Persistent platelet activation in patients with type 2 diabetes treated with low doses of aspirin.

BACKGROUND: The percentage of diabetic patients who do not benefit from the protective effect of aspirin is larger than in other populations at cardiovascular risk. OBJECTIVE: We compared the ability of aspirin to suppress TxA2 and platelet activation in vivo, in type-2 diabetics vs. high-risk non-diabetic patients. METHODS: Urinary 11-dehydro-TXB2, plasma sCD40 L, and sP-selectin were measured, together with indices of low-grade inflammation, glycemic control, and lipid profile, in 82 patients with type-2 diabetes and 39 without diabetes, treated with low doses of aspirin. RESULTS: Urinary 11-dehydro-TxB2, plasma sCD40L and sP-selectin were significantly higher in diabetics than in controls: [38.9 (27.8-63.3) vs. 28.5 (22.5-43.9) ng mmol(-1) of creatinine, P = 0.02], [1.06 (0.42-3.06) vs. 0.35 (0.22-0.95) ng mL(-1); P = 0.0001], [37.0 (16.8-85.6) vs. 20.0 (11.2-35.6) ng mL(-1), P = 0.0001], respectively. The proportion of individuals with diabetes increased across quartiles of 11-dehydro-TxB2, sCD40L, and sP-selectin, with the highest quartiles of 11-dehydro-TxB2, sCD40L and sP-selectin, including 66%, 93.3%, and 93.3% of individuals with diabetes. Markers of platelet activation positively correlated with indices of glycemic control but not with markers of low-grade inflammation. CONCLUSIONS: Platelet dysfunction associated with insufficient glycemic control, may mediate persistent platelet activation under aspirin treatment.

Erythrocyte enzymes catalyze 1-nitropyrene and 3-nitrofluoranthene nitroreduction.

Nitroarenes are environmental contaminants produced during incomplete combustion processes. Nitroreduction, the most important pathway of nitroarene toxification, occurs mainly in the liver and intestine. In the present study, we show that human red cells may also possess the metabolic competence to reduce 1-nitropyrene (NP) and 3-nitrofluoranthene (NF), the nitroarenes chosen as model compounds, to their corresponding amino derivatives, 1-aminopyrene (AP) and 3-aminofluoranthene (AF). The requirement of the cofactor couple NADH/FMN suggests that erythrocyte nitroreductase activity occurs via one electron transfer. The presence of oxygen strongly inhibited the haemolysate-catalyzed nitroarene reduction, whether measured as amine formation or nitroarene disappearance. Intermediate reactive species, that bind covalently to haemoglobin and/or other erythrocyte proteins, are formed during nitroreduction catalyzed by human haemolysate. In fact, the reduced metabolites AP and AF were released after mild acid hydrolysis of red cell proteins exposed to NP and NF, thus suggesting that sulphinamide adducts have been formed.

Erythrocyte-mediated toxification of 1,8-dinitropyrene: I. Reduction.

Nitroarenes are environmental contaminants produced during incomplete combustion processes. It has been reported that nitroreduction, the most important pathway of nitroarene toxification, occurs mainly in the liver and intestine. In the present study we have shown that red cells also possess the metabolic competence to reduce nitroarenes. In particular, 1,8-dinitropyrene, the nitroarene chosen as model compound, was reduced to the corresponding mono- and diamino-derivatives, 1-amino-8-nitropyrene and 1,8-diaminopyrene, by human lysate supplemented with cofactors.

Arylation of sulfhydryl groups in vitro by the naturally occurring sesquiterpenoid benzoquinone avarone.

Avarone (AQ) is a naturally occurring sesquiterpenoid benzoquinone possessing antileukaemic activity. Its reactivity towards glutathione (GSH) and protein sulfhydryl (SH) groups was investigated. The stoichiometry of AQ reaction with GSH at [GSH]/[AQ] ratios lower than unity proved to be 1:2 (thiol:quinone), consistent with the formation of the corresponding hydroquinone (avarol) as well as a quinone-thioether in the reaction. Conversely, when the [GSH]/[AG] ratio was higher than unity, a hydroquinone-thioether was the only reaction product. AQ/protein interaction was also investigated by using bovine serum albumin (BSA) as model compound. As observed with GSH, arylation rather than oxidation of SH groups appeared to be the mechanism responsible for the AQ-induced depletion of protein SH groups. However, AQ proved to be less effective in depleting BSA sulfhydryls than that of GSH. AQ disappearance after BSA addition was greater than expected on the basis of the total SH groups depleted, if a stoichiometric ratio 1:2 (thiol:quinone) was assumed. It also occurred in the presence of BSA with blocked SH groups, thus suggesting that AQ may react with other nucleophilic protein residues, such as amino or imino groups. When HepG2 cells were exposed to AQ, depletion of both protein SH groups and GSH occurred. However, in contrast to the above, AQ proved to be more effective, probably because of its lipophilic nature, in depleting protein SH groups than GSH. Also, in intact cells AQ appeared to arylate both SH and other nucleophilic groups in proteins. This mechanism may play a major role in AQ-induced cytotoxicity.

Effect of avarol and avarone on in vitro-induced microsomal lipid peroxidation.

Lipid peroxidation was employed as an experimental model to study the antioxidant properties of avarol, a sesquiterpenoid hydroquinone and of its quinone, avarone. In the NADPH- or ascorbate-linked lipid peroxidation, avarol and avarone were shown to be more effective as inhibitors than in the t-BuOOH-dependent peroxidative process. However, in all three systems employed avarol was a more powerful inhibitor than avarone. The chemical structure of avarol, having an easily donatable hydrogen atom and its kinetics of inhibition suggested that the hydroquinone acted mainly as a radical scavenger. Conversely avarone appeared to interfere mainly with the initiation phase of lipid peroxidation. However, avarol and the semiquinone intermediate may contribute to the inhibitory action of the quinone. In fact avarone reduction to avarol has been shown to occur in the presence of reducing agents such as ascorbate or Fe(II) and to be catalyzed by NADPH-supplemented microsomes.

Effect of avarol, avarone and nine of their natural and synthetic derivatives on microsomal drug-metabolizing enzymes.

Avarol, a sesquiterpenoid hydroquinone, its quinone avarone, both main secondary metabolites from the marine sponge Dysidea avara and nine of their natural and synthetic derivatives were tested for ability to interact selectively with rat liver microsomal phenobarbital (PB)- or 3-methylcholanthrene (3-MC)-induced cytochrome (cyt.) P-450 isoenzymatic forms. Ethoxy- and pentoxyresorufin, aminopyrine and ethoxycoumarin were the specific substrates used for assaying cyt. P-450-dependent mono-oxygenase activities. All compounds were more effective in inhibiting the enzymatic activities measured in microsomes from PB-induced rat liver than those measured in microsomes from 3-MC-treated rats. Avarol and avarone, which were the most active inhibitors, act as mixed-type inhibitors of pentoxyresorufin-O-dealkylase activity. Mono- and diacetyl-derivatives of avarol, being deacetylated by rat liver microsomes, were almost as effective as the parent compound. Conversely, avarone derivatives, where one or two hydrogen atoms of the quinone ring have been substituted, were less effective inhibitors than the parent compound. The selective inhibition of PB-inducible cyt.P-450IIB by avarol and avarone was confirmed by their ability to inhibit the mutagenic activation of cyclophosphamide, as opposed to that of benzo[a]pyrene, which is activated mainly by the 3-MC-inducible cyt.P-450IA.

Effect of rat liver cytosolic enzymes and cofactors on mutagenicity of 1-amino-8-nitropyrene.

1-Amino-8-nitropyrene (1,8-ANP), a product of 1,8-dinitropyrene metabolism by either bacterial or mammalian enzymes, is weakly mutagenic to the 'classical nitroreductase'-deficient Salmonella tester strain TA98NR. The addition to the test system of rat liver cytosol without cofactors did not produce any effect on the 1,8-ANP mutagenic response toward TA98NR strain. Conversely, when both rat hepatic cytosol and NADPH (1 mM) were added to the mutagenicity assay, a 10-fold increase in 1,8-ANP mutagenic activity was observed. This suggests the involvement of rat hepatic cytosolic NADPH-dependent nitroreductase(s) in 1,8-ANP mutagenic activation. The addition to the mutagenesis assay of pentachlorophenol, an inhibitor of O-acetyltransferase and sulfotransferase, produced a dose-dependent decrease of 1,8-ANP mutagenic activation, whereas 2,6-dichloro-4-nitrophenol, a more specific inhibitor of sulfotransferase than O-acetyltransferase, did not affect the activation of 1,8-ANP to a mutagen at concentrations that selectively inhibit only bacterial sulfotransferase. This indicates that bacterial O-acetyltransferase but not sulfotransferase plays a role in the mutagenic activation of 1,8-ANP. Addition of acetyl co-enzyme A (AcCoA) and adenosine 3'-phosphate 5'-phosphosulfate (PAPS), cofactors for O-acetyl-transferase and sulfotransferase respectively, to the test system caused a dose-dependent inhibition of 1,8-ANP mutagenic activation by rat liver cytosol and NADPH, probably due to the formation of highly reactive O-acetoxy and N-sulfate ester derivatives of 1,8-ANP, which react with nucleophilic sites before reaching bacterial DNA. This hypothesis was confirmed by DNA covalent binding in in vitro experiments showing that both the cofactors AcCoA and PAPS enhanced the NADPH/rat liver cytosol-mediated covalent binding of 1,8-ANP to DNA from calf thymus 10- and 3-fold respectively. It seems likely that rat hepatic cytosolic nitroreductases activate 1,8-ANP to an N-hydroxyarylamine derivative which can be further metabolized to mutagenic species by either bacterial or mammalian O-acetyltransferase.

Effect of enzyme inducers on metabolism of 1-nitropyrene in human hepatoma cell line HepG2.

We measured the response of HepG2 cells to the classic cytochrome (cyt.) P-450 inducers 3-methylcholanthrene (3-MC) and phenobarbital (PB), by evaluating oxidative and/or reductive metabolism of the nitroarenes, 1-NP and 1,6-dinitropyrene (1,6-DNP), in control and induced cells. In HepG2 cells, 3-MC induces ring-hydroxylation of 1-NP, whereas PB stimulates its nitroreduction. PB induces NADPH-cyt. c reductase, but does not affect other cytosolic and microsomal enzymes which contribute to 1-NP nitroreduction in these cells. However, PB-inducible nitroreductase activity seems to be associated primarily with cyt. P-450 isoenzymatic form(s), as indicated by the requirement for NADPH and the response to specific inhibitors such as alpha-naphthoflavone and CO.

Characterization of oxidative and reductive metabolism in vitro of nitrofluoranthenes by rat liver enzymes.

Nitrofluoranthenes (NFs) are mutagenic and carcinogenic environmental pollutants found in incomplete combustion products and urban air particulate. We have studied both oxidative and reductive metabolism in vitro of different NF isomers mediated by subcellular rat liver fractions. Under aerobic conditions only ring hydroxylation of NFs by rat liver microsomes occurred and the isomeric position of the nitro group affected both the amount and the type of phenolic metabolites formed. Liver microsomes from 3-methylcholanthrene-induced rats were most effective in giving ring hydroxylated 7- and 8-nitrofluoranthene, whereas liver microsomes from phenobarbital-pretreated rats were the most active in metabolizing 1- and 3-nitrofluoranthene. Under anaerobic conditions, only reduction of NFs mediated by both cytosolic and microsomal rat liver enzymes occurred. Cofactor requirements and inhibition experiments indicated that the reductase activity in rat liver cytosolic fractions could be ascribed to DT-diaphorase, aldehyde oxidase and/or other unknown enzymes. The microsomal reductase activity was inhibited by oxygen, carbon monoxide, 2-diethylaminoethyl-2,2-diphenylvalerate hydrochloride and n-octylamine, and slightly by cytochrome c; flavin mononucleotide greatly enhanced this activity. 3-Nitrofluoranthene microsomal nitroreductase activity was increased by phenobarbital rat pretreatment and this increment correlated well with the content of cytochrome P450. These results indicate a participation of cytochrome P450 in the reductive metabolism of NFs by rat liver microsomes.

Characterization of the induction of rat hepatic microsomal drug-metabolizing enzymes by 1-nitropyrene metabolites, 1-aminopyrene and N-acetylaminopyrene.

The effect of 1-aminopyrene (1-AP) and N-acetylaminopyrene (1-NAAP) on rat hepatic microsomal monooxygenase system was investigated. Both drugs increased the total content of cytochrome P-450 (cyt. P-450). The substrate specificity and the electrophoretic pattern of 1-AP and 1-NAAP induced cytochrome(s) were compared with those of the major forms of cyt. P-450 induced by 3-methylcholanthrene (3-MC) and phenobarbital (PB). The results suggest that the form of cyt. P-450 induced by 1-AP and 1-NAAP resembles that one induced by 3-MC. Furthermore the abilities of liver microsomes from control or differently induced rats to ring hydroxylate and to activate 1-nitropyrene (1-NP) metabolites to species mutagenic for bacteria were compared. It was observed that: (1) 1-NAAP is a good substrate for microsome-mediated ring hydroxylation, whereas 1-AP is oxygenated only at a low extent; (2) 3-MC, 1-AP and 1-NAAP-stimulated microsomes are more active than control or PB-ones to ring hydroxylate 1-NAAP. As phenolic derivatives of 1-NAAP show high mutagenic activity, these results indicate that 1-AP and 1-NAAP induce toxification pathways of 1-NP in similar way, even if in less extent, as compared to 3-MC.

Induction of hepatic drug-metabolizing enzymes in rats treated with 1-nitropyrene.

The inducing effects of 1-nitropyrene (1-NP) on the microsomal cytochrome P-450 system were studied in rats. Intraperitoneal administration of 1-NP led to increases in cytochrome P-450 content and aryl hydrocarbon hydroxylase, ethoxycoumarin, and ethoxyresorufin-O-deethylase activities. These increases were dose dependent. Cytochrome b5 content and aminopyrine and p-nitroanisole demethylase activities were not affected by treatment of rats with 1-NP. Substrate specificity, sensitivity to mixed-function oxidase inhibitors, and electrophoretic pattern of 1-NP-induced cytochrome(s) P-450 were compared to the major forms of cytochrome P-450 induced by phenobarbital and methylcholanthrene. Furthermore microsomes from 1-NP-induced rats showed greater ability to metabolize the chemical as compared with those from control animals; this result indicates that 1-NP induces a form(s) of cytochrome P-450 especially effective in the metabolism of the substance itself.

Effect of liver enzyme inducers on metabolite excretion in rats treated with 1-nitropyrene.

Non-induced and phenobarbital (PB) or methylcholanthrene (MC) pretreated rats were injected with 1-nitropyrene (1-NP). Mutagenic activity of urine and feces samples were compared by the Salmonella/microsome assay. The highest, indirect-acting mutagenicity was associated with urines from MC-induced rats; HPLC analysis of organic extracts of urine samples showed that the differences in mutagenic response can be ascribed to different amounts of hydroxy derivatives of N-acetylaminopyrene excreted. Monohydroxy derivatives of 1-NP, being detected in the HPLC profiles of urine from PB-induced rats only, could be responsible of the higher direct-acting mutagenic activity of these samples as compared to urine from non-induced or MC-induced rats. The excretion rate of aminopyrene, the main metabolite of 1-NP identified in rat feces samples, was not affected by inducer pretreatment.

Inhibition of cyclophosphamide mutagenicity by beta-carotene.

Cyclophosphamide (CP) metabolites, rather than the parent compound, show mutagenic activity towards Salmonella typhimurium TA 1535 tester strain when S9 fraction from phenobarbital (PB)-induced rat liver is used as in vitro metabolizing system. On the other hand, inhibition of CP in vitro mutagenicity was observed by adding increasing amounts of beta-carotene (beta-C) to the system. A typical dose-dependent mutagenic response was observed by assaying 24 h urine samples of PB-induced rats injected i.p. with different amounts of CP. Addition of beta-C to urines of CP-treated rats failed to inhibit their mutagenicity. Conversely, a marked decrease in urine mutagenicity was observed when rats were simultaneously treated with the two drugs. These data show that beta-carotene partially inhibits, in vitro and in vivo, CP metabolism via hepatic mixed-function oxidase enzymes to mutagenic species.