Effects of RRR-alpha-tocopherol on leukocyte expression of HSP72 in response to exhaustive treadmill exercise.

Previous research revealed an increased expression of HSP72 in leukocytes after vigorous endurance exercise. We questioned whether more intensive but shorter exercise also induces leukocyte HSP72 synthesis. To delineate the role of reactive oxygen species (ROS) in exercise-related HSP72 induction, we additionally examined the effect of RRR-alpha-tocopherol (alpha-toc) on HSP72 expression using a double-blind placebo (P) controlled cross-over design. After supplementation with alpha-toc (500 I.U. daily) or P for 8 days, 9 male subjects performed a combined exhaustive treadmill protocol (total duration 29.4 +/- 2.0 min). HSP72 was assessed on mRNA (RT-PCR) and protein levels (flow cytometry). HSP72 mRNA rose 3 h after exercise only in the P group, but individual differences (alpha-toc - P) did not reveal significant treatment effects. A moderate but significant rise of HSP72 protein occurred in granulocytes up to 48 h after exercise. Three hours post-exercise, granulocyte HSP72 protein was lower when subjects received alpha-toc, but this effect vanished 24 and 48 h post-exercise. Exhaustive treadmill exercise augments HSP72 mRNA in leukocytes and induced a moderate but prolonged response of granulocyte HSP72 protein. These exercise effects are lower when compared to earlier findings obtained after vigorous endurance exercise. ROS seem to be involved, but do not play the major role in the induction of granulocyte HSP72 synthesis after exhaustive exercise.

Oral Mg(2+) supplementation reverses age-related neuroendocrine and sleep EEG changes in humans.

The process of normal aging is accompanied by changes in sleep-related endocrine activity. During aging, an increase in cortisol at its nadir and a decrease in renin and aldosterone concentration occur. In aged subjects, more time is spent awake and slow-wave sleep is reduced: there is a loss of sleep spindles and accordingly a loss of power in the sigma frequency range. Previous studies could show a close association between sleep architecture, especially slow-wave sleep, and activity in the glutamatergic and GABAergic system. Furthermore, recent studies could show that the natural N-methyl-D-aspartate (NMDA) antagonist and GABA(A) agonist Mg(2+) seems to play a key role in the regulation of sleep and endocrine systems such as the HPA system and renin-angiotensin-aldosterone system (RAAS). Therefore, we examined the effect of Mg(2+) in 12 elderly subjects (age range 60-80 years) on the sleep electroencephalogram (EEG) and nocturnal hormone secretion. A placebo-controlled, randomised cross-over design with two treatment intervals of 20 days duration separated by 2 weeks washout was used. Mg(2+) was administered as effervescent tablets in a creeping dose of 10 mmol and 20 mmol each for 3 days followed by 30 mmol for 14 days. At the end of each interval, a sleep EEG was recorded from 11 p.m. to 7 a.m. after one accommodation night. Blood samples were taken every 30 min between 8 p.m. and 10 p.m. and every 20 min between 10 p.m. and 7 a.m. to estimate ACTH, cortisol, renin and aldosterone plasma concentrations, and every hour for arginine-vasopressin (AVP) and angiotensin 11 (ATII) plasma concentrations. Mg(2+) led to a significant increase in slow wave sleep (16.5 +/- 20.4 min vs. 10.1 +/- 15.4 min, < or =0.05), delta power (47128.7 microV(2) +21417.7 microV(2) vs. 37862.1 microV(2) +/- 23241.7 microV(2), p < or =0.05) and sigma power (1923.0 microV(2) + 1111.3 microV(2) vs. 1541.0 microV(2) + 1134.5 microV(2), p< or =0.05 ). Renin increased (3.7 +/- 2.3 ng/ml x min vs. 2.3 +/- 1.0 ng/ml x min, p < 0.05) during the total night and aldosterone (3.6 +/- 4.7 ng/ml x min vs. 1.1 +/- 0.9 ng/ml x min, p < 0.05) in the second half of the night, whereas cortisol (8.3 +/- 2.4 pg/ml x min vs. 11.8 +/- 3.8 pg/ml x min, p < 0.01) decreased significantly and AVP by trend in the first part of the night. ACTH and ATII were not altered. Our results suggest that Mg(2+) partially reverses sleep EEG and nocturnal neuroendocrine changes occurring during aging. The similarities of the effect of Mg(2+) and that of the related electrolyte Li+ furthermore supports the possible efficacy of Mg(2+) as a mood stabilizer.

Inhalation of aerosolized vitamin a: reversibility of metaplasia and dysplasia of human respiratory epithelia -- a prospective pilot study.

The objective of this preliminary uncontrolled study was twofold: First, to assess the feasibility of retinyl palmitate inhalation and second, to analyze the changes of metaplastic lesions of the respiratory epithelium (metaplasia or dysplasia) following retinyl palmitate inhalation. The response to a daily dose of 18.000 I.U. retinyl palmitate by inhalation over a period of 3 month was assessed in 11 subjects (9 smokers, 2 ex-smokers). Using white-light bronchoscopy combined with autofluorescence bronchoscopy, bronchial biopsies were taken before and after a 3 month-period. The biopsy samples were evaluated blind by a referee lung pathologist. The overall response rate (remission or partial remission) was 56% (95% CI 0.30 0.79; p<0.05). These data suggest that inhalation of retinyl esters could be a promising therapeutical approach for chemoprevention of lung cancer. Vitamin A; chemoprevention; lung cancer; squamous metaplasia; dysplasia; retinoids

Physical exercise-induced expression of inducible nitric oxide synthase and heme oxygenase-1 in human leukocytes: effects of RRR-alpha-tocopherol supplementation.

This study evaluated the effects of RRR-alpha-tocopherol (500 IU/day, 8 days) on in vivo cytokine response and cytoplasmic expression of inducible nitric oxide synthase (iNOS) and the antioxidant stress protein heme oxygenase-1 (HO-1) in human leukocytes after exhaustive exercise. Thirteen men were investigated in a double-blind, placebo-controlled, cross-over study with a wash-out period of 28 days. The exercise procedure consisted of an incremental treadmill test followed by a continuous run until exhaustion at 110% of the individual anaerobic threshold (total duration 28.5 +/- 0.8 min). HO-1 and iNOS protein were assessed in mono- (M), lympho-, and granulocytes (G) using flow cytometry. Plasma interleukin-6 (IL-6) and IL-8 were measured by ELISA. IL-6 rose significantly whereas IL-8 did not exhibit significant changes after exercise. Changes of IL-6 were not affected by RRR-alpha-tocopherol. Exercise induced an increase of iNOS protein primarily in M and G. A small, but significant, increase of HO-1 protein was measured in M and G. RRR-alpha-Tocopherol did not show any significant effects on cytoplasmic expression of iNOS and HO-1 at rest and after exercise. In conclusion, exhaustive exercise induces expression of iNOS and HO-1 in human leukocytes by a mechanism that is not sensitive to RRR-alpha-tocopherol supplementation.

Primary aromatic amines: their N-oxidative bioactivation.

There exists a diversity of pathways in mammalian cells serving to activate primary aromatic amines. 1 N-Oxidative mixed-function turnover usually involves participation of the cytochrome P450 superfamily, while catalysis by the flavin-containing monooxygenases is restricted to a few amines capable of forming imine tautomers. Surprisingly, haemoglobin metabolizes cytotoxic and carcinogenic arylamines via a monooxygenase-like mechanism, but peroxygenase activity is also operative. 2 In extrahepatic tissues that exhibit only a low level of monooxygenases, peroxidative transformations, as are brought about by prostaglandin H synthase, myeloperoxidase or lactoperoxidase, predominate in amine activation. Non-mammalian peroxidases frequently used as model systems include horseradish peroxidase and chloroperoxidase. 3 Non-enzymatic, light-induced conversion of aromatic amines to free radical or N-oxy products proceeds either via direct photolysis of the nitrogenous compounds or through attack by lipid-derived reactive intermediates generated during irradiation. 4 The interplay of the various tissue-specific processes of arylamine activation serves to explain differences in susceptibility toward the biological actions of primary aromatic amines.

Chemical modification of Tyr34 and Tyr129 in rabbit liver microsomal cytochrome b5 affects interaction with cytochrome P-450 2B4.

Rabbit liver microsomal cytochrome b5 was allowed to react with tetranitromethane. Up to three tyrosine residues in each cytochrome b5 molecule were found to be accessible to the nitrating agent. Co-modification of tryptophan and histidine residues could be disregarded. CD-spectral measurements disproved gross changes in cytochrome b5 structure as a consequence of derivatization. Introduction of 1.6 nitro groups/polypeptide chain resulted in a fivefold increase in binding affinity for cytochrome P-450 2B4 (P-450 2B4), whereas spectral interaction with cytochrome c remained unaffected. Furthermore, the capacity of nitrated cytochrome b5 to shift the spin equilibrium to the high-spin conformer of P-4502B4 was diminished by 44% compared with the control. This corresponded with the partial disruption of NADH-dependent electron flow to ferric P-450 2B4. Changes in the redox potential of cytochrome b5 could be discounted as being responsible for this effect. The overall oxidative turnover of 4-nitroanisole did not respond to cytochrome b5 modification. MS analysis and sequencing of peptide fragments produced by tryptic digestion of modified cytochrome b5 permitted the detection of three nitrated tyrosine residues located at positions 11, 34 and 129. Derivatization of cytochrome b5 in the presence of a protective amount of P-450 2B4 provided evidence of the involvement of Tyr34 and Tyr129 in complexation of the two hemoproteins. It is proposed that Tyr129 might control docking of cytochrome b5 to P-450 2B4, whereas Tyr34 could be of functional importance in electron transfer.

Inactivation of phenobarbital-inducible rabbit-liver microsomal cytochrome P-450 by allylisopropylacetamide: impact on electron transfer.

Application of a single dose of allylisopropylacetamide (AIA) to phenobarbital-pretreated rabbits resulted in partial destruction of the heme moiety of liver microsomal cytochrome P-450. A minor fraction of chromophore loss was accounted for by heme-derived product(s) covalently attached to microsomal proteins. Interestingly, cytochrome P-450 appeared to have undergone significant drug-mediated alkylation of the apohemoprotein. The modified species was purified to apparent homogeneity and shown to arise from AIA-induced blockage of about 2 histidines in the cytochrome P-450LM2 molecule located close to the heme edge. AIA administration to the animals caused inhibition of hexobarbital-promoted electron flow from NADPH-cytochrome P-450 reductase to phenobarbital-inducible ferricytochrome P-450 both in microsomal particles and reconstituted systems. The impaired interaction between the proteins was shown not to originate from decreased capacity to bind each other but more likely to be due to some defect in a step subsequent to complex formation. In contrast, treatment with the porphyrogenic agent did not affect microsomal electron transmission from cytochrome b5 to the ferric monooxygenase. However, when the intermediate carrier was to donate reducing equivalents to the ferrous oxycytochrome in the presence of benzphetamine, there was a pronounced deceleration of the electron flux observable. These findings were interpreted to mean that there exist multiple reductase- and cytochrome-b5-binding domains in phenobarbital-inducible cytochrome P-450, some of which seem to be common to the two redox proteins. This sheds interesting light on the molecular organization of the catalytic electron transfer complexes.

Chemical modification of lysine residues in cytochrome P450LM2 (P450IIB4): influence on heme liganding of arylamines.

Treatment of cytochrome P450LM2 with fluorescein isothiocyanate to introduce up to two equivalents of fluorophore per polypeptide chain resulted in the selective derivatization of lysine residues. CD-spectral measurements revealed the overall conformation as well as the immediate heme environment of the hemoprotein to remain unaffected by attachment of the label. Modification caused decreased affinity of p-phenylenediamine and other 4-substituted anilines for the heme site, whereas there was a rise in the extent of substrate interaction. Experiments with pigment containing acetylated lysines gave analogous results, suggesting that the observed phenomenon was due to charge neutralization. There was linear correlation between the Hammett sigma P values and both the optical dissociation constants for arylamine binding to intact enzyme and the dipole moments of the anilines, indicating that basicity along with electronic factors controlled heme liganding; lipophilicity appeared to be of minor importance. Introduction of fluorescein isothiocyanate into the oxygenase was found to influence the bond-making process through modulating basicity of the nitrogenous compounds, but perturbation of optimal spacial orientation of the amine nitrogen toward the heme iron also might have been operative. The lysines studied seem to represent metabolically inactive elements of the substrate channel located on the cytosolic surface of the aggregates, as evidenced by steady-state fluorescence measurements. A hydrophilic segment in the cytochrome P450LM2 molecule that would accommodate the critical residues is discussed.

The functional role of cytochrome b5 reincorporated into hepatic microsomal fractions.

Incorporation of detergent-solubilized cytochrome b5 into phenobarbital-induced rabbit liver microsomal fractions decelerates hexobarbital-dependent reduction of ferric cytochrome P-450; this is accompanied by retardation of NADPH utilization and H2O2 formation in the assay media. Integration of manganese-substituted cytochrome b5 into the microsomal preparations fails to affect these parameters. Analysis of the cytochrome P-450 reduction kinetics in the presence of increasing amounts of cytochrome b5 reveals a gradual augmentation of the amplitude of slow-phase electron transfer at the expense of the relative contribution of the fast phase; finally, a slow, apparently monophasic reaction persists. This defect in enzymatic reduction is not due to detergent effects and also does not seem to reflect cytochrome b5-induced perturbation of anchoring of NADPH-cytochrome c(P-450) reductase to cytochrome P-450. Experiments with the highly purified cytochrome P-450 isozyme LM2, in which amino acid residue(s) close to the heme edge had undergone suicidal inactivation through covalent attachment of chloramphenicol metabolite(s) do not exclude the possibility that cytochrome b5 and reductase might compete for a common electron transmission site on the terminal acceptor. Hence, the inhibitory action of cytochrome b5 on the reduction of ferric cytochrome P-450 is tentatively attributed to partial substitution of the former pigment for reductase in direct transport of the first electron to the monooxygenase.

Influence of N,N-dimethylaniline on the association of phenobarbital-induced cytochrome P-450 and NADPH-cytochrome c(P-450) reductase in a reconstituted rabbit liver microsomal enzyme system.

N,N-Dimethylaniline when added to reaction mixtures provokes deviation from Michaelis-Menten law of the interaction kinetics of NADPH-cytochrome c(P-450) reductase (NADPH:ferrihaemoprotein oxidoreductase, EC 1.6.2.4) with highly purified phenobarbital-induced rabbit liver microsomal cytochrome P-450 (P-450LM2). This phenomenon is not associated with the low-to-high spin transition in the iron-coordination sphere of the haemoprotein, as elicited by the arylamine. Substrate-triggered departure from linearity of the kinetics is abolished by inclusion into the assay media of p-chloromercuribenzoate, hinting at a vital role in the process of thiols. Similarly, the parabolic progress curve (nH = 1.7) is transformed to a straight line (nH = 1.01) when the N-terminal reductase-binding domain in the P-450LM2 molecule is selectively blocked through covalent attachment of fluorescein isothiocyanate (FITC); such a modification does not alter the affinity of the haemoprotein for the amine substrate. Steady-state fluorescence polarization measurements reveal that N,N-dimethylaniline perturbs the motional properties of the fluorophore-bearing reductase-binding region, suggesting the induction of a conformational change. Summarizing these results, the data possibly indicate N,N-dimethylaniline-induced cooperativity in the association of reductase with P-450LM2.

Regulative mechanisms in NADH- and NADPH-supported N-oxidation of 4-chloroaniline catalyzed by cytochrome b5-enriched rabbit liver microsomal fractions.

Incorporation into rabbit liver microsomal membranes of detergent-solubilized cytochrome b5 stimulates NADH-supported electron flow to ferric cytochrome P-450, but impairs NADPH-dependent reduction of the pigment such as to make the rates of both reactions equivalent; yet, in the enriched preparations NADPH-driven N-oxidation of 4-chloroaniline proceeds at considerably higher rate than does the NADH-supported process. Analysis of transfer of the second electron to oxyferrous cytochrome P-450, as assessed by measuring substrate-induced reoxidation of ferrous cytochrome b5, reveals faster flow with NADH than with NADPH as the source of reducing equivalents. Quantification of the pools of cytochrome P-450 active in attack on the amine substrate in the presence of either reduced pyridine nucleotide, as well as measurements of maximum arylamine turnover suggest that the cofactor-dependent discrepancy in N-oxidase activity reflects differences in the rates of breakdown of the intermediary enzyme complexes. The NADH- and NADPH-supported pathway of N-oxidation in the cytochrome b5-supplemented microsomal fractions thus probably involves distinct forms of cytochrome P-450. Alternatively, functional linkage of the cofactor-specific electron-transfer chains to a single cytochrome P-450 species might yield aggregates of differing conformational state and catalytic capacity. The latter concept receives support from experiments with individually reconstituted enzyme systems.

N-Oxidation of 4-chloroaniline by prostaglandin synthase. Redox cycling of radical intermediate(s).

4-Chloroaniline undergoes N-oxidation in ram seminal-vesicle microsomal preparations supplemented with arachidonic acid to yield N-(4-chlorophenyl)-hydroxylamine and 1-chloro-4-nitrosobenzene. H2O2 also supports metabolism of the amine substrate to the same organic-solvent-extractable products, suggesting that the hydroperoxidase activity of prostaglandin synthase is responsible for the co-oxidation. Analysis of the reaction mixtures by e.s.r. spectrometry reveals the formation of a radical intermediate bearing the characteristics of a strongly immobilized nitroxide. Arylamine-stimulated O2.- release can be observed when the arachidonic acid-containing incubation media are supplemented with NADPH. Redox cycling of the nitroxide/hydroxylamine couple is presumed to represent the major source of O2.-, but additional mechanisms, such as redox changes of nitro anion radicals resulting from potential further metabolism of 1-chloro-4-nitrosobenzene, cannot be excluded. The concerted action of carrier-bound nitroxides and O2.- in initiating damage of cellular macromolecules is discussed.

The role of lipid peroxidation in the N-oxidation of 4-chloroaniline.

Irradiation with u.v. light of aerobic aqueous media containing both rabbit liver microsomal fraction and 4-chloroaniline results in N-oxidation of the arylamine. The reaction is severely blocked by exhaustive extraction with organic solvents of the microsomal membranes to remove lipids. Further, scavengers of OH. and O2.-impair the photochemical process. These findings suggest that the observed phenomenon may be closely associated with light-induced lipid peroxidation. Indeed, N-oxidation of 4-chloroaniline is fully preserved when either phospholipid liposomes or dispersed linoleic acid substitute for intact microsomal fraction. Co-oxidation of the amine substrate occurs during iron/ascorbate-promoted lipid peroxidation also, but H2O2 or free OH. radicals do not appear to be involved. Cumene hydroperoxide-sustained rabbit liver microsomal turnover of the amine generates N-oxy product via O2-dependent and -independent pathways; propagation of lipid peroxidation is presumed to govern the former route. Lipid hydroperoxides, either exogenously added to rabbit liver microsomal suspensions or enzymically formed from arachidonic acid in ram seminal-vesicle microsomal preparations, support N-oxidation of 4-chloroaniline. The significance, in arylamine activation, of lipid peroxidation in certain extrahepatic tissues exhibiting but low mono-oxygenase activity is discussed.

The role of hemoglobin in the N-oxidation of 4-chloroaniline.

Rabbit hemoglobin effects reduced pyridine nucleotide-dependent N-oxidation of 4-chloroaniline in the presence of NADPH-cytochrome c (P-450) reductase (EC 1.6.2.4). The reaction is blocked by the addition of CO, superoxide dismutase (EC 1.15.1.1) and catalase (EC 1.11.1.6). The apparent Km value for the amine is 5.9 mM. The substrate interacts with hemoglobin in a non-cooperative manner; highly purified alpha- and beta-subunits mediate amine oxidation with kinetic constants close to those of the intact tetramer. Metabolism of 4-chloroaniline is associated with the formation of a 421 nm absorbing spectral complex, which might represent a ferryl species or a product adduct. Rapid reaction measurements suggests that either transfer of the second electron or product dissociation limits the overall rate of hemoglobin cycling. Erythrocyte reductases, such as 'NADPH-methemoglobin reductase' or soluble NADH-cytochrome b5 reductase (EC 1.6.2.2), also sustain amine oxidation in the presence of an appropriate electron carrier. Similarly, intact rabbit erythrocytes generate low amounts of N-oxy product when incubated with the parent amine. These findings support the notion that the red blood cell might be a site of bioactivation of aromatic amines, some of which, after being N-oxidized, become potent mutagens and carcinogens.

Comparative studies on the cumene hydroperoxide- and NADPH-supported N-oxidation of 4-chloroaniline by cytochrome P-450.

The present study confirms that cytochrome P-450 can act as a catalyst in the cumene hydroperoxide-supported N-oxidation of 4-chloroaniline. Analogous to the NADPH/O2-driven N-oxidation process, product dissociation is likely to limit the overall rate of cytochrome P-450 cycling also in the peroxidatic pathway. The oxy complexes involved in either metabolic route differ with respect to stability, spectral properties and need for thiolate-mediated resonance stabilization. With the organic hydroperoxide, the metabolic profile is shifted from the preponderant production of N-(4-chlorophenyl)hydroxylamine to the formation of 1-chloro-4-nitrobenzene. This finding suggests that the peroxide-sustained N-oxidation mechanism differs in several ways from that functional in the NADPH/O2-dependent oxenoid reaction. Thus one-electron oxidation, triggered by homolytic cleavage of the oxygen donor, is proposed as the mechanism of peroxidatic transformation of 4-chloroaniline.