Xenobiotic-metabolizing cytochromes P450 convert prostaglandin endoperoxide to hydroxyheptadecatrienoic acid and the mutagen, malondialdehyde

Cyclooxygenases catalyze the oxygenation of arachidonic acid to prostaglandin endoperoxides. Cyclooxygenase-2- and the xenobiotic-metabolizing cytochrome P450s 1A and 3A are all aberrantly expressed during colorectal carcinogenesis. To probe for a role of P450s in prostaglandin endoperoxide metabolism, we studied the 12-hydroxyheptadecatrienoate (HHT)/malondialdehyde (MDA) synthase activity of human liver microsomes and purified P450s. We found that human liver microsomes have HHT/MDA synthase activity that is concentration-dependent and inhibited by the P450 inhibitors, ketoconazole and clotrimazole with IC(50) values of 1 and 0.4 microM, respectively. This activity does not require P450 reductase. HHT/MDA synthase activity was present in purified P450s but not in heme alone or other heme proteins. The catalytic activities of various purified P450s were determined by measuring rates of MDA production from prostaglandin endoperoxide. At 50 microM substrate, the catalytic activities of purified human P450s varied from 10 +/- 1 to 0.62 +/- 0.02 min(-1), 3A4 > 2E1 > 1A2. Oxabicycloheptane analogs of prostaglandin endoperoxide, U-44069 and U-46619, induced spectral changes in human P450 3A4 with K(s) values of 240 +/- 20 and 130 +/- 10 microM, respectively. These results suggest that co-expression of cyclooxygenase-2 and P450s in developing cancers may contribute to genomic instability due to production of the endogenous mutagen, MDA.     

Inhibition of glucose uptake in murine cardiomyocyte cell line HL-1 by cardioprotective drugs dilazep and dipyridamole

Inhibition of adenosine reuptake by nucleoside transport inhibitors, such as dipyridamole and dilazep, is proposed to increase extracellular levels of adenosine and thereby potentiate adenosine receptor-dependent pathways that promote cardiovascular health. Thus adenosine can act as a paracrine and/or autocrine hormone, which has been shown to regulate glucose uptake in some cell types. However, the role of adenosine in modulating glucose transport in cardiomyocytes is not clear. Therefore, we investigated whether exogenously applied adenosine or inhibition of adenosine transport by S-(4-nitrobenzyl)-6-thioinosine (NBTI), dipyridamole, or dilazep modulated basal and insulin-stimulated glucose uptake in the murine cardiomyocyte cell line HL-1. HL-1 cell lysates were subjected to SDS-PAGE and immunoblotting to determine which GLUT isoforms are present. Glucose uptake was measured in the presence of dipyridamole (3-300 microM), dilazep (1-100 microM), NBTI (10-500 nM), and adenosine (50-250 microM) or the nonmetabolizable adenosine analog 2-chloro-adenosine (250 microM). Our results demonstrated that HL-1 cells possess GLUT1 and GLUT4, the isoforms typically present in cardiomyocytes. We found no evidence for adenosine-dependent regulation of basal or insulin-stimulated glucose transport in HL-1 cardiomyocytes. However, we did observe a dose-dependent inhibition of glucose transport by dipyridamole (basal, IC(50) = 12.2 microM, insulin stimulated, IC(50) = 13.09 microM) and dilazep (basal, IC(50) = 5.7 microM, insulin stimulated, IC(50) = 19 microM) but not NBTI. Thus our data suggest that dipyridamole and dilazep, which are widely used to specifically inhibit nucleoside transport, have a broader spectrum of transport inhibition than previously described. Moreover, these data may explain previous observations, in which dipyridamole was noted to be proischemic at high doses.     

Mutation of residue 33 of human equilibrative nucleoside transporters 1 and 2 alters sensitivity to inhibition of transport by dilazep and dipyridamole.

Human equilibrative nucleoside transporters (hENT) 1 and 2 differ in that hENT1 is inhibited by nanomolar concentrations of dipyridamole and dilazep, whereas hENT2 is 2 and 3 orders of magnitude less sensitive, respectively. When a yeast expression plasmid containing the hENT1 cDNA was randomly mutated and screened by phenotypic complementation in Saccharomyces cerevisiae to identify mutants with reduced sensitivity to dilazep, clones with a point mutation that converted Met33 to Ile (hENT1-M33I) were obtained. Characterization of the mutant protein in S. cerevisiae and Xenopus laevis oocytes revealed that the mutant had less than one-tenth the sensitivity to dilazep and dipyridamole than wild type hENT1, with no change in nitrobenzylmercaptopurine ribonucleoside (NBMPR) sensitivity or apparent uridine affinity. To determine whether the reciprocal mutation in hENT2 (Ile33 to Met) also altered sensitivity to dilazep and dipyridamole, hENT2-I33M was created by site-directed mutagenesis. Although the resulting mutant (hENT2-I33M) displayed >10-fold higher dilazep and dipyridamole sensitivity and >8-fold higher uridine affinity compared with wild type hENT2, it retained insensitivity to NBMPR. These data established that mutation of residue 33 (Met versus Ile) of hENT1 and hENT2 altered the dilazep and dipyridamole sensitivities in both proteins, suggesting that a common region of inhibitor interaction has been identified.     

Mutagenicity of the malondialdehyde oligomerization products 2-(3'-oxo-1'-propenyl)-malondialdehyde and 2,4-dihydroxymethylene-3-(2,2-dimethoxyethyl)glutaraldehyde in Salmonella

Malondialdehyde (MDA), a byproduct of non-enzymatic lipid peroxidation and prostaglandin biosynthesis, has been shown to be a weak frameshift mutagen in Salmonella mutagenicity assays. Because it is a dialdehyde, MDA can undergo self condensation to form polymeric products. It is possible that these condensation products are highly mutagenic and have contributed to previously reported estimates of MDA mutagenicity. We synthesized two major MDA polymerization products, (1) 2-(3'-oxo-1'-propenyl)-malondialdehyde [(MDA)2] and (2) 2,4-dihydroxymethylene-3-(2,2-dimethoxyethyl)glutaraldehyde [(MDA)3Me2] and tested their mutagenicity in the Salmonella frameshift tester strains hisD3052 and TA94 (hisD3052/pKM101). Analysis of the reversion rates revealed both (MDA)2 and (MDA)3Me2 to be weak mutagens, approximately equipotent to MDA. Although both (MDA)2 and (MDA)3Me2 are mutagenic, the fact that their formation is thermodynamically unfavorable under physiological conditions suggests they do not contribute significantly to the mutagenicity of MDA solutions.     

Dilazep analogues for the study of equilibrative nucleoside transporters 1 and 2 (ENT1 and ENT2)

As ENT inhibitors including dilazep have shown efficacy improving oHSV1 targeted oncolytic cancer therapy, a series of dilazep analogues was synthesized and biologically evaluated to examine both ENT1 and ENT2 inhibition. The central diamine core, alkyl chains, ester linkage and substituents on the phenyl ring were all varied. Compounds were screened against ENT1 and ENT2 using a radio-ligand cell-based assay. Dilazep and analogues with minor structural changes are potent and selective ENT1 inhibitors. No selective ENT2 inhibitors were found, although some analogues were more potent against ENT2 than the parent dilazep.     

Interactions of Malondialdehyde and 4-Hydroxynonenal with Rat Liver Plasma Membranes and their Effect on Binding of Prostaglandin E2 by Specific Receptors

We investigated effect of aldehydic products of lipid peroxidation, malondialdehyde (MDA) and 4-hydrox-ynonenal (HNE) on prostaglandin (PG) E₂ receptors of liver plasma membranes. The modification of the membranes by MDA diminished PGE₂ binding, decreasing receptor affinity for PGE₂ and receptor density whereas HNE increased PGE₂ binding, enhanced receptor density but did not changed receptor affinity. ESR study showed the decrease of the whole membrane fluidity after modification by MDA whereas HNE lowered membrane fluidity only in the internal zone of lipid bilayer and increased it in the surface area. The possible effects of membrane changes caused by MDA and HNE on PGE₂ receptor parameters are discussed.     

Dilazep-induced vasodilation is mediated through adenosine receptors

Administration of dilazep, an inhibitor of adenosine uptake, significantly reduced systemic arterial blood pressure and increased superior mesenteric arterial conductance without affecting the plasma adenosine levels of femoral arterial or portal venous blood. Administration of a bolus dose of 8-phenyltheophylline (8-PT), an antagonist of adenosine receptors, blocked adenosine-mediated autoregulation of the superior mesenteric artery. After the blockade of adenosine receptors by 8-PT, dilazep did not produce vasodilation. These data suggest that dilazep has a vasodilating effect in vivo that is mediated by adenosine.     

Powerful mutagenicity of a bipyridylium herbicide in a nitrogen-fixing blue-green alga Nostoc muscorum

Malondialdehyde (MDA), an in vivo metabolite of lipid peroxidation and prostaglandin biosynthesis, is mutagenic in Salmonella typhimurium. It is a reactive electrophile that can form interstrand cross-links in DNA. To explore the possibility that MDA-induced interstrand cross-links are the pre-mutagenic lesion, we have quantitated the ability of highly purified preparations of MDA to form interstrand cross-links when reacted with linear plasmid DNA. At physiological temperature and pH, MDA did not form DNA cross-links as determined by DNA denaturation followed by agarose gel electrophoresis. DNA cross-links were formed, however, when incubations with MDA were carried out at either pH 4.2 or temperatures exceeding 60 degrees. alpha-Methylmalondialdehyde (CH3MDA) was found to cross-link DNA more efficiently than MDA, but was not mutagenic in any tester strain of Salmonella. MDA polymers, formed by acid incubation of MDA, also were capable of inducing cross-links. However, an inverse relationship was observed between mutagenicity and extent of polymerization. The pattern of mutagenic response for MDA in different strains of Salmonella was compared with mitomycin C, an established mutagenic cross-linking agent. Error-prone repair and a UvrB+ phenotype, which are needed for the induction of mutations by mitomycin C, were not required for MDA mutagenesis. These findings, taken together, dissociate the mutagenicity of MDA from its ability to form interstrand cross-links with DNA.     

Dilazep, a nucleoside transporter inhibitor, modulates cell cycle progression and DNA synthesis in rat mesangial cells in vitro

The direct effects of the nucleoside transporter inhibitor dilazep on the cell cycle of mesangial cells have not before been investigated. The purpose of this study was to elucidate whether dilazep can inhibit the proliferation of mesangial cells and how it interferes with the cell cycle of these cells. DNA histograms were used and BrdUrd uptake rate was measured by flow cytometry. There was no significant difference in the cell numbers among the untreated group and the 10⁻⁵M, 10⁻⁶M or 10⁻⁷M dilazep-treated groups at 24 h of incubation. However, at 48 and 72 h, the cell numbers in the dilazep-treated groups were significantly lower compared with that of the untreated group (P0.005). The DNA histograms of cultured rat mesangial cells at 12, 24, and 48 h of incubation with 10⁻⁵ M dilazep showed that the ratio of the S phase population in the dilazep-treated group decreased by 2.2% at 12 h, by 9.6% at 24 h, and by 18.9% at 48 h compared with the untreated group. The ratio of the G₀/G₁ phase population in the dilazep-treated group significantly increased: 6.8% at 12h (P 0.05), 13.9% at 24 h (P 0.001), and 76.5% at 48 h (P 0.001) compared with the untreated group. A flow cytometric measurement of bivariate DNA/BrdUrd distribution demonstrated that the DNA synthesis rate in the S phase decreased after 6 h (P 0.005) and 12 h (P 0.05) of incubation compared with the untreated group. These results suggest that dilazep inhibits the proliferation of cultured rat mesangial cells by suppressing the G₁/S transition by prolonging G₂/M and through decreasing the DNA synthesis rate     

High affinity uptake of cAMP in rat brain: Inhibition by coronary vasodilators dilazep and hexobendine

High affinity uptake of cAMP by slices of rat cerebral cortex was found to be inhibited by specific coronary vasodilators dilazep and hexobendine, but it was insensitive to a wide variety of centrally-acting drugs. Experiments using subcellular fractionation of cortical homogenates indicated preferential association of cAMP uptake with fractions rich in synaptosomes. Furthermore, autoradiographical studies showed that cAMP uptake by cerebellar slices was particularly strong in the molecular layer which is known to have a relatively high density of synaptic terminals.The present results suggest that high affinity uptake of cAMP is located in the vicinity of synaptic contacts, probably in nerve terminals. The data on the effects of drugs, especially the inhibition by hexobendine and dilazep, offer a pharmacological means to investigate a possible role of cAMP uptake in synaptic transmission.     

Mutation of Trp29 of human equilibrative nucleoside transporter 1 alters affinity for coronary vasodilator drugs and nucleoside selectivity

hENT1 (human equilibrative nucleoside transporter 1) is inhibited by nanomolar concentrations of various structurally distinct coronary vasodilator drugs, including dipyridamole, dilazep, draflazine, soluflazine and NBMPR (nitrobenzylmercaptopurine ribonucleoside). When a library of randomly mutated hENT1 cDNAs was screened using a yeast-based functional complementation assay for resistance to dilazep, a clone containing the W29G mutation was identified. Multiple sequence alignments revealed that this residue was highly conserved. Mutations at Trp29 were generated and tested for adenosine transport activity and inhibitor sensitivity. Trp29 mutations significantly reduced the apparent V(max) and/or increased the apparent K(m) values for adenosine transport. Trp29 mutations increased the IC50 values for hENT1 inhibition by dipyridamole, dilazep, NBMPR, soluflazine and draflazine. NBMPR and soluflazine displayed remarkably similar trends, with large aromatic substitutions at residue 29 resulting in the lowest IC50 values, suggesting that both drugs could interact via ring-stacking interactions with Trp29. The W29T mutant displayed a selective loss of pyrimidine nucleoside transport activity, which contrasts with the previously identified L442I mutant that displayed a selective loss of purine nucleoside transport. W29T, L442I and the double mutant W29T/L442I were characterized kinetically for nucleoside transport activity. A helical wheel projection of TM (transmembrane segment) 1 suggests that Trp29 is positioned close to Met33, implicated previously in nucleoside and inhibitor recognition, and that both residues line the permeant translocation pathway. The data also suggest that Trp29 forms part of, or lies close to, the binding sites for dipyridamole, dilazep, NBMPR, soluflazine and draflazine.     

Haptoglobin and lipoperoxides in rabbit plasma under turpentine- and endotoxin-induced inflammation

Inflammation was induced in rabbits by injection of turpentine or/and endotoxin. Haptoglobin (Hp) and lipoperoxide (as malondialdehyde - MDA) concentrations in rabbit plasma, were measured. In some experiments indomethacin was administered, as a specific control of anti-inflammatory action. In the course of the reagents-induced inflammation there existed positive correlation between changes in Hp and MDA levels. Indomethacin was found to eliminate this effect. Under the model system of experiments, Hp as presumed endogenous inhibitor of prostaglandin synthesis, even in concentrations in rabbits' blood 15-20 times higher than normal, did not exert any protective action against inflammation.     

Biological and methodological implications of prostaglandin involvement in mouse brain lipid peroxidation measurements

Enhanced cyclooxygenase-mediated prostaglandin (PG) turnover occurring during sacrifice and biochemical processing of tissues also generates malondialdehyde (MDA), a product of lipid peroxidation (LPO). Studies reporting on LPO estimated by thiobarbituric acid reactive substances (TBARS) have failed to consider such artefactual increases. This study reports the relative proportion of PG metabolism-derived MDA (PG-MDA) in mouse brain regions during the TBARS assay. The cyclooxygenase inhibitor indomethacin significantly lowered MDA in fronto-parietal cortex and corpus striatum. Indomethacin (50–800 g/ml, in vitro) increased estimated TBARS in whole brain. Such enhancement was absent when indomethacin (20–80 g/sample) was added to the MDA standard curve, reflecting its interaction with TBARS other than MDA. PG-MDA contributes as much as 15% to the total estimated value of MDA in fronto-parietal cortex and corpus striatum and must be corrected for in LPO studies.     

Effect of factor VIII concentrate on malondialdehyde release by normal human platelets]

It is well known that Factor VIII concentrates affect platelet function both "in vitro" and "in vivo" but the mechanism of their action is poorly understood. Therefore the aim of the present work was to investigate a possible effect of these concentrates on prostaglandin synthesis by platelets, measured as the amount of released malondialdehyde (MDA), which is an index of Thromboxane A2 production. Our data suggest that Factor VIII concentrates have no effect on MDA release by platelets and on its inhibition by aspirin and heparin. In conclusion the effect of Factor VIII concentrates on platelets is not mediated by an increased synthesis of prostaglandins.     

Modification of platelet proteins by malondialdehyde: prevention by dicarbonyl scavengers

The thromboxane synthase converts prostaglandin H₂ to thromboxane A₂ and malondialdehyde (MDA) in approximately equimolar amounts. A reactive dicarbonyl, MDA forms covalent adducts of amino groups, including the ε-amine of lysine, but the importance of this reaction in platelets was unknown. Utilizing a novel LC/MS/MS method for analysis of one of the MDA adducts, the dilysyl-MDA cross-link, we demonstrated that dilysyl-MDA cross-links in human platelets are formed following platelet activation via the cyclooxygenase (COX)-1/thromboxane synthase pathway. Salicylamine and analogs of salicylamine were shown to react with MDA preferentially, thereby preventing formation of lysine adducts. Dilysyl-MDA cross-links were measured in two diseases known to be associated with increased platelet activation. Levels of platelet dilysyl-MDA cross-links were increased by 2-fold in metabolic syndrome relative to healthy subjects, and by 1.9-fold in sickle cell disease (SCD). In patients with SCD, the reduction of platelet dilysyl-MDA cross-links following administration of nonsteroidal anti-inflammatory drug provided evidence that MDA modifications of platelet proteins in this disease are derived from the COX pathway. In summary, MDA adducts of platelet proteins that cross-link lysines are formed on platelet activation and are increased in diseases associated with platelet activation. These protein modifications can be prevented by salicylamine-related scavengers.     

Reaction conditions affecting the relationship between thiobarbituric acid reactivity and lipid peroxides in human plasma.

The thiobarbituric acid (TBA) reactivity of human plasma was studied to evaluate its adequacy in quantifying lipid peroxidation as an index of systemic oxidative stress. Two spectrophotometric TBA tests based on the use of either phosphoric acid (pH 2.0, method A) or trichloroacetic plus hydrochloric acid (pH 0.9, method B) were employed with and without sodium sulfate (SS) to inhibit sialic acid (SA) reactivity with TBA. To correct for background absorption, the absorbance values at 572 nm were subtracted from those at 532 nm, which represent the absorption maximum of the TBA:MDA adduct. Method B gave values of TBA-reactive substances (TBARS) 2-fold higher than those detected with method A. SS lowered TBARS by about 50% with both methods, indicating a significant involvement of SA in plasma TBA reactivity. Standard SA, at a physiologically relevant concentration of 1.5 mM, reacted with TBA, creating interference problems, which were substantially eliminated by SS plus correction for background absorbance. When method B was carried out in the lipid and protein fraction of plasma, SS inhibited by 65% TBARS formation only in the latter. Protein TBARS may be largely ascribed to SA-containing glycoproteins and, to a minor extent, protein-bound MDA. Indeed, EDTA did not affect protein TBARS assessed in the presence of SS. TBA reactivity of whole plasma and of its lipid fraction was instead inhibited by EDTA, suggesting that lipoperoxides (and possibly monofunctional lipoperoxidation aldehydes) are involved as MDA precursors in the TBA test. Pretreatment of plasma with KI, a specific reductant of hydroperoxides, decreased TBARS by about 27%. Moreover, aspirin administration to humans to inhibit prostaglandin endoperoxide generation reduced plasma TBARS by 40%. In conclusion, reaction conditions affect the relationship between TBA reactivity and lipid peroxidation in human plasma. After correction for the interfering effects of SA in the TBA test, 40% of plasma TBARS appears related to in vivo generated prostaglandin endoperoxides and only about 60% to lipoperoxidation products. Thus, the TBA test is not totally specific to oxidant-driven lipid peroxidation in human plasma.     

二甲双胍联合西格列汀对2型糖尿病患者氧化应激、胰岛素抵抗的影响

目的:探讨二甲双胍联合西格列汀对2型糖尿病患者氧化应激、胰岛素抵抗的影响。方法:收集我院就诊或住院治疗的80例2型糖尿病患者,随机分为实验组和对照组,每组40例。两组患者入院后均给予相应的治疗措施,对照组患者给予二甲双胍250 mg/次,2次/d;实验组患者在对照组的基础上给予西格列汀100 mg/次,1次/d,治疗均连续8周。治疗结束后对患者血清丙二醛(MDA)、8异前列腺素F2α(8-iso-PGF2α)、空腹血糖(FBG)、空腹胰岛素(FINS)、胰岛素抵抗指数(HOMA-IR)以及患者临床治疗效果进行检测并比较。结果:与治疗前相比,治疗后两组患者MDA、8-iso-PGF2α、FBG、FINS以及HOMA-IR水平均下降(P0.05);与对照组相比,实验组患者MDA、8-iso-PGF2α、FBG、FINS以及HOMA-IR水平较低(P0.05),临床治疗总有效率较高(P0.05)。结论:二甲双胍联合西格列汀能够降低2型糖尿病患者血糖水平,降低MDA、8-iso-PGF2α水平,减轻氧化应激反应,降低胰岛素抵抗,临床疗效较好。     

The kinetics of dissociation of the inhibitor of nucleoside transport, nitrobenzylthioinosine, from the high-affinity binding sites of cultured hamster cells.

Nucleoside transport in various types of animal cells is inhibited by the binding of nitrobenzylthioinosine (NBMPR) to a set of high-affinity sites on the plasma membrane. This work examined the binding of [3H]NBMPR to the nucleoside transporters of cultured Nil 8 hamster fibroblasts and of cells of a virus-transformed clone (Nil SV) derived from Nil 8. Experiments conducted with intact Nil 8 and Nil SV cells and with membrane preparations indicated that the two lines differed significantly in the cellular content of binding sites and only slightly in the affinities of these sites for NBMPR. Nil 8 and Nil SV cells possessed (4.2-8.0) X 10(5) and (2.0-4.0) X 10(6) sites per cell respectively, whereas the dissociation constants of site-bound NBMPR obtained with intact cells and with membrane preparations were similar, ranging from 0.29 to 1.5 nM. Dilazep, a potent inhibitor of nucleoside transport that is structurally unrelated to NBMPR, appeared to compete with NBMPR for binding to the high-affinity sites when tested under equilibrium conditions with Ki values for inhibition of NBMPR binding to Nil 8 and Nil SV cells respectively of 15 +/- 4 and 32 +/- 4 nM. The dissociation of NBMPR from the binding site--NBMPR complex of Nil SV membrane preparations was a first-order decay process with a rate constant of 0.68 +/- 0.26 min-1. The rate of dissociation of NBMPR from the binding-site complex of membrane preparations and intact cells was decreased significantly in the presence of dilazep and increased in the presence of the permeant uridine. These results suggest that the apparent competitive-inhibition kinetics obtained for dilazep under equilibrium conditions should not be interpreted as binding of dilazep to the same site as NBMPR but rather as binding of the two inhibitors to closely associated sites on the nucleoside transporter. Similarly, uridine also appears to bind to a site separate from the NBMPR-binding site.     

Residues 334 and 338 in transmembrane segment 8 of human equilibrative nucleoside transporter 1 are important determinants of inhibitor sensitivity, protein folding, and catalytic turnover

Equilibrative nucleoside transporters (ENTs) are important for the metabolic salvage of nucleosides and the cellular uptake of antineoplastic and antiviral nucleoside analogs. Human equilibrative nucleoside transporter 1 (hENT1) is inhibited by nanomolar concentrations of structurally diverse compounds, including dipyridamole, dilazep, nitrobenzylmercaptopurine ribonucleoside (NBMPR), draflazine, and soluflazine. Random mutagenesis and screening by functional complementation for inhibitor-resistant mutants in yeast revealed mutations at Phe-334 and Asn-338. Both residues are predicted to lie in transmembrane segment 8 (TM 8), which contains residues that are highly conserved in the ENT family. F334Y displayed increased V(max) values that were attributed to increased rates of catalytic turnover, and N338Q and N338C displayed altered membrane distributions that appeared to be because of protein folding defects. Mutations of Phe-334 or Asn-338 impaired interactions with dilazep and dipyridamole, whereas mutations of Asn-338 impaired interactions with draflazine and soluflazine. A helical wheel projection of TM 8 predicted that Phe-334 and Asn-338 lie in close proximity to other highly conserved and/or hydrophilic residues, suggesting that they form part of a structurally important region that influences interactions with inhibitors, protein folding, and rates of conformational change during the transport cycle.