On the mode of action and biochemical properties of anti-inflammatory drugs. I

The horse-radish peroxidase-hydrogen peroxide (HRP-H₂O₂) catalyzed oxidation of o-dianisidine was inhibited by indomethacin, oxyphenbutazone, mefenamic acid, phenylbutazone and p-acetamidophenol. The greatest inhibitory effect (id₅₀ 0.014 mM) was obtained with indomethacin, and its mode of action in this system has been examined further. The results obtained suggest that indomethacin acted by competing with o-dianisidine for peroxidase and hydrogen peroxide or peroxidase hydrogen peroxide complexes.     

Inhibition of peroxidase activity by some non-steroidal anti-inflammatory drugs.

The non-steroidal anti-inflammatory drugs, indomethacin, flufenamic acid and naproxen inhibited thyroid peroxidase-catalyzed iodination of BSA in vitro. Inhibition by all three drugs was affected more effectively in a hydrogen peroxide generating system than in an incubation system in which hydrogen peroxide was added. Naproxen differed from the other two drugs in so far as it inhibited mainly hydrogen peroxide generation while its comparatively low inhibitory influence (>20%) on TPO was not dose-dependent. The inhibitory influence of these anti-inflammatory drugs was also observed when other peroxidases, such as lactoperoxidase, chloroperoxidase and horseradish peroxidase were used for catalyzing BSA iodination in a hydrogen peroxide generating system. No iodination of BSA was obtained with horseradish peroxidase when hydrogen peroxide was added instead of generated so that the inhibitory nature of these drugs could not distinguish between their direct effect on horseradish peroxidase or on hydrogen peroxide generation. However, in lactoperoxidase and chloroperoxidase-catalyzed BSA iodinations in non-H₂O₂-generating systems naproxen had no appreciable inhibitory influence below a 1 millimolar concentration. On another thyroid peroxidase activity, namely its catalyzing influence on the exchange reaction between inorganic iodide and organic iodine in diiodotyrosine, indomethacin and naproxen showed unappreciable effects lower than 1 mM concentrations. Similarly, on thyroid peroxidase and lactoperoxidasecatalyzed deiodination of thyroxine they were ineffective inhibitors. The data suggest that these antiinflammatory drugs are effective inhibitors of iodination reactions but ineffective inhibitors of deiodination reactions.     

Effect of Peroxide- Versus Alkoxyl-Induced Chemical Oxidation on the Structure,Stability, Aggregation,and Function of a Therapeutic Monoclonal Antibody

Current guidelines indicate that the effects of oxidation should be included as part of forced degradation studies on protein drugs. We probed the effect of 3 commonly used oxidants, hydrogen peroxide, tert-butyl hydroperoxide, and 2,2'-Azobis(2-amidinopropane) dihydrochloride (AAPH), on a therapeutic monoclonal IgG1 antibody (mAb8). Upon oxidation, mAb8 did not show noticeable changes in its secondary structure but showed minor changes in tertiary structure. Significant decrease in conformational stability was observed for all the 3 oxidized forms. Both hydrogen peroxide and tert-butyl hydroperoxide destabilized mainly the C_H2 domain, whereas AAPH destabilized the variable domain in addition to C_H2. Increased aggregation was found for AAPH-oxidized mAb8. In addition, a significant decrease in Fc receptor binding was observed for all 3 oxidized forms. Antibody dependent cell-mediated cytotoxicity, binding to target protein receptor, and cell proliferation activity were significantly reduced in the case of AAPH-oxidized mAb8. The presence of free methionine in the formulation buffer seems to alleviate the effect of oxidation. The results of this study show that the 3 oxidants differ in terms of their effects on the structure and function of mAb8 because of chemical modification of different sets of residues located in Fab versus Fc.     

Selenium- and non-selenium-dependent glutathione peroxidases in mouse liver

About 70% of the activity of mouse liver peroxidase utilizing H₂O₂ as well as t-butyl hydroperoxide as substrates is the selenium-dependent glutathione peroxidase. With cumene hydro-peroxide as substrate the sum of non-Se-dependent and Se-dependent enzymes has been measured. Se-deficient mice have about 30% of the total activity of Se-adequate mice, with cumene hydroperoxide as substrate. Glutathione S-transferase activity was not affected by Se deficiency. Fractionation on DEAE-Sepharose and subsequently on CM-Sepharose showed at least four transferase activities, and besides the Se-GSH-Px, two non-Se-GSH-Px activities detectable with cumene hydroperoxide that did not coincide with any of the transferases. For the major activity the apparent Michaelis constants at 1 mmole/l GSH were found to be 0.07mM for linoleic acid hydroperoxide, 10 mM for t-butyl hydro-peroxide, and 0.11 mM for cumene hydroperoxide. The ratios of the apparent maximum velocities of these hydroperoxides were 1 (linoleic): 100 (butyl) :3000 (cumene). This has the practical consequence that the cumene hydroperoxide-supported reaction is extremely favored under standard assay conditions. The results suggest that data based on only a single organic peroxide and hydrogen peroxide can lead to invalid conclusions as to the nature of the enzymes that are capable of catalyzing GSH-linked reduction of hydroperoxides.Subcellular fractionation showed that cytosol, mitochondria and microsomes contain both types of peroxidase, although in different proportions. The distribution as well as the kinetic properties suggest the Se-GSH-Px is exceedingly faster in reducing organic, including fatty acid, hydroperoxides.     

Peroxidative xenobiotic oxidation by partially purified peroxidase and lipoxygenase from human fetal tissues at 10 weeks of gestation

• 1.1. Present study reports the ability of partially purified peroxidase and lipoxygenase from human fetal tissues at 10 weeks of gestation to oxidize selected xenobiotics in vitro.
• 2.2. Peroxidase was found to oxidize four different chemicals in the presence of H₂O₂. Sodium azide and potassium cyanide inhibited peroxidase activity towards guaiacol in a concentration-dependent manner.
• 3.3. The dioxygenase and co-oxidase activities of lipoxygenase towards linoleic acid and four model xenobiotics, respectively, were observed. Both the catalytic activities of lipoxygenase were significantly inhibited by < 1.0 μM nordihydroguaiaretic acid.
• 4.4. These findings suggest that peroxidase and lipoxygenase may be important pathways for peroxidative xenobiotic oxidation in human fetal tissues.

     

Stability of some phenolic antioxidants in fatty preparations

Stability of butylated hydroxyanisole (BHA), propyl gallate (PG), trihydroxy butytophenone (THBP), tert-butyl hydroquinone (TBHQ) and a mixture of PG and BHA (1:1) in fatty preparations stored under three different conditions was studied. Each antioxidant was added to a fatty model to determine its degradation kinetics and its evolution during the oxidation of the fatty product. Their degradation was adequately described by a first-order kinetic process. An opposite relationship between the stability of antioxidants and their rate protection against oxidation of the fatty product was obtained. At 25°C, t₉₀ values ranged between 10 days for TBHQ and 61 days for BHA. On the other hand, compared when they were formulated alone, the increase of PG and the diminution of BHA degradation kinetics were observed. A degradation product arising from TBHQ was found in samples and stock solutions; an HPLC method for its identification is proposed with detection at 254 nm.     

The interaction of 5-hydroxytryptamine and related hydroxyindoles with horseradish and mammalian peroxidase systems.

5-Hydroxytryptamine (5-HT) was found to be a competitive inhibitor with respect to guaiacol, and a non-competitive inhibitor with respect to H₂O₂ in a guaiacol-H₂O₂-horseradish peroxidase system. Other indolic compounds possessing a hydroxyl group in the C5 position behaved similarly to 5-HT, but those devoid of a nuclear substituted hydroxyl group were poor inhibitors. 5-HT altered the spectral characteristics of peroxidase, a shift from 417 to 399 nm occurring when an equimolar ratio of 5-HT and H₂O₂ is present. During such interaction 5HT fluorescence is lost and a yellow-brown product is formed. Attempts to characterize this product were only partially successful but it is suggested that it may possibly possess a dimeric structure. The formation of this product from ¹⁴C 5-HT could be demonstrated in disrupted mammalian spleen preparations but not in spleen slices or brain synaptosomes.     

Mediation of the antilipolytic and lipogenic effects of insulin in adipocytes by intracellular accumulation of hydrogen peroxide

Exposure of rat adipocytes to insulin causes activation of a pyridine nucleotide oxidase at the internal side of the plasma membrane, generating hydrogen peroxide (S. P. Mukherjee and W. S. Lynn, Fedn Proc.35, 1694 (1976); S. P. Mukherjee and W. S. Lynn, Archs Biochem. Biophys.184, 69 (1977). Evidence was also presented that intracellular utilization of H₂O₂ by the peroxidative pathways of glutathione peroxidase and catalase is coupled with glucose oxidation via the pentose phosphate pathway [S. P. Mukherjee, R. H. Lane and W. S. Lynn, Biochem. Pharmac.27, 2589 (1978)]. The relationship between the glucose-independent insulin effect on H₂O₂ production and its metabolic role is evaluated on the basis of formate oxidation, lipogenesis, antagonism of lipase activity and lowering of cellular levels of cyclic 3',5'-adenosine monophosphoric acid (cAMP). These measures of the effects of insulin, observed at low concentrations of glucose, were reversed at higher concentrations of glucose (over 0.3 mM). Exogenous H₂O₂ had metabolic effects similar to insulin. Addition of H₂O₂ (10^?4 M and higher) to the extracellular medium caused a substantial inhibition of epinephrine-stimulated and adrenocorticotropin-stimulated depot-fat lipase activity in these cells, measured by net glycerol production. H₂O₂ also increased lipogenesis by increasing the provision of the substrates and cofactor (NADPH) and activating pyruvate dehydrogenase in the same manner as insulin. Exogenous catalase (16 μg/ml) abolished the insulin-like effects of H₂O₂ (but not of insulin itself) on glucose oxidation, the contribution of glucose carbons to glyceride fatty acids and glyceride glycerol, the inhibition of lipolysis, and the time-dependent decline in the cAMP content of the cells. The data suggest that, while insulin-stimulated H₂O₂ production from the plasma membrane may bring about some glucose-independent metabolic effects through a lowering of cytoplasmic redox potential, increasing availability of d-glucose may counterbalance these effects by replenishing the reducing equivalents. Endogenous H₂O₂ appears, therefore, to satisfy the manifold criteria for a ‘second messenger’ of insulin.     

Pharmacokinetic Dose Proportionality Between Two Strengths (12.5?mg and 25?mg) of Doxylamine Hydrogen Succinate Film-Coated Tablets in Fasting State: A Single-Dose,Randomized, Two-Period Crossover Study in Healthy Volunteers

Background

Doxylamine succinate, an ethanolamine-based antihistamine, is used in the short-term management of insomnia because of its sedative effects. No data on the dose proportionality of the pharmacokinetics of doxylamine are available, although this drug has been marketed in European countries for more than 50 years.

Objective

The objective of this study was to evaluate and compare the dose proportionality between two marketed strengths (12.5 mg and 25 mg) of doxylamine hydrogen succinate after a single oral dose administration under fasting conditions in healthy human subjects.

Study Design

This was a single-center, randomized, single dose, laboratory-blinded, two-period, two-sequence, crossover study.

Setting

The study was conducted in a phase I clinical unit.

Subjects and Methods

A single oral dose of doxylamine hydrogen succinate of 12.5 mg (equivalent to 8.7 mg of doxylamine base) or 25 mg (equivalent to 17.4 mg of doxylamine base) was administered to healthy volunteers under fasting conditions in each study period. The drug administrations were separated by a wash-out period of 7 calendar days. Blood samples were collected for up to 60 h post-dose, and plasma doxylamine levels were determined by an ultra high-performance liquid chromatography method with tandem mass spectrometry detection. Pharmacokinetic parameters were calculated using non-compartmental analysis. Dose proportionality was assessed based on the parameter area under the concentration–time curve (AUC_{t normalized}). Safety was evaluated through assessment of adverse events, standard laboratory evaluations, vital signs and 12-lead electrocardiogram (ECG).

Results

In total, 12 healthy volunteers (3 male; 9 female) were included in the study. Mean maximum observed plasma concentration (C_max) and area under the concentration–time curve from time zero to time t (AUC_t) of doxylamine hydrogen succinate 12.5 mg and 25 mg tablets increased linearly and dose-dependently [12.5 mg: mean C_max 61.94 ng/mL, coefficient of variation (CV) 23.2 %; mean AUC_t 817.33 ng·h/mL, CV 27.4 %; and 25 mg: mean C_max 124.91 ng/mL, CV 18.7 %; mean AUC_t 1630.85 ng·h/mL, CV 22.8 %]. Mean AUC_{t normalized} was 815.43 ng·h/mL, CV 22.8 % for 25 mg. The dose-normalized geometric mean ratio (%, 12.5 mg/25 mg) of AUC_t was 98.92 (90 % CI: 92.46, 105.83). The most common adverse event was somnolence.

Conclusions

Exposure to doxylamine was proportional over the therapeutic dose range of 12.5–25 mg in healthy volunteers. Based on the results, a predictable and linear increase in systemic exposure can be expected. Doxylamine hydrogen succinate was safe and well tolerated.     

Prostamide F2α receptor antagonism combined with inhibition of FAAH may block the pro-inflammatory mediators formed following selective FAAH inhibition

Background and PurposeProstamides are lipid mediators formed by COX-2-catalysed oxidation of the endocannabinoid anandamide and eliciting effects often opposed to those caused by anandamide. Prostamides may be formed when hydrolysis of anandamide by fatty acid amide hydrolase (FAAH) is physiologically, pathologically or pharmacologically decreased. Thus, therapeutic benefits of FAAH inhibitors might be attenuated by concomitant production of prostamide F_2α. This loss of benefit might be minimized by compounds designed to selectively antagonize prostamide receptors and also inhibiting FAAH.Experimental ApproachInhibition of FAAH by a series of selective antagonists of prostamide receptors, including AGN 204396, AGN 211335 and AGN 211336, was assessed using rat, mouse and human FAAH in vitro, together with affinity for human recombinant CB₁ and CB₂ receptors. Effects in vivo were measured in a model of formalin-induced inflammatory pain in mice.Key ResultsThe prostamide F_2α receptor antagonists were active against mouse and rat FAAH in the low μM range and behaved as non-competitive and plasma membrane-permeant inhibitors. AGN 211335, the most potent inhibitor of rat FAAH (IC₅₀ = 1.2 μM), raised exogenous anandamide levels in intact cells and also bound to cannabinoid CB₁ receptors. Both AGN 211335 and AGN 211336 (0.25–1 mg·kg⁻¹, i.p.) inhibited the formalin-induced nociceptive response in mice.Conclusions and ImplicationsSynthetic compounds with indirect agonist activity at cannabinoid receptors and antagonist activity at prostamide receptors can be developed. Such compounds could be used as alternatives to selective FAAH inhibitors to prevent the possibility of prostamide F_2α-induced inflammation and pain.Linked ArticlesThis article is part of a themed section on Cannabinoids 2013. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2014.171.issue-6     

Inhibition of human erythrocyte orotidylate decarboxylase

Ribonucleotide derivatives of allopurinol and oxipurinol are potent inhibitors of human erythrocyte orotidylate decarboxylase. The inhibition constants are dependent upon the aggregation state of the enzyme, much tighter binding being observed with higher molecular weight forms. The trend was similar to that observed for K_mvalues for orotidine-5'-phosphate, the substrate of the enzyme. Of the compounds tested, 1-oxipurinol-5'-phosphate, with a K_i, value of 0.3 nM for the 250,000 M.W. species, was the most effective inhibitor. This was some two orders of magnitude tighter than 7-oxipurinol-5'-phosphate, which in turn was two orders of magnitude tighter than 1-allopurinol-5'-phosphate. A similar trend of K_i estimates with molecular weight of the enzyme was observed with a number of other inhibitors, including 3-XMP, 9-XMP, 6-azaUMP, UMP and inorganic phosphate (HPO₄^2?).     

Pharmacokinetic optimization of four soluble epoxide hydrolase inhibitors for use in a murine model of inflammation

Background and purpose:

Early soluble epoxide hydrolase inhibitors (sEHIs) such as 12-(3-adamantan-1-yl-ureido)-dodecanoic acid (AUDA) are effective anti-hypertensive and anti-inflammatory agents in various animal models. However, their poor metabolic stability and limited water solubility make them difficult to use pharmacologically. Here we present the evaluation of four sEHIs for improved pharmacokinetic properties and the anti-inflammatory effects of one sEHI.

Experimental approach:

The pharmacokinetic profiles of inhibitors were determined following p.o. (oral) administration and serial bleeding in mice. Subsequently the pharmacokinetics of trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (t-AUCB), the most promising inhibitor, was further studied following s.c. (subcutaneous), i.v. (intravenous) injections and administration in drinking water. Finally, the anti-inflammatory effect of t-AUCB was evaluated by using a lipopolysaccharide (LPS)-treated murine model.

Key“ results:

Better pharmacokinetic parameters (higher C_max, longer t_1/2 and greater AUC) were obtained from the tested inhibitors, compared with AUDA. Oral bioavailability of t-AUCB (0.1 mg·kg⁻¹) was 68 ± 22% (n = 4), and giving t-AUCB in drinking water is recommended as a feasible, effective and easy route of administration for chronic studies. Finally, t-AUCB (p.o.) reversed the decrease in plasma ratio of lipid epoxides to corresponding diols (a biomarker of soluble epoxide hydrolase inhibition) in lipopolysaccharide-treated mice. The in vivo potency of 1 mg·kg⁻¹ of t-AUCB (p.o.) was better in this inflammatory model than that of 10 mg·kg⁻¹ of AUDA-butyl ester (p.o) at 6 h after treatment.

Conclusions and implications:

t-AUCB is a potent sEHI with improved pharmacokinetic properties. This compound will be a useful tool for pharmacological research and a promising starting point for drug development.     

Efficient Degradation of Zearalenone by Dye-Decolorizing Peroxidase from Streptomyces thermocarboxydus Combining Catalytic Properties of Manganese Peroxidase and Laccase

Ligninolytic enzymes, including laccase, manganese peroxidase, and dye-decolorizing peroxidase (DyP), have attracted much attention in the degradation of mycotoxins. Among these enzymes, the possible degradation pathway of mycotoxins catalyzed by DyP is not yet clear. Herein, a DyP-encoding gene, StDyP, from Streptomyces thermocarboxydus 41291 was identified, cloned, and expressed in Escherichia coli BL21/pG-Tf2. The recombinant StDyP was capable of catalyzing the oxidation of the peroxidase substrate 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), phenolic lignin compounds 2,6-dimethylphenol, and guaiacol, non-phenolic lignin compound veratryl alcohol, Mn²⁺, as well as anthraquinone dye reactive blue 19. Moreover, StDyP was able to slightly degrade zearalenone (ZEN). Most importantly, we found that StDyP combined the catalytic properties of manganese peroxidase and laccase, and could significantly accelerate the enzymatic degradation of ZEN in the presence of their corresponding substrates Mn²⁺ and 1-hydroxybenzotriazole. Furthermore, the biological toxicities of the main degradation products 15-OH-ZEN and 13-OH-ZEN-quinone might be remarkably removed. These findings suggested that DyP might be a promising candidate for the efficient degradation of mycotoxins in food and feed.     

Protective effects of compound FLZ,a novel synthetic analogue of squamosamide,on β-amyloid-induced rat brain mitochondrial dysfunction in vitro

Aim:

The aim of the present study was to assess the effects of N-[2-(4-hydroxyphenyl)ethyl]-2-(2,5-dimethoxyphenyl)-3-(3-methoxy-4-hydroxyphenyl) acrylamide (compound FLZ), a novel synthetic analogue of squamosamide, on the dysfunction of rat brain mitochondria induced by Aβ_25–35 in vitro.

Methods:

Isolated rat brain mitochondria were incubated with aged Aβ_25–35 for 30 min in the presence and absence of FLZ (1–100 μmol/L). The activities of key mitochondrial enzymes, the production of hydrogen peroxide (H₂O₂) and superoxide anion (O₂^·-), and the levels of glutathione (GSH) in mitochondria were examined. Mitochondrial swelling and the release of cytochrome c from mitochondria were assessed by biochemical and Western blot methods, respectively.

Results:

Incubation of mitochondria with aged Aβ_25–35 inhibited the activities of α-ketoglutarate dehydrogenase (α-KGDH), pyruvate dehydrogenase (PDH) and respiratory chain complex IV. It also resulted in increased H₂O₂ and O₂^·- production, and decreased the GSH level in mitochondria. Furthermore, it induced mitochondrial swelling and cytochrome c release from the mitochondria. The addition of FLZ (100 μmol/L) prior to treatment with Aβ_25–35 significantly prevented these toxic effects of Aβ_25–35 on the mitochondria.

Conclusion:

FLZ has a protective effect against Aβ_25–35-induced mitochondrial dysfunction in vitro.     

Role of catalase in the oxidation of mercury vapor

In human red blood cells the slow rate of production of hydrogen peroxide limits the rate of oxidation of mercury vapor. When H₂O₂ was added to blood samples, the rate of mercury uptake increased six times, but the inhibitory effect of KCN was less pronounced than in samples without H₂O₂, possibly because H₂O₂ tends to destroy cyanide. Only in the absence of exogenous H₂O₂ did the inhibition of oxidation of mercury vapor by different concentrations of KCN parallel the inhibition of catalase. Addition of peroxide to liver homogenates was without effect. When 20% (w/v) liver homogenates were exposed to mercury vapor at 37°, neither acatalasemia in mice nor pretreatment of rats with 2 g/kg of aminotriazole produced an appreciable decrease in the uptake of mercury during the 90-min incubation period. However, it was clear that catalase was responsible for the oxidation of mercury from the correlation (r² = 0.85) between catalase activity and mercury uptake when 0.4 mg liver in 2 ml of incubation medium was exposed to mercury vapor. With higher concentrations of homogenates the availability of mercury limited the oxidation process. Experiments with horseradish peroxidase, beef liver catalase and inorganic catalysts of H₂O₂ decomposition indicate that the elemental mercury atom serves as an electron donor for complex I of catalase which is formed from the first reaction of catalase with H₂O₂. Other possible pathways of oxidation such as oxidation of elemental mercury by nascent oxygen released from H₂O₂ do not appear to be important.     

Effects of exogenous female sex-steroid hormones on lymphocyte β2-adrenoceptors in normal females

We have previously shown that lymphocyte β₂-adrenoceptors (AR) are under cyclical control of sex-steroid hormones with greater receptor density during the luteal phase of the menstrual cycle. It has also been postulated that abnormal cyclical regulation of β₂-AR might be a possible mechanism for premenstrual asthma. The effects of exogenous female sex-steroid hormones on lymphocyte β₂-AR function were studied in eight normal healthy females. They were evaluated at two successive menstrual cycles, during the follicular phase (day 1–6). They were randomized to receive single oral doses of either ethinyloestradiol 50 μg or medroxyprogesterone 10 mg in a cross-over study. Lymphocyte β₂-AR parameters were evaluated at baseline (t₀), 24 h (t₂₄) and 72 h (t₇₂) after ingestion. Baseline levels of progesterone and oestradiol were comparable on both cycles. Receptor density (B_max) increased significantly (P<0.01) from t₀ after progesterone but not oestradiol at t₂₄: a 1.39-fold geometric mean difference (95% CI 0.96–2.00) between t₂₄vs t₀. Receptor affinity (K_d) and maximal cAMP response to isoprenaline (E_max) were not altered by either treatment. These results show that exogenous progesterone but not oestradiol, given during the follicular phase, significantly increased β₂-AR. This, therefore, suggests that endogenous progesterone is probably responsible for previously observed increase in B_max during the luteal phase of the female menstrual cycle. These findings may suggest possible therapeutic strategies for modulation of β₂-AR in premenstrual asthma.     

DNA cleavage by metabolites of butylated hydroxytoluene

The effect of butylated hydroxytoluene (BHT) and its metabolites on DNA cleavage in vitro was studied with supercoiled plasmid DNA, pUC18, by agarose gel electrophoresis. Among several BHT metabolites, 2,6-di -t-butyl-p-benzoquinone (BHT-quinone) caused cleavage of supercoiled DNA (form I) at a concentration as low as 1 × 10^–6 M. The relative amount of linear form (form III) was increased with increasing concentration of BHT-quinone. 2,6-Di-t-butyl-4-hydroperoxy-4-methyl-2,5-cyclohexadienone (BHT-peroxyquinol) and 3,5-di-t-butyl-4-hydroxybenzaldehyde (BHT-CHO) also cleaved DNA, but to a lesser extent than BHT-quinone. No DNA cleavage was detected by BHT, 2,6-di-t-butyl-4-hydroxymethyl phenol (BHT-OH), 3,5-di-t-butyl-4-hydroxybenzoic acid (BHT-COOH), 2,6-di-t-butyl-4-hydroxy-4-methyl-2,5-cyclohexadienone (BHT-quinol) or 2,6-di-t-butyl-4-methylene-2,5-cyclohexadienone (BHT-quinone methide). The DNA cleavage by BHT-quinone was inhibited by oxygen radical scavengers including Superoxide dismutase (SOD), catalase, polyethylene glycol,t-butyl alcohol, dimethyl sulfoxide, sodium azide, sodium benzoate, bovine serum albumin and methionine, while it was enhanced by the addition of FeCl₂. The production of Superoxide radical in a solution of BHT-quinone was confirmed by cytochrome c reduction assay. Superoxide was not produced by BHT or other BHT metabolites except for BHT-quinone. These results suggest that BHT-quinone, one of the principal metabolites of BHT, cleaves DNA strands via its generation of oxygen radicals. Such modification of DNA observed in vitro may be relevant to genotoxicity by BHT after metabolic activation in vivo.     

Endogenous hydrogen peroxide and peroxidative metabolism in adipocytes in response to insulin and sulfhydryl reagents

Evidence is presented that glucose oxidation in adipocytes via the pentose phosphate pathway, known to be regulated essentially by the supply of NADP⁺ in the presence of oxidized glutathione, is increased in response to insulin due to a peroxidative catabolism of endogenously produced hydrogen peroxide. A similar response was observed with sulfhydryl reagents, e.g. dithiothreitol (DTT) and p-chloromercuribenzoate (pCMB). both of which inhibit d-glucose transport and glucose oxidation by the glycolytic pathway, but like insulin enhance the pentose phosphate pathway. A catalase-dependent peroxidative reaction, viz. ¹⁴CO₂ production from sodium [¹⁴C]formate, was increased about 2-fold in cells exposed to insulin, or DTT and pCMB. Its partial inhibition in the presence of 50 mM 3-amino- l,2,4-triazole was greater in the insulin- DTT- or pCMB-treatcd cells than in the control. This indicated intracellular accumulation of catalase: H₂O₂ complexes. The data on the catalatic and peroxidative activities of catalase in the adipocytes under these experimental conditions, coupled with the preferential stimulation of the pentose phosphate pathway in the presence of inhibitors of catalase, e.g. aminotriazole and azide, indicate a major role of glutathione peroxidase in the disposal of H₂O₂. These findings support our previous report that insulin or —SH reactive agents activate a pyridine nucleotide oxidase in the plasma membrane of these cells [S. P. Mukherjee and W. S. Lynn, Fedn Proc.35, 1694 (1976); Archs Biochem. Biophys.184, 69 (1977)]. A close relationship was observed between the cellular content of glutathione (GSH) and glucose utilization. Thus, induction of a peroxidative metabolism within these cells, coupled with glucose oxidation, appears to be a major effect of insulin.     

Xenobiotic metabolism in humans during early pregnancy: peroxidase-mediated oxidation and bioactivation of 2-aminofluorene

1. Arylamines such as 2-aminofluorene (2-AF) are known teratogens and transplacental carcinogens in laboratory animal species. Although exposure of women to arylamines is likely to occur during pregnancy, how these chemicals are metabolized by the enzymes from the human conceptual tissues is currently unknown.

2. Highly purified preparations of peroxidase isolated from human intrauterine conceptual tissues at 8 weeks of gestation were used to study in vitro metabolism of 2-AF. The oxidation of 2-AF was examined spectrophotometrically whereas the bioactivation was assessed from the covalent binding to protein and DNA using [³H] 2-AF.

3. Using guaiacol as a model substrate, the purified preparations of peroxidase used exhibited a specific activity of 15–20 μmol/min/mg protein. 2-AF oxidation was found to be enzymatic in nature. Kinetic data obtained under optimal assay conditions yielded a K_m = 41 μM for 2-AF, 8.33 μM for H₂O₂, and a V_max = 1.2 μmol 2-AF oxidized/min/mg protein.

4. Under optimal assay conditions, the covalent binding of reactive intermediate(s) to protein and DNA (nmol equivalent/min/mg enzyme/mg bovine serum albumin or calf thymus DNA) was observed at the rate of about 3.75 ± 0.39 and 1.90 ± 0.11 respectively.

5. A significant decline in the rate of both oxidation and bioactivation of 2-AF was observed in the presence of classical peroxidase inhibitors, KCN and NaN₃.