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Inhibitors of Cytochrome P450 Suppress Tumor Necrosis Factor Production
Abstract
We tested the effect of different inhibitors of cytochrome P450 on tumor necrosis factor (TNF) production. Metyrapone and SKF525A (100 and 50 mg/kg, ip, respectively) suppressed serum TNF induced by cotreatment with endotoxin (LPS), (2.5 micrograms/mouse). Inhibition was independent of endogenous corticosteroids since it was also observed in adrenalectomized mice. In vitro production of TNF by endotoxin-stimulated human monocytes was also inhibited by metyrapone and SKF525A. Since lipoxygenase (LO) inhibitors also block TNF production and metyrapone was reported to inhibit LO, we suggest that inhibition by metyrapone and SKF525A might be due to inhibition of either LO or a cytochrome P450 implicated in the oxidation of endogenous substrates involved in the inflammatory response.
... The observed ameliorating effects of metyrapone may be related to ACTH increase [57]. Metyrapone exhibits antioxidants properties by inhibiting cytochrome P450 activity [58,59] and suppressed injury-induced production of tumor necrosis factor [58] that can protect hippocampal and cortical neurons against excitotoxic and metabolic insults [60]. The above evidences tempted us to conclude that the neuroprotective effects of systemic administration of metyrapone, as documented in the present study, resulted solely from such central actions. ...
... The observed ameliorating effects of metyrapone may be related to ACTH increase [57]. Metyrapone exhibits antioxidants properties by inhibiting cytochrome P450 activity [58,59] and suppressed injury-induced production of tumor necrosis factor [58] that can protect hippocampal and cortical neurons against excitotoxic and metabolic insults [60]. The above evidences tempted us to conclude that the neuroprotective effects of systemic administration of metyrapone, as documented in the present study, resulted solely from such central actions. ...
Chronic exposure to hypobaric hypoxia causes oxidative stress and neurodegeneration leading to memory impairment. The present study aimed at investigating the role of corticosterone in hypoxia induced neurodegeneration and effect of metyrapone, a corticosterone synthesis inhibitor that reduces the stress induced elevation of corticosterone without affecting the basal level, in ameliorating chronic hypobaric hypoxia induced cognitive decline. Rats were exposed to simulated altitude of 25,000 ft for 0, 3, 7, 14 and 21 days to determine the temporal alterations in corticosterone and its receptors following exposure to hypobaric hypoxia. Our results showed an elevation of corticosterone in plasma and hippocampal tissue following 7 days of exposure, which declined on prolonged hypoxic exposure for 21 days. A concomitant increase in ROS and lipid peroxidation was observed along with depletion of intracellular antioxidants. Glucocorticoid and mineralocorticoid receptors were upregulated on 3 and 7 days of hypoxic exposure. Though expression of Glut1 and Glut3 were upregulated on 3 days of hypoxic exposure, sharp decline in Glut1 expression following 7 days of hypoxic exposure leads to reduced neuronal glucose uptake. Administration of metyrapone from 3rd to 7th day of hypoxic exposure to suppress hypoxia induced increase in corticosterone levels resulted in reduced oxidative damage, neurodegeneration and improvement of intracellular energy status. The metyrapone treated hypoxic animals performed better in the Morris Water Maze. Further, administration of exogenous corticosterone along with metyrapone during hypoxic exposure blunted the neuroprotective effect of metyrapone indicating a role for corticosterone in mediating hypobaric hypoxia induced neurodegeneration and memory impairment.
... Schedule-dependent effect of DEX on TNF and CS response to LPS Figure I shows the effect of DEX pretreatment (10 mg/kg; 0 5, 12, 18, 24, 48 or 72 h before LPS, 16 Mg/kg, intraperitoneally) on the induction of serum TNF and CS 1-5 h after LPS. DEX pretreatment from 0 5 h up to 12 h almost completely inhibited TNF production; this effect disappeared when LPS was given 18 h after DEX, and the 24-48 h DEX pretreatment actually potentiated LPS-induced TNF production. ...
... Other activities of GC might be taken into account. For instance, DEX was reported to induce a cytochrome of the P-450 family (P-4503A) [15], and we previously reported that inhibitors of cytochrome P-450 can suppress TNF production in vivo and in vitro [16], an effect probably associated with a lipooxygenase-like activity of cytochrome P-450. On the other hand, DEX has a series of activities that can take place in vivo, and might be important in the regulation of cytokine production. ...
Glucocorticoids (GC) are well known inhibitors of tumour necrosis factor (TNF) production. We investigated the role of endogenous GC in the regulation of TNF production in mice treated with lipopolysaccharide (LPS) using a pretreatment with dexamethasone (DEX) to down-regulate the hypothalamus-pituitary-adrenal axis (HPA). Short-term DEX pretreatment (up to 12 h before LPS) inhibited TNF production, but earlier (24-48 h) pretreatments potentiated it. This up-regulating effect was not observed in adrenalectomized mice or when GC synthesis was inhibited with cyanoketone (CK). This effect could not be explained only by the suppression of LPS-induced corticosterone (CS) levels induced by DEX, since a 48-h pretreatment potentiated TNF production without affecting LPS-induced CS levels. On the other hand, mice chronically pretreated with DEX were still responsive to its inhibitory effect on TNF production, thus ruling out the possibility of a decreased responsiveness to GC.
... Metyrapone, a bipyridyl compound, is a reversible inhibitor of cytochrome P450. It contributes to inflammatory responses by suppressing endogenous adrenal corticosteroid synthesis and lowering glucocorticoid levels (Fantuzzi et al., 1993). TG-101348 is an ATP-competitive Janus kinase 2 (JAK2) inhibitor with antitumor activity by inducing cancer cell apoptosis (Wernig et al., 2008;Verstovsek 2009). ...
Tuberculosis (TB) is a common infectious disease linked to host genetics and the innate immune response. It is vital to investigate new molecular mechanisms and efficient biomarkers for Tuberculosis because the pathophysiology of the disease is still unclear, and there aren’t any precise diagnostic tools. This study downloaded three blood datasets from the GEO database, two of which (GSE19435 and 83456) were used to build a weighted gene co-expression network for searching hub genes associated with macrophage M1 by the CIBERSORT and WGCNA algorithms. Furthermore, 994 differentially expressed genes (DEGs) were extracted from healthy and TB samples, four of which were associated with macrophage M1, naming RTP4, CXCL10, CD38, and IFI44. They were confirmed as upregulation in TB samples by external dataset validation (GSE34608) and quantitative real-time PCR analysis (qRT-PCR). CMap was used to predict potential therapeutic compounds for tuberculosis using 300 differentially expressed genes (150 downregulated and 150 upregulated genes), and six small molecules (RWJ-21757, phenamil, benzanthrone, TG-101348, metyrapone, and WT-161) with a higher confidence value were extracted. We used in-depth bioinformatics analysis to investigate significant macrophage M1-related genes and promising anti-Tuberculosis therapeutic compounds. However, more clinical trials were necessary to determine their effect on Tuberculosis.
... LPS-induced NO formation in animals was potentiated by dexamethasone induction of CYP3A, and the potentiation was inhibited by troleandomycin, an antibiotic inhibitor of CYP3A enzymes [209] . NO stimulates TNF-α production, and inhibition of NO generation blocked TNF-α release [210] . Thus, all these biomolecular metabolic alterations may be a basis of NO-induced cytotoxicity in disease states associated with excessive NO production. ...
This work for the first time provide evidence that T. gondii may be an environmental factor responisble for inducing development of autism spectrum disorders
... Metyrapone also inhibits P450 [26] in numerous species [27]. Its inhibitory effect is not specific as it targets the CYP11B1 [28] and the CYP3A [29], but also other enzymes such as lipooxygenase [30], guanylate cyclase [31] or nitric oxide synthetase as a member of cytochrome P450-like hemoprotein [32]. Some works have focused on the mechanisms by which metyrapone interacts with P450. ...
Metyrapone (2-methyl-1,2-di-3-pyridyl-1-propanone) is a drug largely used as inhibitor of glucocorticoid synthesis. Although its binding to various proteins has been well indentified, its accurate molecular mechanism of action remains unknown. Therefore, the interactions of metyrapone (MET) with various membrane components such as phospholipids, cholesterol, their corresponding polar heads and a model serine containing peptide have been investigated by NMR and ESR methods. It was found that neither cholesterol nor most of the phospholipids tested, nor dimyristin exhibit any in-teraction with MET, except phosphatidylserine (DMPS). Furthermore, only serine bearing polar head (O-phosphoser-ine) showed an association with MET (stoechiometry 1:1, Kd = 3200M-1). As similar observations were also performed on serine alone and in the presence of the serine containing model peptide, (NASDSDGQDL), a possible implication of these interactions in the binding recognition of MET on serine-containing active site was finally tested and discussed.
... This would be another effect of MET administration apart from the already described blockade of glucocorticoids (GC) synthesis. 57,58 Furthermore, in the present study, we observed other alterations on innate effector mechanisms, such as an enhancement of the macrophage phagocytic capacity. Several reports have shown an exacerbated phagocytic capacity during endotoxin tolerance status using different cell models. ...
Prior exposure to endotoxins renders the host temporarily refractory to subsequent endotoxin challenge (endotoxin tolerance). Clinically, this state has also been pointed out as the initial cause of the non-specific humoral and cellular immunosuppression described in these patients. We recently demonstrated the restoration of immune response with mifepristone (RU486), a receptor antagonist of glucocorticoids. Here we report the treatment with other modulators of glucocorticoids, i.e. dehydroepiandrosterone (DHEA), a hormone with anti-glucocorticoid properties, or metyrapone (MET) an inhibitor of corticosterone synthesis. These drugs were able to partially, but significantly, restore the humoral immune response in immunosuppressed mice. A significant recovery of proliferative responsiveness was also observed when splenocytes were obtained from DHEA- or MET-treated immunosuppressed mice. In addition, these treatments restored the hypersensitivity response in immunosuppressed mice. Finally, although neither DHEA nor MET improved the reduced CD4 lymphocyte count in spleen from immunosuppressed mice, both treatments promoted spleen architecture reorganization, partially restoring the distinct cellular components and their localization in the spleen. The results from this study indicate that DHEA and MET could play an important role in the restoration of both adaptive humoral and cellular immune response in LPS-immunosuppressed mice, reinforcing the concept of a central involvement of endogenous glucocorticoids on this phenomenon.
... , 1993). Furthermore, metyrapone can inhibit cytochrome P450 (Fantuzzi et al., 1993)-a mechanism that gives it some antioxidant properties (Montoliu et aI. , 1994). ...
Increasing evidence indicates that glucocorticoids (GCs), produced in response to physical/emotional stressors, can exacerbate brain damage resulting from cerebral ischemia and severe seizure activity. However, much of the supporting evidence has come from studies employing nonphysiological paradigms in which adrenalectomized rats were compared with those exposed to constant GC concentrations in the upper physiological range. Cerebral ischemia and seizures can induce considerable GC secretion. We now present data from experiments using metyrapone (an 11-β-hydroxylase inhibitor of GC production), which demonstrate that the GC stress-response worsens subsequent brain damage induced by ischemia and seizures in rats. Three different paradigms of brain injury were employed: middle cerebral artery occlusion (MCAO) model of focal cerebral ischemia; four-vessel occlusion (4VO) model of transient global forebrain ischemia; and kainic acid (KA)-induced (seizure-mediated) excitotoxic damage to hippocampal CA3 and CA1 neurons. Metyrapone (200 mg/kg body wt) was administered systemically in a single i.p. bolus 30 min prior to each insult. In the MCAO model, metyrapone treatment significantly reduced infarct volume and also preserved cells within the infarct. In the 4VO model, neuronal loss in region CA1 of the hippocampus was significantly reduced in rats administered metyrapone. Seizure-induced damage to hippocampal pyramidal neurons (assessed by cell counts and immunochemical analyses of cytoskeletal alterations) was significantly reduced in rats administered metyrapone. Measurement of plasma levels of corticosterone (the species-typical GC of rats) after each insult showed that metyrapone significantly suppressed the injury-induced rise in levels of circulating corticosterone. These findings indicate that endogenous corticosterone contributes to the basal level of brain injury resulting from cerebral ischemia and excitotoxic seizure activity and suggest that drugs that suppress glucocorticoid production may be effective in reducing brain damage in stroke and epilepsy patients.Keywords: Alzheimer's; Corticosterone; Excitotoxicity; Hippocampus; Kainic acid; Microtubule-associated proteins; Middle cerebral artery occlusion; Transient global forebrain ischemia
... Among the other activities of DEX that might account for the upregulation of TNF production, we considered its ability to induce some cytochrome P450 isoforms (9). In fact, we had observed that inhibitors of cytochrome P450 (metyrapone, SKF 525A) suppressed LPS-induced TNF production in mice (10), suggesting that a cytochrome of the P450 family might be involved in the signaling pathway leading to TNF production. ...
Dexamethasone (DEX) is a well-known inhibitor of tumor necrosis factor (TNF) production when given shortly before lipopolysaccharide (LPS). However, DEX (10 mg/kg, ip) potentiates TNF production when administered 24-48 hr before LPS (16 micrograms/kg, ip). We have found that this is probably due to DEX induction of cytochrome P450 3A, which is known to produce nitric oxide (NO). The upregulating effect of DEX on TNF production is associated with increased NO production. Both the upregulation of NO and of TNF production by DEX are inhibited by co-administration of the P450 3A inhibitor troleandomycin (TAO, 40 mg/kg, ip). These data suggest that P450 3A-generated NO might be involved in TNF induction.
... NO stimulates TNF␣ production, and inhibition of NO production blocks TNF␣ release. In accordance with a physiological role for P450s in NO generation, nonspecific P450 inhibitors block LPS-induced TNF␣ production in mice (Fantuzzi et al., 1993). These studies should be interpreted with caution because of possible nonspecific effects of the drugs used. ...
Expression and activities of cytochrome P450 enzymes are down-regulated in the liver during the host response to inflammation or infection, leading to alterations in drug clearance and toxin activation. This review focuses on recent studies on the mechanisms of this down-regulation, as well as the cytokines and cell types involved. Possible reasons for cytochrome P450 down-regulation are discussed.
1. The effects of NG-nitro-L-arginine (l-NNA; 20mg/kg bodyweight (BW), i.v.) and metyrapone (300 mg/kg BW, s.c.) on acetylcholine (ACh)-induced depressor responses were investigated in anaesthetized rats.
2. Acetylcholine (0.05,0.5,5 μg/kg BW, i.v.) dose-dependently evoked a sharp fall in mean blood pressure (BP) followed by a slow recovery under control conditions.
3. Basal BP level was elevated when rats were treated with l-NNA, indicating endogenous nitric oxide (NO) participated in BP regulation. However, pretreatment with l-NNA did not attenuate but rather augmented the ACh-induced maximum vasodilation. In contrast, the time for recovery of mean BP to the pre-ACh administration level was shortened by l-NNA. These observations suggested that ACh-induced vasodilation consisted of two phases: a sharp and transient fall (phase 1) that was resistant to l -NNA followed by a longer depressor response (phase 2) that was suppressed by l-NNA.
4. To examine whether augmentation of phase 1 by l-NNA resulted from the elevation of basal BP, an appropriate dose of phenylephrine was infused to obtain similar BP elevation. Phenylephrine infusion augmented the phase 1 in a similar manner to l-NNA pretreatment but showed little effect on phase 2, supporting the selective inhibition of phase 2 by l-NNA.
5. The s.c. pretreatment with metyrapone for 3 days failed to attenuate phase 1. Thus, the involvement of endothelium-derived hyperpolarizing factor that could be formed by a metyrapone-sensitive oxidase in phase 1 was unlikely.
6. These results suggest that some factor(s), which is not inhibitable by l-NNA or metyrapone, may induce the phase 1 depressor response to ACh while NO is responsible for the phase 2 response. The mechanism inducing the phase 1 response remains to be identified.
Glucocorticoids (GCs) are released in response to immune activation by the bacterial endotoxin, lipopolysaccharide (LPS). However, GC secretion in response to immune activation and other stressors is attenuated at term of pregnancy. GCs are important modulators of the immune response, and both pro- and anti-inflammatory effects are described. Here, we examined whether GC secretion in response to LPS is maintained in earlier pregnancy before term, and investigated the role of endogenous GCs in modulating LPS-induced circulating cytokines, interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), in pregnant compared to non-pregnant female rats. Plasma corticosterone (Cort) and ACTH responses to LPS were well maintained in pregnant rats at embryonic days 15/16 (E15/16) and E18/19 compared to non-pregnant rats. At E19, maternal LPS administration increased fetal plasma Cort and decreased testosterone in male fetuses. In non-pregnant animals, pretreatment with the GC synthesis inhibitor, metyrapone, inhibited the LPS-induced increase in IL-6, and the IL-6 response was restored by Cort replacement, indicating that LPS induction of IL-6 is Cort-dependent. In E15 pregnant animals, metyrapone had no effect on LPS-induced IL-6 levels, indicating that LPS-induction of IL-6 is not dependent on Cort. These contrasting patterns of IL-6 induction in non-pregnant and pregnant animals were reflected in levels of hypothalamic Socs3 mRNA, an indicator of IL-6 signaling pathway activation. In both non-pregnant and pregnant rats, LPS-induced plasma TNF-α responses were inhibited by metyrapone but not re-instated by Cort replacement. It is suggested that altered GC regulation of IL-6 may be required to sustain specialized functions of IL-6 during pregnancy.
Murine cytomegalovirus (MCMV) induces adrenalitis in BALB/c mice but does not compromise adrenal function, assessed by levels of circulating adrenocorticotropic hormone (ACTH) and by the response to challenge with synthetic ACTH. Levels of corticosterone increased 2 days after infection in mice of this strain, consistent with previously established interactions between mediators of acute inflammation and activation of the hypothalmus-pituitary-adrenal axis. Moreover, an adrenocortical response was critical to survival of BALB/c (but not C57BL/6) mice 3 days after infection, as pharmacologically or surgically adrenalectomized BALB/c mice died when given doses of virus up to fivefold lower, than they could normally tolerate. However, death could not be prevented by the administration of soluble cytokine receptors to inhibit the action of interleukin 1 (IL-1) or tumour necrosis factor alpha (TNF alpha). The corticosteroid response did not mediate.MCMV-induced thymic atrophy. As the above traits were all less evident in C57BL/6 mice, a common genetic basis is discussed.
Dihydrocodeine is increasingly used in slow-release preparations for the treatment of chronic pain on step 2 of the "analgesic ladder" of the World Health Organization. Dihydrocodeine is suggested to act after O-demethylation to dihydromorphine. To test this possibility, experiments were carried out on rats under urethane anesthesia in which nociceptive activity was evoked by electrical stimulation of afferent C fibers in the sural nerve and recorded from neurons in the ventrobasal complex of the thalamus. Dihydrocodeine administered by intravenous injection reduced the evoked nociceptive activity in a dose-dependent manner. Like morphine, dihydrocodeine was capable of completely suppressing the evoked activity. Maximum depression was caused by 2 mg/kg, and the ED50 is 0.47 mg/kg. Naloxone (0.2 mg/kg) reversed the effect of dihydrocodeine (2 mg/kg). To inhibit O-demethylation of dihydrocodeine to dihydromorphine, metyrapone or cimetidine (50 mg/kg) was injected intraperitoneally 20 min before dihydrocodeine (1 and 2 mg/kg). This failed to markedly reduce the effect of dihydrocodeine. Dihydromorphine injected intravenously also reduced the evoked activity in a dose-dependent way. Maximum depression occurred at a dose of 4 mg/kg, and the ED50 is 0.97 mg/kg. Dihydrocodeine and dihydromorphine were equieffective when administered by intrathecal injection at a dose of 100 microg. It is concluded that dihydrocodeine causes analgesia independent of biotransformation to dihydromorphine.
Hepatic P450 activities are profoundly affected by various infectious and inflammatory stimuli, and this has clinical and toxicological consequences. Whereas the expression of most P450s in the liver is suppressed, some are induced. Many of the effects observed in vivo can be mimicked by pro-inflammatory cytokines and IFNs, and P450s are differentially regulated by these agents. Therefore, different cytokine profiles and concentrations in the vicinity of the hepatocyte in different models of inflammation may result in qualitatively and quantitatively different effects on populations of P450s. In addition to cytokines, glucocorticoids may have an important role in P450 regulation in stress conditions, including that caused by inflammatory stimuli. Although in many cases the decreases in activity are due primarily to a down-regulation of P450 gene transcription, it is likely that modulation of RNA and protein turnover, as well as enzyme inhibition, contributes to some of the observed effects. The mechanisms whereby these effects are produced may also vary with both the P450 under study and the time course of the effect. The complexity of the P450 response to inflammation and infection means that all of the above factors must be considered when trying to predict the effect of a given infectious or inflammatory condition on the clinical or toxic response of humans or animals to an administered drug or toxin. The question of whether the down-regulation of the hepatic P450 system to inflammation or infection is a homeostatic or pathological response cannot be answered at present. It is difficult to discern the physiological benefit of reducing hepatic P450 activities, unless it is to prevent the generation of reactive oxygen species generated by uncoupled catalytic turnover of the enzymes. On the other hand, as we proposed some years ago [64], the suppression of P450 may be due to the liver's need to utilize its transcriptional machinery and energy for the synthesis of APPs involved in the inflammatory response. In that case, one could ask why the organism has gone to the trouble of employing differential mechanisms for suppression of P450. One answer could be that the response evolved after the divergence of many of the P450 genes, necessitating the evolution of multiple redundant mechanisms for P450 suppression. In contrast to the down-regulation of P450s in the liver, the induction of several forms in this and other tissues suggests a more specific homeostatic role of these effects, e.g., in generation or catabolism of bioactive metabolites.
Until recently, studies on the role of the metabolites of arachidonic acid (AA), eicosanoids in fever have primarily focused on prostaglandins, prostaglandin E2 (PGE2) in particular, derived from the pathway related to cyclooxygenases (COX). COX exists in two known isoforms; a constitutive COX-1, and COX-2, which is inducible upon the action of pyrogens. Data accumulated in our laboratories suggest a thermoregulatory role for two other pathways of arachidonate metabolism; 5-lipoxygenase (5-LOX) and cytochrome P-450 (epoxygenase). We have demonstrated that leukotrienes (LTs; 5-LOX-derived eicosanoids) and various isomers of epoxyeicosatrienoic acids (EETs; epoxygenase-derived eicosanoids) contribute to the process of endogenous antipyresis or cryogenesis, which limits the height of fever. In support of this are several lines of evidence based on both in vivo and in vitro experiments. 1) Intracerebroventricular (icv) injections of LTC4 at nanomolar concentrations cause a dose-dependent decrease of body temperature (Tb) in mice. 2) Lipopolysaccharide (LPS)-induced anapyrexia in mice is preceded and accompanied by elevation in hypothalamic cysteinyl-LT (CysLT) production. 3) The inhibitor of LT synthesis MK-886 suppresses both of these processes. 4) EETs as well as inducers of the epoxygenase attenuate, whereas inhibitors of epoxygenase enhance the LPS-induced fever in rats. 5) One of the isomers of EET, 11,12-EET, in in vitro studies inhibited both the generation of PGE2 and IL-6 in monocytes stimulated with LPS. These results, together with a well-established pyrogenic role of PGE2, indicate that AA cascade may be regarded as an endogenous system to regulate the temperature response upon disease. COX, 5-LOX, and epoxygenase products may act at the level of hypothalamus as proximal mediators of, respectively, fever (PGE2) or cryogenesis (CysLTs and EETs), or indirectly by influencing the other endogenous cryogens and pyrogens.
Inflammation and infection have long been known to downregulate the activity and expression of cytochrome P450 (CYP) enzymes involved in hepatic drug clearance. This can result in elevated plasma drug levels and increased adverse effects. Recent information on regulation of human CYP enzymes is presented, as are new developments in our understanding of the mechanisms of regulation. Experiments to study the effects of modulating CYP activities on the inflammatory response have yielded possible insights into the physiological consequences, if not the purpose, of the downregulation. Regulation of hepatic flavin monooxygenases, UDP-glucuronosyltransferases, sulfotransferases, glutathione S-transferases, as well as of hepatic transporters during the inflammatory response, exhibits similarities and differences with regulation of CYPs.
Cytochrome P450 (CYP) enzymes are the major phase I enzymes in drug metabolism but may also exert important functions in physiological and pathophysiological processes. This becomes especially evident, when genetic variations, drug interactions, pathophysiological or environmental factors cause reduced, absent, or increased enzymatic activity. CYP enzymes and myocardial infarction (MI) are intertwined at basically two levels: 1. Atherosclerosis, the major underlying pathophysiological substrate in MI, has a multifactorial etiology involving vascular cells, lipid metabolism, inflammatory mediators, and tissues and processes influenced by CYP activity. Thus, various CYP enzymes may influence pathogenesis or progression of MI. 2. Patients at risk for MI or included in secondary prevention regimes usually take a multitude of different drugs that are primarily metabolized by hepatic or intestinal CYPs. Inherited differences or acquired changes in enzyme activity may, therefore, influence therapeutic outcome or the incidence of adverse drug reactions. This chapter will provide an overview of both aspects of CYP interaction with MI and summarizes the current knowledge on variable CYP activities and their potential impact on pathogenesis, progression, risk assessment, and therapy of cardiovascular disease.
Although tumor necrosis factor (TNF) is undoubtedly a major mediator of the antitumor and shock-inducing activities of lipopolysaccharide (LPS), the outcome of a challenge with TNF is highly dependent on the presence or absence of other substances or conditions. We have previously shown that to obtain lethality in mice after TNF administration both TNF receptor (TNF-R) types have to be triggered. This is illustrated by the fact that recombinant human (rh) TNF, which is a selective murine (m) TNF-R55 agonist, is not lethal, whereas mTNF, which binds both mTNF-R55 and mTNF-R75, is lethal in mice. Triggering of TNF-R75 is, however, no longer needed when sensitizers such as galactosamine or low doses of LPS or interleukin (IL)-1 are also present. Here, we report that this selective species specificity of TNF is also reflected in patterns of induced IL-6: both rmTNF and rhTNF could induce considerable IL-6 peak levels in the plasma (up to 10 ng/ml) 2 to 3 h after TNF administration. However, only rmTNF was capable of inducing the same pattern of sustained IL-6 levels previously observed after lethal LPS doses, while rhTNF only caused induction of transient IL-6 levels, as found after nonlethal LPS doses. We also observed that the sensitizer IL-1 could complement rhTNF to induce such a sustained IL-6 induction. Since we were interested in sensitizers with a defined mechanism of action, we further investigated the effects of the glucocorticoid and progesterone inhibitor RU38486 on the lethal and IL-6-inducing properties of TNF. We observed that RU38486 closely mimicked IL-1: both had similar effects on IL-6 induction and sensitized mice to the lethal effects of TNF with comparable efficiency and kinetics. Using a monoclonal anti-IL-1R antibody, we finally observed that the effects of RU38486 were most probably not mediated by IL-1. These observations suggest that a glucocorticoid-antagonistic activity might be a key factor in the pathways leading to septic shock and that such activity could be a key target for the pharmacological manipulation of sepsis.
TNF is a major mediator in the pathogenesis of endotoxic shock, and its inhibition has a protective effect in various animal models of sepsis or endotoxin (lipopolysaccharide, LPS) toxicity. LPS treatment also induces an oxidative damage mediated by increased production of reactive oxygen intermediates. N-Acetylcysteine (NAC) is an antioxidant and a precursor of the synthesis of glutathione (GSH) and was reported to protect against LPS toxicity and LPS-induced pulmonary edema. In this study we investigated the effect of NAC on TNF production and LPS lethality in mice. The results indicated that oral administration of NAC protects against LPS toxicity and inhibits the increase in serum TNF levels in LPS-treated mice. The inhibition was not confined to the released form of TNF, since NAC also inhibited LPS-induced spleen-associated TNF. On the other hand, the inhibitor of GSH synthesis, DL-buthionine-(SR)-sulfoximine (BSO), had the opposite effect of potentiating LPS-induced TNF production, and this was associated with a decrease in liver GSH levels. Repletion of liver GSH with NAC reversed this effect. NAC was also active in inhibiting TNF production and hepatotoxicity in mice treated with LPS in association with a sensitizing dose of Actinomycin D. These data indicate that GSH can be an endogenous modulator of TNF production in vivo. On the other hand, NAC pretreatment did not inhibit other effects of LPS, particularly induction of serum IL-6, spleen IL-1 alpha, and corticosterone, in the same experimental model, suggesting that the observed effect could be specific for TNF.
Lipoxygenase inhibitors have been shown to exert beneficial effects in experimental models of endotoxin shock. In the present study it was found that lipoxygenase inhibitors prevented LPS, but not tumor necrosis factor alpha (TNF alpha)-evoked leukopenia in mice. These inhibitors protected against endotoxin lethality but not against TNF alpha induced lethality. When the protective potency of the specific 5-lipoxygenase inhibitors (MK 886, CGS 81585) was tested in endotoxin-induced leukopenia and shock, they were found to be ineffective. Site specificity of the inhibitors was assessed by comparison of their effects on the formation of LTC4 and the conversion of linoleic acid to 13-hydroxyoctadecadienoic acid (13-HODD) by macrophages. The 5-lipoxygenase inhibitors interfered with LTC4 formation in macrophages, however, they did not affect endotoxin-induced TNF alpha formation, neither in cell cultures nor in mice. The inhibitory strength of other, less specific lipoxygenase blockers to suppress TNF alpha formation correlated quantitatively with their ability to interfere with 13-HODD synthesis. From these findings it is concluded that lipoxygenase inhibitors interfere with endotoxic effects because they block TNF alpha formation. Since 5-lipoxygenase inhibitors neither prevented the formation of TNF alpha nor endotoxin leukopenia and lethality, it is suggested that a lipoxygenase product distinct from the leukotrienes is involved in TNF alpha synthesis. Based on the fact that a tight correlation exists between inhibition of TNF alpha synthesis and 13-HODD formation, activation of 15-lipoxygenase might be important for TNF alpha formation.
Currently, the major recognized biochemical functions of members of the large superfamily of P450 hemoproteins (referred to commonly as the cytochromes P450) include catalyses of the monooxygenations of a wide variety of endogenous and exogenous lipophilic chemicals. Substrates that have attracted the greatest attention thus far are steroids, fatty acids, eicosanoids, retinoids, other endogenous lipids, therapeutic agents, pesticides/herbicides, chemical carcinogens, industrial chemicals and other environmental contaminants and toxic xenobiotic organics of low molecular weight. Commonly, monooxygenation of such substrates results in the generation of metabolites capable of producing biological effects that are profoundly different (qualitatively as well as quantitatively) from those elicitable by the parent chemical per se. P45OXIX-dependent conversion of testosterone to estradiol-17 beta provides a dramatic example. Thus, these hemoproteins serve as extremely important but, as yet, largely unpredictable regulators of the biological effects producible by endobiotics as well as by xenobiotics. Current focus is on the identification and acquisition of sequence information on hereto unidentified and/or uncharacterized P450 isoforms and ascertainment of the specific functions of specific, individual isoforms. The regulation of quantities and activities of such isoforms in specific species/tissues, understandably, is also of great current interest. This interest has been further intensified by recent results indicating that substrate specificity associated with one P450 may not be the same as the corresponding isoform derived from a different animal species. Recent technological advances promise to greatly hasten the acquisition of knowledge concerning the functions of these important hemoproteins.
Intraperitoneal injection of a sublethal dose of lipopolysaccharide (LPS) into mice resulted in the appearance of tumor necrosis factor (TNF) in the serum within 45 min. Maximal serum TNF was detected by 1 h, and by 3-4 h TNF levels were no longer significantly above baseline. Injection of mice with an additional dose of LPS at 4 h resulted in no further increase in serum TNF. The in vivo kinetics of TNF appearance correlated with in vitro studies in which TNF mRNA was detected in murine peritoneal macrophages 30 min after LPS stimulation. The increase in serum TNF was not detected in mice treated with dexamethasone, 3 mg/kg, prior to LPS stimulation. The decrease in TNF correlated with the appearance of significant amounts of endogenous serum corticosterone which were maximal by 3 h. Further evidence for the role of endogenous steroids in the modulation of serum TNF levels was obtained in studies with adrenalectomized or hypophysectomized mice. Compared to sham-operated animals, serum TNF levels remain elevated 5 h post LPS stimulation in adrenalectomized or hypophysectomized mice. In contrast with the transient increase in TNF, serum IL 1 was maximal 4 h post LPS injection and remained elevated at 24 h. In vitro studies with primary cultures of human peripheral blood monocytes and human umbilical cord vein endothelial cells demonstrated that LPS-induced monocyte IL 1 levels were reduced approximately 5-fold by 10(-7) M dexamethasone while dexamethasone had only minimal effects on endothelial cell IL 1. Therefore, the in vitro data would suggest that the maintenance of elevated IL 1 levels coincident with the appearance of endogenous corticosteroids during LPS shock is related to the synthesis of IL 1 by both monocyte-macrophages and non-myeloid cell populations including endothelial cells.
A mouse pituitary tumor cell line (AtT-20) releases corticotropin (ACTH) in response to a number of secretagogues, including corticotropin-releasing factor (CRF), beta-adrenergic agents, N6,O2'-dibutyryladenosine 3',5'-cyclic monophosphate (Bt2 cAMP), and potassium. The stimulation of ACTH secretion induced by the secretagogues can be blocked by inhibitors of the enzymes that generate (phospholipase A2) and metabolize (lipoxygenase and epoxygenase) arachidonic acid. The phospholipase A2 blockers mepacrine and p-bromophenacylbromide inhibited the ACTH release induced by secretagogues. The lipoxygenase inhibitors nordihydroguaieretic acid, butylated hydroxytoluene, and icosatetraynoic acid abolished the ACTH secretion induced by secretagogues, whereas indomethacin, a cycloxygenase inhibitor, did not. Blockers of the cytochrome P-450 epoxygenase, such as SKF 525A and piperonyl butoxide, compounds that have different molecular structures, also suppressed secretagogue-induced ACTH release. These findings suggest that metabolites of arachidonic acid formed via the epoxygenase and/or the lipoxygenase pathway are involved in the stimulation of ACTH release caused by secretagogues.
Ketoconazole inhibits in vitro (IC50:2.6 X 10(-5) M) the formation of 5-HETE and LTB4 by isolated, carrageenin-elicited rat peritoneal PMN leukocytes, challenged with the Ca2+-ionophore A23187 in the presence of [14C]-arachidonic acid ([14C]-AA). The relative potency of various compounds tested in this respect is NDGA greater than nafazatrom greater than phenidone greater than ketoconazole greater than BW 755C. In contrast to the other compounds studies, ketoconazole in vitro, up to 1 X 10(-4) M, has no effect on the fatty acid cyclo-oxygenase or the 12-lipoxygenase-mediated metabolism of [14C]-AA by isolated human platelets; however, it stimulates the 15-lipoxygenase activity in phenylhydrazine-induced rabbit reticulocytes. After oral administration (10-40 mg/kg, -2 hr), ketoconazole inhibits in a dose-dependent way, the leukotriene-mediated anaphylactic bronchoconstriction in guinea pigs. This study demonstrates that ketoconazole is a comparatively specific and orally active inhibitor of the 5-lipoxygenase activity bearing on the production of leukotrienes derived from arachidonic acid.
Prostaglandin E2 (PGE2) can modulate the actions of a number of hormones in liver. PGE2 is rapidly metabolized in liver tissue, and thus alterations in the rate of PGE2 catabolism might exert a short-term influence on the concentration of PGE2 in liver. The present study examined the effects of inhibitors of oxidative metabolism on PGE2 catabolism and action in isolated rat hepatocytes. [3H]-PGE2 was metabolized to three major products by the hepatocyte system as assessed by reverse-phase high performance liquid chromatography. Metyrapone (5 mM), aminopyrine (5 mM), SKF-525A (20 microM) and alpha-naphthoflavone (20 microM) each inhibited the breakdown of [3H]-PGE2. The inhibition of oxidative metabolism by these compounds was not limited to action at cytochrome P-450, and metyrapone, aminopyrine and SKF-525A each was shown to inhibit [1-14C]-palmitate beta-oxidation in the hepatocyte system. To determine the contribution of beta-oxidation to the rapid catabolism of [3H]-PGE2, studies were performed using [1-14C]-PGE2 as substrate. Two major product peaks seen with [3H]-PGE2 as substrate lacked radioactivity when [1-14C]-PGE2 was the substrate, and thus these two products did not contain the 1-position carbon, consistent with their identity as beta-oxidation products. Furthermore, [1-14C]-PGE2 also yielded 14CO2 and a [14C]-PGE2 metabolite not seen with [3H]-PGE2. It was calculated that 60% of the rapid PGE2 inactivation in the hepatocyte system occurred via beta-oxidation. An additional, non-beta-oxidation, metyrapone-sensitive, pathway accounted for 26% of PGE2 disappearance. The effect of PGE2 to inhibit glucagon-stimulated glycogenolysis was potentiated when metyrapone was included in the incubation, consistent with increased survival of intact PGE2. In summary, PGE2 was rapidly inactivated by intact hepatocytes via oxidative metabolism, primarily beta-oxidation. Inhibition of prostaglandin catabolism can have short-term effects on PGE2 concentrations and result in potentiation of PGE2 effects on hepatic glucose metabolism.
Human blood monocytes were purified by a new method capable of handling 3 X 10(9) mononuclear leukocytes, which does not involve adherence of the cells and takes about 3 h to perform. The yield of monocytes is 70%, the purity about 90% and the viability 99%. Monocytes purified by this method were cryopreserved at -196 degrees C. The function of the cells was tested before freezing and after thawing. We found that the capacity of cryo-preserved monocytes to move to the source of a chemotactic gradient, to ingest particles, to mount a respiratory burst during phagocytosis, to kill intracellular bacteria, to lyse anti-D sensitized erythrocytes and to help autologous lymphocytes in a proliferative response to mitogens or antigens, was preserved by 70% or more as compared with non-frozen cells. Thus, cryopreservation of human blood monocytes is possible with maintenance of functional capacities.