the average distance run/day reaching up to about 12 kilometers. These runners are not outstanders, but they represent the majority of the population, with important scientific and economic fallouts to be taken into account during experimental planning. Spontaneous activity persists in pathological conditions, such as cancer-associated cachexia. This important amount of physical activity results in a minor muscle adaptation to endurance exercise over a three-week period; indeed, only a nonsignificant increase in NADH transferase+ fibers occurs in this time frame.

Maturity-dependent chilling tolerance regulated by the antioxidative capacity in postharvest cucumber (Cucumis sativus L.) fruits.

BACKGROUND: The aim of the present study was to reveal the effect of fruit maturity on the chilling tolerance of cucumber (Cucumis sativus L.) fruit and the oxidative and antioxidative mechanisms involved. Chinese mini-cucumber (cv. Hangcui-1) fruits were harvested at four developmental stages: Immature (3-8 days after anthesis (DAA)), Mature (9-16 DAA), Breaker (17-22 DAA) and Yellow (35-40 DAA). All fruits were stored at 2 °C for 9 days and rewarmed at 20 °C for 2 days. RESULTS: The chilling injury index declined with advancing fruit maturity. High superoxide anion radical production rate and hydrogen peroxide content were observed in Immature fruits after cold storage and rewarming. Under chilling stress, superoxide dismutase showed an early response. Fruits at earlier maturity stages exhibited higher catalase, ascorbate peroxidase and monodehydroascorbate reductase activities and glutathione content as well as its redox state, and lower peroxidase, dehydroascorbate reductase and glutathione reductase activities and ascorbate content as well as its redox state. CONCLUSION: Fruits at the earlier developmental stage are more susceptible to chilling injury, which is related to increased oxidative stress. High peroxidase activity and ascorbate content and maintenance of the latter's redox state appear critical to the chilling tolerance of cucumber fruits at later developmental stages.

Macrophage deactivation. Altered kinetic properties of superoxide-producing enzyme after exposure to tumor cell-conditioned medium.

Incubation of activated mouse peritoneal macrophages with tumor cell-conditioned medium (TCM) results in their deactivation, as measured by ability to release reactive oxygen intermediates and kill protozoal pathogens. The mechanism of suppression by macrophage deactivation factor (MDF) was studied. Inhibition of H2O2 release could not be overcome by increasing the concentration of phorbol diesters used to trigger the respiratory burst. Deactivated macrophages consumed H2O2 at the same rate as activated cells (t1/2, 35-40 min for 25 nmol H2O2 per 10(6) peritoneal cells). They transported glucose with the same kinetics (Km, 1 mM; Vmax, approximately 100 nmol per 6 min per milligram cell protein), and maintained similar intracellular concentrations of NADPH and NADP (approximately 0.62 mM and approximately 0.11 mM, respectively), as measured by enzymatic cycling methods and determinations of the volume of cell water (3.6 microliter/mg cell protein). To study the kinetics of the PMA-triggered NADPH oxidase in cell lysates, mixed detergents were used (deoxycholate and Tween 20). These stabilized the oxidase for approximately 3.3-fold longer than deoxycholate alone, which was used in previous studies. Incubation of activated macrophages in MDF resulted in a marked increase in the Km of the oxidase for NADPH, from 0.06 mM to 0.67 mM. The Vmax fell approximately 1.7-fold. These kinetic changes, together with the measured intracellular concentration of NADPH, account quantitatively for the suppression of H2O2 release by deactivated macrophages, and are nearly the mirror image of the kinetic changes observed during macrophage activation.

Redox modifier genes in amyotrophic lateral sclerosis in mice.

Amyotrophic lateral sclerosis (ALS), one of the most common adult-onset neurodegenerative diseases, has no known cure. Enhanced redox stress and inflammation have been associated with the pathoprogression of ALS through a poorly defined mechanism. Here we determined that dysregulated redox stress in ALS mice caused by NADPH oxidases Nox1 and Nox2 significantly influenced the progression of motor neuron disease caused by mutant SOD1(G93A) expression. Deletion of either Nox gene significantly slowed disease progression and improved survival. However, 50% survival rates were enhanced significantly more by Nox2 deletion than by Nox1 deletion. Interestingly, female ALS mice containing only 1 active X-linked Nox1 or Nox2 gene also had significantly delayed disease onset, but showed normal disease progression rates. Nox activity in spinal cords from Nox2 heterozygous female ALS mice was approximately 50% that of WT female ALS mice, suggesting that random X-inactivation was not influenced by Nox2 gene deletion. Hence, chimerism with respect to Nox-expressing cells in the spinal cord significantly delayed onset of motor neuron disease in ALS. These studies define what we believe to be new modifier gene targets for treatment of ALS.

Colour characteristics of beef longissimus thoracis during early 72 h postmortem.

This study aimed to investigate the characteristics of beef meat colour during the initial 72 h postmortem to assess the possible effects of mitochondria on meat colour development. Bovine longissimus thoracis muscles (n = 5) were collected from one side of carcasses at 0.5, 4, 8, 12, 24, and 72 h postmortem and displayed in air for 6 days to measure colour and detect mitochondrial morphology and function. The results showed that beef had higher L⁎, a⁎, and b⁎ at 24 and 72 h postmortem and less colour change during 6 days of display in comparison with meat from 0.5, 4, and 8 h postmortem. Changes in mitochondrial morphology were observed at 24 and 72 h postmortem. Mitochondria presented a metabolic pattern early postmortem in that the MRA and NADH content did not change. Both the increase in beef colour stability and tissue oxygen consumption were observed within 72 h postmortem.

Human retinol dehydrogenase 13 (RDH13) is a mitochondrial short-chain dehydrogenase/reductase with a retinaldehyde reductase activity.

Retinol dehydrogenase 13 (RDH13) is a recently identified short-chain dehydrogenase/reductase related to microsomal retinoid oxidoreductase RDH11. In this study, we examined the distribution of RDH13 in human tissues, determined its subcellular localization and characterized the substrate and cofactor specificity of purified RDH13 in order to better understand its properties. The results of this study demonstrate that RDH13 exhibits a wide tissue distribution and, by contrast with other members of the RDH11-like group of short-chain dehydrogenases/reductases, is a mitochondrial rather than a microsomal protein. Protease protection assays suggest that RDH13 is localized on the outer side of the inner mitochondrial membrane. Kinetic analysis of the purified protein shows that RDH13 is catalytically active and recognizes retinoids as substrates. Similar to the microsomal RDHs, RDH11, RDH12 and RDH14, RDH13 exhibits a much lower Km value for NADPH than for NADH and has a greater catalytic efficiency in the reductive than in the oxidative direction. The localization of RDH13 at the entrance to the mitochondrial matrix suggests that it may function to protect mitochondria against oxidative stress associated with the highly reactive retinaldehyde produced from dietary beta-carotene.

Lactate-upregulation of lactate oxidation complex-related genes is blunted in left ventricle of myocardial infarcted rats.

Lactate modulates the expression of lactate oxidation complex (LOC)-related genes and cardiac blood flow under physiological conditions, but its modulatory role remains to be elucidated regarding pathological cardiac stress. The present study evaluated the effect of lactate on LOC-related genes expression and hemodynamics of hearts submitted to myocardial infarction (MI). Four weeks after MI or sham operation, isolated hearts of male Wistar rats were perfused for 60 min with Na+-lactate (20 mM). As expected, MI reduced cardiac contractility and relaxation with no changes in perfusion. The impaired cardiac hemodynamics were associated with increased reactive oxygen species (ROS) levels (Sham: 19.3±0.5 vs MI: 23.8±0.3 µM), NADPH oxidase (NOX) activity (Sham: 42.2±1.3 vs MI: 60.5±1.5 nmol·h-1·mg-1) and monocarboxylate transporter 1 (mct1) mRNA levels (Sham: 1.0±0.06 vs MI: 1.7±0.2 a.u.), but no changes in superoxide dismutase (SOD), catalase, NADH oxidase (NADox), and xanthine oxidase activities. Lactate perfusion in MI hearts had no additional effect on ROS levels, NADox, and NOX activity, however, it partially reduced mct1 mRNA expression (MI-Lactate 1.3±0.08 a.u.). Interestingly, lactate significantly decreased SOD (MI-Lactate: 54.5±4.2 µmol·mg-1·min-1) and catalase (MI: 1.1±0.1 nmol·mg-1·min-1) activities in MI. Collectively, our data suggest that under pathological stress, lactate lacks its ability to modulate the expression of cardiac LOC-related genes and the perfused pressure in hearts submitted to chronic MI. Together, these data contribute to elucidate the mechanisms involved in the pathogenesis of heart failure induced by MI.

Neutrophil nicotinamide adenine dinucleotide phosphate oxidase assembly. Translocation of p47-phox and p67-phox requires interaction between p47-phox and cytochrome b558.

Two of the cytosolic NADPH oxidase components, p47-phox and p67-phox, translocate to the plasma membrane in normal neutrophils stimulated with phorbol myristate acetate (PMA). We have now studied the translocation process in neutrophils of patients with chronic granulomatous disease (CGD), an inherited syndrome in which the oxidase system fails to produce superoxide due to lesions affecting any one of its four known components: the gp91-phox and p22-phox subunits of cytochrome b558 (the membrane-bound terminal electron transporter of the oxidase), p47-phox, and p67-phox. In contrast to normal cells, neither p47-phox nor p67-phox translocated to the membrane in PMA-stimulated CGD neutrophils which lack cytochrome b558. In one patient with a rare X-linked form of CGD caused by a Pro----His substitution in gp91-phox, but whose neutrophils have normal levels of this mutant cytochrome b558, translocation was normal. In two patients with p47-phox deficiency, p67-phox failed to translocate, whereas p47-phox was detected in the particulate fraction of PMA-stimulated neutrophils from two patients deficient in p67-phox. Our data suggest that cytochrome b558 or a closely linked factor provides an essential membrane docking site for the cytosolic oxidase components and that it is p47-phox that mediates the assembly of these components on the membrane.

Isoprenoid metabolism is required for stimulation of the respiratory burst oxidase of HL-60 cells.

The formation of oxygen radicals by phagocytic cells occurs through the activation of a multiple-component NADPH oxidase system. An unidentified low molecular weight GTP-binding protein has been proposed to modulate the activity of the NADPH oxidase. The low molecular weight GTP-binding proteins undergo posttranslational processing, including an initial covalent incorporation of an isoprenyl group. To test whether such an isoprenylation reaction might be required for the activity of the oxidase, we utilized compactin and lovastatin as inhibitors of the isoprenylation pathway. Treatment of DMSO-differentiated HL-60 cells with compactin produced a concentration-dependent inhibition of O2- formation in response to FMLP or phorbol myristate acetate. Cell viability was not affected nor was normal differentiation of the HL-60 cells into a neutrophil-like cell. The inhibitory effect of compactin was specifically prevented by addition of exogenous mevalonic acid to the HL-60 cells, indicating that the inhibitory effects of the drug were due to blockade of the pathway leading to isoprenoid synthesis. Addition of cholesterol, ubiquinone, or dolichol, which are also downstream products of the isoprenoid pathway, did not override the inhibitory effects of the drug. Subcellular fractions were prepared from compactin-treated cells, and the location of the compactin-sensitive factor was determined by complementation analysis in a cell-free NADPH oxidase system. The inhibited factor was localized to the HL-60 cytosol. These data suggest that an isoprenoid pathway intermediate is necessary for activation of the phagocyte NADPH oxidase. This is likely to represent the requirement for an isoprenoid moiety in the posttranslational modification of a low molecular weight GTP-binding protein. Our studies provide support for the involvement of such a low molecular weight GTP-binding protein in NADPH oxidase activation.

Breaking down the wall: fractionation of mycobacteria.

Mycobacterium spp. possess a complex cell envelope that consists of a plasma membrane, a peptidoglycan-arabinogalactan complex which in turn is esterified by mycolic acids that form with other non-bound lipids an asymmetric permeability barrier and an outer layer, also called a capsule in the case of pathogenic species. In order to investigate the functional roles of the cell envelope components, especially those of the major pathogens Mycobacterium tuberculosis and Mycobacterium leprae, it is necessary to fractionate the envelope by breaking the unusual wall that covers these bacteria. To this aim we first compared the efficiency of high pressure (cell disrupter/French press) with those of pathogen-compatible breakage methods such as sonication, bead beater and lysozyme treatment using the non-pathogenic Mycobacterium smegmatis. When the distribution of various specific markers of the cell envelope compartments, which include mycolic acids, arabinose, NADH oxidase activity, cell wall and cytosolic proteins, were determined sonication combined with lysozyme treatment was found to be the best option. The protocol of subcellular fractionation was then validated for pathogenic species by applying the method to Mycobacterium bovis BCG cells, an attenuated strain of the M. tuberculosis complex.

The effect of temperature in the range of -0.8 to 4°C on lamb meat color stability.

This study investigated effects of controlled freezing point storage (CFPS, -0.8°C) on lamb color stability compared with storage at 4°C (control). The muscle samples (n=5) of longissimus thoracis et lumborum from both carcass sides were assigned randomly to the two storage treatments and stored for 10days. The a⁎, b⁎, R630/580 and Chroma values of samples stored in CFPS were significantly higher than that of samples in control from day 2 to day 10 (P<0.05). Higher relative content of oxymyoglobin but lower relative content of metmyoglobin were observed in samples stored in CFPS treatment than those in control over 10days of storage (P<0.05). Meat samples stored in CFPS group had a significantly higher NADH content and metmyoglobin reductase activity than that in control group. In conclusion, ovine muscle stored in CFPS treatment for 10days demonstrated better color stability in comparison with those in 4°C storage.

Expression of NOX-I, gp91phox, p47phox and P67phox in the aorta segments above and below coarctation.

Aorta coarctation results in hypertension (HTN) in the arterial tree proximal to stenosis and, as such, provides an ideal model to discern the effects of different levels of blood pressure on the vascular tissue in the same animal. Compelling evidence has emerged supporting the role of oxidative stress as a cause of HTN. However, whether or not HTN (independent of the circulating humoral factors) can cause oxidative stress is less certain. NAD(P)H oxidase isoforms are the main source of reactive oxygen species (ROS) in the vascular tissues. We therefore compared the expressions of NOX-I, gp91phox and the regulatory subunits of the enzyme in the aorta segments residing above and below coarctation in rats with abdominal aorta banding. Rats were studied 4 weeks after aorta banding above the renal arteries or sham operation. Subunits of NAD(P)H oxidase and its NOX-I isoform as well as endothelial NO synthase (eNOS) and nitrotyrosine (footprint of NO oxidation by superoxide) were measured in the aorta segments above and below coarctation. The gp91phox, p47phox, and p67phox subunits of NAD(P)H oxidase, NOX-I isoform, eNOS and nitrotyrosine were markedly increased in the aorta segment above coarctation (hypertensive zone), but were virtually unchanged in the segment below coarctation. Since, excepting blood pressure, all other conditions were constant, the upregulation of NAD(P)H oxidase isoforms and the increased NO oxidation in the aorta segment above, but not below, coarctation prove that HTN, per se, independent of circulating mediators can cause oxidative/nitrosative stress in the arterial wall. These observations suggest that HTN control may represent a specific form of antioxidant therapy for hypertensive disorders.

Complex I deficiency in Persian multiple sclerosis patients.

Multiple sclerosis (MS) is a demyelinating disease of the central nervous system characterized by the morphological hallmarks of inflammation, demyelination and axonal loss. Until now, little attention has been paid to the contribution of mitochondrial respiratory chain enzyme activities to MS. In this study, kinetic analysis of mitochondrial respiratory chain complex I enzyme (measured as NADH-ferricyanide reductase) was performed on intact mitochondria isolated from fresh skeletal muscle in MS patients (n = 10) and control subjects (n = 11). Mitochondrial DNA common deletion and deletions were also tested in MS patients. Our findings showed that complex I activities were significantly reduced (P = 0.007) in patients compared with control. However, we could not find deletion in mtDNA of patients with MS. The presupposition of relationship between MS and mitochondrial disorders is due to predominant maternal transmission of MS in affected parent-child pairs, pathoaetiological role of respiratory chain dysfunction in multisystem disorders and important role of it in neurodegenerative disorders, a number of patients such as LHON or other mtDNA abnormality with developed neurological symptoms indistinguishable from MS and similarity of clinical symptoms in mitochondrial disorders to those of MS. This study suggested that a biochemical defect in complex I activity may be involved in pathogenesis of MS.

Metabolism of 3-tert-butyl-4-hydroxyanisole by microsomal fractions and isolated rat hepatocytes.

3-tert-Butyl-4-hydroxyanisole is oxidatively metabolized in the presence of rat liver microsomes, reduced nicotinamide adenine dinucleotide phosphate, and oxygen to yield tert-butylhydroquinone, tert-butylquinone, and a polar metabolite(s). In the presence of human and rat liver microsomes or eight purified cytochrome P-450 isozymes reconstituted with NADPH-cytochrome P-450 reductase, this phenolic antioxidant is converted to the oxidoreduction-active metabolite, tert-butylquinone, that can stimulate the NADPH oxidase activities of these preparations by 2- to 7-fold. The rate of formation of each of the metabolites of 3-tert-butyl-4-hydroxyanisole was increased by pretreatment of rats with either 5,6-benzoflavone or phenobarbital. In addition the tert-butylhydroquinone and tert-butylquinone concentrations in solution reached apparent steady-state levels during metabolism; the steady-state concentrations were also increased by various animal pretreatment regimens. Furthermore it was shown that the metabolism of 3-tert-butyl-4-hydroxyanisole yielded material which was covalently bound to protein. In the presence of glutathione the rates of formation of the polar metabolite(s) were enhanced 3- to 4-fold, while covalently bound products were nearly stoichiometrically decreased. The increase in the amount of polar metabolite was due to the formation of a 3-tert-butyl-4-hydroxyanisole-glutathione conjugate. 3-tert-Butyl-4-hydroxyanisole was also oxidatively metabolized by rat lung microsomes to yield the polar metabolite(s) and tert-butylhydroquinone. The polar metabolite(s), tert-butylquinone, and tert-butylhydroquinone were also shown to be formed in isolated hepatocyte suspensions. They could be found as either the free hydroquinone, the sulfate conjugate, the glucuronide conjugate, and polar metabolites, presumedly the 3-tert-butyl-4-hydroxyanisole-glutathione conjugate. The total tert-butylhydroquinone concentration attained a steady-state level in a manner similar to that seen with the microsomal suspensions. In addition 3-tert-butyl-4-hydroxyanisole itself formed sulfate and glucuronide conjugates, the glucuronide being the major product.