Biochemical observations relating to oxidant stress injury in Chernobyl clean-up workers ("liquidators") from Latvia.

AIMS: To establish if there is further evidence for the long-term oxidant stress injury (as reported previously--Kumerova et al. in Biol Trace Elem Res 77:1-12, 2000) in surviving Chernobyl nuclear power plant (NPP) workers from Latvia. The overall objectives of this study have been to establish if there have been long-term systemic changes in the oxidant/antioxidant status of clean-up workers that might reflect adaptation to the progression of oxidative stress injury. METHODS: Biochemical analyses of the circulating levels of endogenous oxidants and anti-oxidants were undertaken over two periods (Stage 1 in 1998-1999 and Stage 2 in 2005-2006) at approximately 6-7 years time interval, in order to establish if there have been time-dependent changes in the parameters that may be important for the health of the clean-up workers. The biochemical analyses included (a) plasma levels of the anti-oxidant, selenium, (b) blood and plasma levels of glutathione peroxidase, (c) red blood cell catalase, (d) plasma total oxidant status as lipid peroxides and hydroperoxides, (e) plasma ceruloplasmin, and (f) total blood levels of zinc and copper. RESULTS: The circulating content of lipid peroxides, plasma oxidisability, lipid peroxides, catalase, Zn, and Cu were elevated above normal values at both the stages of this study. Glutathione peroxidase was increased above normal values at Stage 1 but not at Stage 2. The most pronounced changes between Stage 1 and Stage 2 were (a) a reduction by about (1/2) in the content of lipid peroxides and lipid peroxidation, but not in the blood oxidisability and (b) increased plasma selenium. The data show that there may be a partial improvement in the anti-oxidant/oxidant status of the Chernobyl NPP workers over the 7-year period of investigation. CONCLUSIONS: The NPP patients may be undergoing progressive reduction in blood oxidants accompanied by adaptation to oxidant stress injury due to the increased anti-oxidant activity measured in their plasma and blood.

Effect of the blood substitute diaspirin crosslinked hemoglobin in rat mesenteric and human radial collateral arteries.

The actions of the blood substitute diaspirin crosslinked hemoglobin (DCLHb) were investigated in rat (small mesenteric artery) and human (radial collateral artery) resistance vessels mounted in a wire myograph for isometric tension recording. DCLHb did not contract resting vessels from rats, but vasoconstrictor responses were observed in isolated arteries and perfused mesenteric beds prestimulated with threshold concentrations of methoxamine. The DCLHb contractile responses were greatly attenuated by N(G)-nitro-L-arginine methyl ester hydrochloride (L-NAME) or endothelial removal, whereas BQ-123 (endothelin A receptor antagonist), prazosin (alpha1-adrenoceptor antagonist), or indomethacin (cyclooxygenase inhibitor) had no effect. Endothelium-dependent relaxations to carbachol in both rat mesenteric and human radial collateral artery were inhibited by DCLHb. Relaxations to carbachol were studied in the presence of L-NAME or 25 mM KCl to investigate the effect of DCLHb on endothelium-derived hyperpolarizing factor (EDHF) and nitric oxide, respectively. In both rat and human vessels, EDHF-mediated relaxations were not affected by DCLHb preincubation, whereas the nitric oxide component of carbachol-induced relaxations was practically abolished. In conclusion, inhibition of the effects of basal nitric oxide release underpins the vasoconstrictor effects of DCLHb. DCLHb effectively abolishes the nitric oxide component of carbachol-induced relaxation, with no effect on the EDHF-mediated component in both isolated rat mesenteric and human radial collateral arteries.

A comparison of the inotropic effects of milrinone and piroximone.

Patients with low cardiac output syndrome often have a reduced sensitivity to inotropes acting via the beta-adrenergic receptors. In this situation, drugs such as milrinone and piroximone may have a therapeutic advantage. Strips of human right atrial appendage were used to examine the inotropic actions of phosphodiesterase (PDE) inhibitors, measured as an increase in isometric tension in response to electrical stimulation at a frequency of 1 Hz. Cumulative concentration response curves were established for piroximone (an imidazalone derivative) and Milrinone (a bipyridine derivative.) Concentration response curves were then recorded to adrenaline in the presence or absence of the EC50 of each PDE inhibitor. The results indicate that these drugs, under the conditions employed in these experiments, have a significant inotropic effect when used alone. They also have a significant additive and potentiating effect when used in combination with adrenaline. Milrinone produced a significantly greater maximum tension as a percentage of basal tension, and has a lower EC50 than Piroximone.

The action of diaspirin cross-linked haemoglobin blood substitute on human arterial bypass conduits.

BACKGROUND: Immediately available blood substitutes could transform medicine. In coronary artery surgery, vasoconstriction induced by some of these agents could have serious implications. We have examined some of the vasoactive effects of one of these blood substitute, diaspirin cross-linked haemoglobin (DCLHb), on isolated rings of human arterial conduits. METHODS: Sections of human left internal mammary artery (LIMA) and radial artery (RA) were cut into 3-mm rings, mounted in individual organ baths containing aerated (95% O(2)/5% CO(2)) Krebs-Heinseleit solution at 37 degrees C and attached to isometric strain gauge for measurements of tension. All rings were tested for the presence of endothelium by addition of carbachol to rings pre-contracted with phenylephrine. The relative importance of nitric oxide (NO) in contraction mediated by the addition of DCLHb was studied. RESULTS: Carbachol relaxed phenylephrine precontracted LIMA by 72.3+/-1.7% and RA by 97+/-0.7% confirming the presence of a functional endothelium. Sodium nitroprusside (SNP) caused complete relaxation of LIMA with an EC(50) value of 2.0+/-0.1x10(-8) M and RA with an EC(50) value of 1. 9+/-0.1x10(8) M. In the presence of DCLHb (10(-7) M), carbachol-induced relaxation was significantly reduced to 46.3+/-0. 7% (P<0.01) and the BC(50) value for SNP relaxation increased to 1. 2+/-0.1x10(-7) M (P<0.01). DCLHb caused rings to contract in the absence of phenylephrine with EC(50) values of 1.6+/-0.1x10(-7) M (LIMA) and 1.8+/-0.1x10(-7) M (RA). Presence of L-NAME (300 microM) caused no alteration in DCLHb-induced contraction. CONCLUSION: In this study of isolated rings of human vessels, DCLHb causes a significant reduction in relaxation mediated by carbachol and SNP, which is likely to be due to its ability to bind NO. However, it is possible that other mechanisms might contribute to the vasoconstrictor effects of DCLHb and these might be amenable to anti-vasospastic strategies.

Inhibitory effects of glibenclamide on the contraction of human arterial conduits used in coronary artery bypass surgery.

Glibenclamide has been shown to inhibit prostanoid-induced contraction in a number of blood vessel types. In this study, the effects of glibenclamide on the contraction of human peripheral arteries in response to both prostanoid and non-prostanoid agonists were compared and possible mechanisms of action were investigated. Segments of left internal mammary artery (LIMA) and radial artery, taken from patients undergoing coronary artery bypass graft (CABG) surgery, were mounted in organ baths containing physiological saline solution aerated with 95% O2/5% CO2 at 37 degrees C. Contractions were obtained by either the use of a thromboxane analogue (U46619), L-phenylephrine, KCl or CaCl2. The effects of glibenclamide on these contractions were observed and pEC50 values were determined after manipulation of a logistic curve-fitting equation. Concentration-dependent relaxation of U46619-contracted LIMA and radial artery was observed in the presence of glibenclamide, with calculated pEC50 values of 4.2+/-0.17 (n = 7) for LIMA and 3.26+/-0.48 (n = 5) for radial artery. Incubation of both LIMA and radial artery with glibenclamide (50 microM) caused the concentration-response curves for U46619 and L-phenylephrine to shift significantly to the right. Similarly the KCl tension relationship was caused to shift to the right. Finally, glibenclamide (100 microM) also had an inhibitory effect on Ca2+-induced tension in radial artery. These results show that the inhibitory effects of glibenclamide on human peripheral blood vessels are not restricted to prostanoid-induced contractions. Furthermore, evidence has been provided to suggest that these effects might be mediated through an interaction with voltage-sensitive Ca2+ channels.

Monoamine oxidase activities in human cystic and colonic arteries--influence of age.

The deamination of 5-hydroxytryptamine, phenylethylamine and benzylamine by monoamine oxidases (MAO-A and B) and semicarbazide sensitive amine oxidase (SSAO) respectively has been studied in homogenates of human cystic and colonic arteries by radiochemical assays. In cystic artery the deamination is mainly carried out by SSAO with a lower participation of MAO-B. The kinetic parameters were: to MAO-B the Vmax = 15.11 +/- 0.51 nmol/mg protein.h and the Km = 78.51 +/- 5.16 microM (+/- SE) and to SSAO the Vmax = 211.70 +/- 8.75 nmol/mg protein.h and the Km = 211.51 +/- 23.27 microM (+/- SE). We could not measure MAO-A activity in our experimental conditions and also the levels of catecholamines are very low and the histological studies show a poor innervation in these tissues. In colonic artery the kinetic parameters were: to MAO-B the Vmax = 5.09 +/- 0.31 nmol/mg protein.h and the Km = 29.12 +/- 4.55 microM (+/- SE) and to SSAO the Vmax = 273.67 +/- 8.35 nmol/mg protein.h and the Km = 197.89 +/- 21.81 microM (+/- SE). In this artery we could find MAO-A in five among the nine samples studied and the kinetic parameters were: the Vmax = 14.48 +/- 0.82 nmol/mg protein.h and the Km = 136.40 +/- 25.46 microM. As we have performed the experiments with human vessels from donors with different age we could not find any relationship between the activity or affinity, in MAO-B and SSAO, with age. Nevertheless, the results show in cystic artery an increase in the affinity of MAO-B with age when we consider the female group which suggests a possible role of the hormonal condition in this behaviour.

Further studies on the ex-vivo effects of procarbazine and monomethylhydrazine on rat semicarbazide-sensitive amine oxidase and monoamine oxidase activities.

Following administration of the anticancer agent, procarbazine, or one of its metabolites, monomethylhydrazine, to rats, activities of monoamine oxidases A and B (MAO A and MAO B) and of semicarbazide-sensitive amine oxidase (SSAO) were measured ex-vivo. Both compounds were found to be potent inhibitors of SSAO in tissue homogenates, exhibiting ID50 values in most tissues of approximately 8 mg kg-1 (procarbazine) and 0.08 mg kg-1 (monomethylhydrazine). Concurrent dose-dependent inhibition of MAO activities did not occur. However, in liver, potentiation of MAO B activity, to 140% of that in controls, was apparent following monomethyl-hydrazine and this effect was independent of the drug dose. Both compounds produced a dose-dependent potentiation of MAO A in brown adipose tissue, the elevation being more pronounced following monomethylhydrazine, with activity rising to 350% of that in control homogenates. In a parallel in-vitro study, monomethylhydrazine was without effect on MAO A in brown adipose tissue homogenates. By perfusing the SSAO substrate, benzylamine, through the isolated mesenteric arterial bed of the rat, it was found that pretreatment of animals with procarbazine or monomethylhydrazine reduced metabolism of this amine by a similar degree as had been determined ex-vivo in blood vessel homogenates. The results presented suggest that these compounds would be suitable for use as selective inhibitors in pharmacological examinations of SSAO function in isolated tissues and organs.

Some aspects of the pathophysiology of semicarbazide-sensitive amine oxidase enzymes.

The widespread distribution of enzymes classed as semicarbazide-sensitive amine oxidases (SSAO enzymes) throughout a very wide range of eukaryotic as well as prokaryotic organisms encourages the aspirations of those who wish to demonstrate physiological, pathological or pharmacological importance. Such enzymes are found in several tissues of mammals, both freely soluble, as in blood plasma, and membrane-bound, for example, in smooth muscle and adipose tissue. While they are capable of deaminating many amines with the production of an aldehyde and hydrogen peroxide, doubt still surrounds the identity of the most important endogenous substrates for these enzymes. At present, methylamine and aminoacetone appear to head the list of candidates. The possibility that SSAO enzymes can convert amine substrates to highly toxic metabolites is illustrated by the production of acrolein from the xenobiotic amine, allylamine and formaldehyde and methylglyoxal from methylamine and aminoacetone, respectively. Activities of SSAO enzymes may be influenced by physiological changes, such as pregnancy or pathologically by disease states, including diabetes, tumours and burns. Increased deamination of aminoacetone by tissue and plasma SSAO enzymes as a result of its increased production from L-threonine in conditions such as exhaustion, starvation and diabetes mellitus may be harmful. Such dangers could be mitigated either physiologically by a compensatory reduction in SSAO activity or pharmacologically by treatment with inhibitors of SSAO.

Semicarbazide-sensitive amine oxidases in sheep plasma: interactions with some substrates and inhibitors.

The present study has examined the affinities of sheep plasma semicarbazide-sensitive amine oxidase (SSAO) enzymes for a range of aliphatic amines and also the effects of two inhibitory compounds, beta-aminopropionitrile (BAPN) and mexiletine. Two kinetically separable enzyme activities appeared to be responsible for the metabolism of amines containing 2-5 carbon atoms while the deamination of higher amines and methylamine and allylamine produced kinetic plots characteristic of only one enzyme activity. When benzylamine metabolism was used as an indication of enzyme activity, the two inhibitors had different effects. BAPN exhibited predominantly a mixed pattern of inhibition while the effects of low concentrations of mexiletine were largely competitive. These results present evidence confirming the presence of two kinetically separable SSAO activities in sheep plasma, although we must await the development of highly selective inhibitors before these two activities can be fully resolved.

Location of the active site of rat vascular semicarbazide-sensitive amine oxidase.

Semicarbazide-sensitive amine oxidase (SSAO) activity in rat vascular smooth muscle cells is associated extensively with the plasmalemma. To determine which side of the plasmalemma the active sites of these enzymes face, the non-permeating agent, diazotised sulphanilic acid (DSA; 4.4 mM) was perfused through the isolated mesenteric arterial bed of the rat, in an attempt to inactivate only those active sites facing extracellularly. DSA perfusion abolished the pressor responses to noradrenaline via inactivation of extracellular alpha 1 receptors but had no effect on cytosolic lactate dehydrogenase activity. SSAO activity, estimated by perfusing [14C] benzylamine, was reduced following DSA perfusion to 55.9 +/- 4.9% of that in control beds and to 52.4 +/- 6.0% in homogenates of these vessels. These results suggest that almost half of SSAO active sites in rat mesenteric arteries face outwards.

The ex vivo effects of procarbazine and methylhydrazine on some rat amine oxidase activities.

Monoamine oxidase (MAO) and semicarbazide-sensitive amine oxidase (SSAO) activities were examined in homogenates of various rat tissues following i.p. administration of procarbazine or methylhydrazine. Both compounds inhibited SSAO in a dose-dependent manner in all tissues examined, with methylhydrazine the more potent agent in this respect. Little inhibition of MAO could be detected in most cases. However, hepatic MAO-B activity was potentiated significantly in rats receiving methylhydrazine and both drugs caused a dose-dependent potentiation of MAO-A in homogenates of brown adipose tissue. The potential use of these compounds in vivo as selective SSAO inhibitors is discussed.

Drug interactions with reversible monoamine oxidase-A inhibitors.

The use of monoamine oxidase inhibitors (MAOIs) in the treatment of depression has been bedeviled by reports of potentially dangerous or even fatal interactions with dietary amines and other drugs together with reports of more conventional toxicity to organs such as the liver. As a result, these otherwise very important and useful drugs have not been employed clinically as much as their effectiveness as antidepressants would indicate. Many of these unwanted actions were due partially or entirely to the fact that the older MAOIs all inhibited MAO irreversibly. The introduction of reversible inhibitors of monoamine oxidase-A (RIMAs) has greatly reduced both the number and severity of these interactions and, in particular, the risk of hypertensive crises following the ingestion of tyramine (the "cheese effect"). Potential interactions may remain with inhibitors of the uptake of 5-hydroxytryptamine, including pethidine and rapid release formulations of nasal decongestant amines. Potential interactions between RIMAs and other drugs that compete for the same metabolic pathways may still occur but should only result in changes in duration of action.

Inhibition of chick embryo lysyl oxidase by various lathyrogens and the antagonistic effect of pyridoxal.

Lysyl oxidase, which cross-links collagen and elastin, was obtained from chick embryo bone and cartilage and its substrate, elastin, from aorta. The enzyme was studied using an improved assay which enabled the stability of the substrate to be monitored. The enzyme was fully inhibited in vivo by beta-aminopropionitrile, semicarbazide, thiosemicarbazide and isoniazid and in vitro by beta-aminopropionitrile and semicarbazide but only partially by thiosemicarbazide and isoniazid. Penicillamine, which solubilizes collagen by labilizing Schiff base cross-links in vivo and which prevents stable cross-link formation in vitro indirectly by binding to aldehyde groups on collagen, was shown to have no direct inhibitory effect on lysyl oxidase in vivo or in vitro. Homocysteine, which also solubilizes collagen by a mechanism similar to penicillamine does not inhibit lysyl oxidase either in vivo or in vitro. Pyridoxal reversed the inhibition of lysyl oxidase by semicarbazide and isoniazid in vivo but was unable to reverse that produced by either beta-aminopropionitrile or thiosemicarbazide. These results can be explained by the presence of a sulphydryl group near the active site of lysyl oxidase, which can form a complex with the nitrile group on beta-aminopropionitrile or with the thiol group on thiosemicarbazide leading to irreversible inhibition.

Characteristics of procarbazine as an inhibitor in-vitro of rat semicarbazide-sensitive amine oxidase.

Procarbazine (N-isopropyl-alpha-(2-methyl hydrazino)-p-toluamide hydrochloride) inhibited more powerfully the deamination of benzylamine by semicarbazide-sensitive amine oxidase (SSAO) of rat brown adipose tissue than the deamination of 5-hydroxytryptamine and benzylamine by rat liver monoamine oxidase-A or -B activities, respectively. Inhibition of SSAO, but not monoamine oxidase, was time-dependent. Use of metabolic inhibitors, and an enzyme dilution technique, suggested that any conversion of procarbazine to an active species must be as a result of the action of SSAO itself and not of any other enzyme. The non-competitive kinetics and the time-dependence of inhibition were indicative of a suicide interaction between procarbazine and SSAO. The slow reversal of inhibition by dialysis was evidence in favour of the involvement of tight binding, rather than covalent bonding. High concentrations of benzylamine afforded the enzyme significant protection from the action of procarbazine, indicating that the interaction is at or near the active site. If the properties of procarbazine, evident in in-vitro studies, are retained in-vivo, these data suggest that procarbazine might be suitable for the examination of SSAO activities, both in-vivo and ex-vivo.

Effects in-vitro of procarbazine metabolites on some amine oxidase activities in the rat.

The effects were examined of four metabolites of the anticancer agent, procarbazine (N-isopropyl-alpha-(2-methyl hydrazino)-p-toluamide hydrochloride) on semicarbazide-sensitive amine oxidase (SSAO) and monoamine oxidase-A and -B (MAO-A and -B) activities in rat brown adipose tissue and liver homogenates, respectively. Azoprocarbazine (AZO) and monomethylhydrazine (MMH) inhibited selectively the deamination of benzylamine by SSAO, when compared with their effects on MAO activities. The IC50 values against SSAO, of 32.7 nM (AZO) and 7.0 nM (MMH), were more than three orders of magnitude lower than those exhibited against MAO. Neither isomer of azoxyprocarbazine was an effective inhibitor of rat amine oxidase activities. The inhibition of SSAO by AZO was reversed very slowly by dialysis, in contrast to results seen for MMH. The non-competitive kinetics of MMH and the ability of B24, a rapidly reversible SSAO inhibitor, to protect SSAO against inhibition by MMH are consistent with the view that this compound binds to the enzyme cofactor at, or near, the active site.

Amine oxidase enzymes of sheep blood vessels and blood plasma: a comparison of their properties.

1. A comparison between the biochemical properties of semicarbazide-sensitive amine oxidase (SSAO) activities has been made in sheep blood plasma and arterial wall. 2. The metabolism of benzylamine (BZ) by blood plasma was resolved into high affinity (Km 2.76 +/- 0.24 microM) and low affinity (Km 743 +/- 49 microM) activities. Spermidine metabolism was by a single component (Km 174 +/- 22 microM) and this amine reduced the metabolism of high concentrations of BZ. 3. A single component metabolised BZ in arterial homogenates (Km 11.3 +/- 1.3 microM) and which metabolised spermidine at a very slow rate. 4. Dopamine was deaminated by plasma SSAO and competed with both low and high concentrations of BZ. Dopamine also interfered with arterial metabolism of BZ. 5. These results suggest that there are two SSAO enzymes in sheep blood plasma, with one having similar properties to SSAO in the arterial wall.

Effect of benzylamine and its metabolites on the responses of the isolated perfused mesenteric arterial bed of the rat.

1. Semicarbazide-sensitive amine oxidase (SSAO) is an enzyme activity which can be found in the plasma membrane of rat vascular smooth muscle cells. We have investigated the possibility that the products of deamination by this enzyme, namely ammonia, hydrogen peroxide and the aldehyde, may be important in the modulation of the responses of vascular smooth muscle to extracellular stimuli. 2. The isolated perfused mesenteric arterial bed of the rat was used and dose-pressure response curves (DRC) to bolus injections of adrenaline (Ad) or ATP were plotted by non-linear curve fitting. The relaxant effects of carbachol (CCh), which releases endothelium dependent relaxing factor (ERDF), were studied by co-administering CCh with Ad. The effects of including the preferred SSAO substrate, benzylamine (BZ; 25 microM), in the perfusion fluid throughout the experiment and of inhibition of SSAO by treatment of rats with (E)-2-(3',4'-dimethoxyphenyl)-3-fluoroallylamine (MDL 72145; 1 mg kg-1) 1 h before dissection, have been studied. 3. Neither BZ nor SSAO inhibition affected the DRC to ATP. BZ shifted Ad responses to the left, inhibition of SSAO increased this shift indicating that the amine, but not its metabolites, were responsible for the potentiation of the responses to Ad. DRC to CCh showed a shift to the left and a significant decrease in the Hill slope with BZ, indicative of a potentiation of low doses of CCh more than high doses. Inhibition of SSAO prevented this change and so the metabolites of BZ deamination appeared to be involved in the potentiation. 4. Ammonia generated by SSAO may contribute to the production of EDRF or hydrogen peroxide may sensitize guanylate cyclase to stimulation by EDRF and so explain these findings.