P450-linked Monooxygenase Research Articles

The Customizable E-cigarette Resistance Influences Toxicological Outcomes: Lung Degeneration, Inflammation, and Oxidative Stress-Induced in a Rat Model.

Despite the knowledge gap regarding the risk-benefit ratio of the electronic cigarette (e-cig), its use has grown exponentially, even in teenagers. E-cig vapor contains carcinogenic compounds (eg, formaldehyde, acetaldehyde, and acrolein) and free radicals, especially reactive oxygen species (ROS) that cause toxicological effects, including DNA damage. The role of e-cig voltage customization on molecule generation has been reported, but the effects of the resistance on e-cig emissions and toxicity are unknown. Here, we show that the manipulation of e-cig resistance influences the carbonyls production from nonnicotine vapor and the oxidative and inflammatory status in a rat model. Fixing the voltage at the conventional 3.5 V, we observed that the amount of the selected aldehydes increased as the resistance decreased from 1.5 to 0.25 Ω. Under these conditions, we exposed Sprague Dawley rats to e-cig aerosol for 28 days, and we studied the pulmonary inflammation, oxidative stress, tissue damage, and blood homeostasis. We found a perturbation of the antioxidant and phase II enzymes, probably related to the increased ROS levels due to the enhanced xanthine oxidase and P450-linked monooxygenases. Furthermore, frames from scanning electron microscope showed a disorganization of alveolar and bronchial epithelium in 0.25 Ω group. Overall, various toxicological outcomes, widely recognized as smoke-related injuries, can potentially occur in e-cig consumers who use low-voltage and resistance device. Our study suggests that certain "tips for vaping safety" cannot be established, and encourages further independent investigations to help public health agencies in regulating the e-cig use.

Effects of chlorinated drinking water on the xenobiotic metabolism in Cyprinus carpio treated with samples from two Italian municipal networks.

Drinking water (DW) disinfection represents a milestone of the past century, thanks to its efficacy in the reduction of risks of epidemic forms by water micro-organisms. Nevertheless, such process generates disinfection by-products (DBPs), some of which are genotoxic both in animals and in humans and carcinogenic in animals. At present, chlorination is one of the most employed strategies but the toxicological effects of several classes of DBPs are unknown. In this investigation, a multidisciplinary approach foreseeing the chemical analysis of chlorinated DW samples and the study of its effects on mixed function oxidases (MFOs) belonging to the superfamily of cytochrome P450-linked monooxygenases of Cyprinus carpio hepatopancreas, was employed. The experimental samples derived from aquifers of two Italian towns (plant 1, river water and plant 2, spring water) were obtained immediately after the disinfection (A) and along the network (R1). Animals treated with plant 1 DW-processed fractions showed a general CYP-associated MFO induction. By contrast, in plant 2, a complex modulation pattern was achieved, with a general up-regulation for the point A and a marked MFO inactivation in the R1 group, particularly for the testosterone metabolism. Together, the toxicity and co-carcinogenicity (i.e. unremitting over-generation of free radicals and increased bioactivation capability) of DW linked to the recorded metabolic manipulation, suggests that a prolonged exposure to chlorine-derived disinfectants may produce adverse health effects.

Modulation of cytochrome P450 and induction of DNA damage in Cyprinus carpio exposed in situ to surface water treated with chlorine or alternative disinfectants in different seasons

Epidemiological studies have shown an association between consumption of disinfected drinking water and adverse health outcomes. The chemicals used to disinfect water react with occurring organic matter and anthropogenic contaminants in the source water, resulting in the formation of disinfection by-products (DBPs). The observations that some DBPs are carcinogenic in animal models have raised public concern over the possible adverse health effects for humans. Here, the modulation of liver cytochrome P450-linked monooxygenases (MFO) and the genotoxic effects in erythrocytes of Cyprinus carpio fish exposed in situ to surface drinking water in the presence of disinfectants, such as sodium hypochlorite (NaClO), chlorine dioxide (ClO2) and peracetic acid (PAA), were investigated in winter and summer. A complex induction/suppression pattern of CYP-associated MFOs in winter was observed for all disinfectants. For example, a 3.4- to 15-fold increase was recorded of the CYP2B1/2-linked dealkylation of penthoxyresorufin with NaClO (10 days) and PAA (20 days). In contrast, ClO2 generated the most notable inactivation, the CYP2E1-supported hydroxylation of p-nitrophenol being decreased up to 71% after 10 days’ treatment. In summer, the degree of modulation was modest, with the exception of CYP3A1/2 and CYP1A1 supported MFOs (62% loss after 20 days PAA).The micronucleus (MN) induction in fish circulating erythrocytes was also analysed as an endpoint of genotoxic potential in the same fish population. Significant increases of MN induction were detected at the latest sampling time on fish exposed to surface water treated with chlorinate-disinfectants, both in winter (NaClO) and summer (NaClO and ClO2), while no effect was observed in fish exposed to PAA-treated water.These results show that water disinfection may be responsible for harmful outcomes in terms of MFO perturbation and DNA damage; if extrapolated to humans, they ultimately offer a possible rationale for the increased urinary cancer risk recorded in regular drinking water consumers.

Cytochrome P450 species involved in the metabolism of quinoline.

Quinoline is a hepatocarcinogen in rats and mice and a well-known mutagen in bacteria after incubation with rat liver microsomes. The specific cytochrome P450 enzymes involved in quinoline metabolism in human and rat liver microsomes were determined using cDNA-expressed cytochrome P450s, correlations with specific cytochrome P450-linked monooxygenase activities in human liver microsomes and inhibition by specific inhibitors and antibodies. CYP2A6 is the principal cytochrome P450 involved in the formation of quinoline-1-oxide in human liver microsomes (correlation coefficient r = 0.95), but is formed in only minute quantities in rat liver microsomes. CYP2E1 is the principal cytochrome P450 involved in the formation of 3-hydroxyquinoline (r = 0.93) in human liver microsomes and is involved in the formation in rat liver microsomes. A high correlation coefficient (r = 0.91) between CYP2A6 activity and quinoline-5,6-diol formation in human liver microsomes was observed, but this most likely reflects the involvement of CYP2A6 in the formation of quinoline-5,6-epoxide, from which the quinoline-5,6-diol is formed, as conversion of quinoline-5,6-epoxide to quinoline-5,6-diol on incubation of the epoxide with CYP2A6 could not be demonstrated. A cDNA-expressed human microsomal epoxide hydrolase, however, efficiently converted the epoxide to the diol and the microsomal epoxide inhibitor cyclohexene oxide inhibited quinoline-5,6-diol formation in rat liver microsomes. A preliminary kinetic analysis of quinoline metabolism in human liver microsomes was carried out and Eadie-Hofstee plots indicate that the formation of quinoline-5,6-diol is monophasic, while that of quinoline-1-oxide and 3-hydroxyquinoline is biphasic.

Possible role of the cytochrome P450-linked monooxygenase system in preventing δ9-tetrahydrocannabinol-induced stimulation of tuberoinfundibular dopaminergic activity in female rats

The administration of δ 9-tetrahydrocannabinol (THC) or related cannabinoids markedly affected neurobehavioral and neuroendocrine indices in male rodents but usually failed to affect those indices in females. We examined whether inhibition of the cytochrome P450-linked monooxygenase system in female rats is able to elicit the effects of THC on one of the most characteristic targets of cannabinoid action, tuberoinfundibular dopaminergic neurons, whose activity is known to increase after cannabinoid exposure in males. It was found that the administration of THC to ovariectomized rats acutely replaced with estradiol (to discard problems derived from differences in the estrogenic status) did not affect either dopamine and l-3,4-dihydroxyphenylacetic acid (DOPAC) contents and tyrosine hydroxylase activity in the medial basal hypothalamus or the density of D2-dopaminergic receptors in the anterior pituitary. However, the administration of THC to estrogen-replaced ovariectomized rats that had been pretreated with two separately administered inhibitors of cytochrome P450, piperonyl butoxide or metyrapone, significantly increased DOPAC content in the medial basal hypothalamus, with no changes in the other parameters. Collectively, these results indicate that the metabolism of THC to inactive compounds might play a protective role in females, counteracting the effects of this cannabinoid on tuberoinfundibular dopaminergic activity because pharmacological inhibition of cytochrome P450-linked monooxygenase system elicited a significant stimulation of these neurons by THC.

In vivo administration of H 2 blockers, cimetidine and ranitidine, reduced the contents of the cytochrome P450IID (CYP2D) subfamily and their activities in rat liver microsomes

1. 1. The effects of in vivo administration of H 2 blockers, cimetidine and ranitidine (0.6 mmol/kg body weight/day, for 5 days), on several P450 isozymes, the P450IID (CYP2D) subfamily, and their monooxygenase activities in rat liver microsomes were investigated. 2. 2. In vivo administration of cimetidine and ranitidine decreased the contents of P450 isozymes and the activities of P450-linked monooxygenase systems; i.e., benzphetamine N-demethylase, aminopyrine N-demethylase, 7-ethoxycoumarine O-deethylase, debrisoquine 4-hydroxylase and bufuralol 1'-hydroxylase. 3. 3. The inhibitory effect on the enzymatic activities of the P450IID (CYP2D)-linked monooxygenase systems was studied by Western blot analysis with serum containing antiCYP2D6 IgG, i.e., LKM1 autoantibody. The amount of P450IID (CYP2D) in liver microsomes decreased more remarkably in the group administered ranitidine or cimetidine in vivo than in controls. 4. 4. The effects of cimetidine and ranitidine on the activities of the P450IID (CYP2D)-linked monooxygenase systems were investigated in vitro. The activities of debrisoquine 4-hydroxylase and bufuralol 1'-hydroxylase were inhibited in vitro by cimetidine or ranitidine at a higher concentration than that on in vivo administration of either H 2 blocker. 5. 5. The kinetic parameters for cimetidine or ranitidine as to the activities of debrisoquine 4-hydroxylase and bufuralol 1'-hydroxylase in liver microsomes were determined by means of Lineweaver-Burk plots. 6. 6. The suppressive effects of cimetidine and ranitidine on the activities of the P450IID (CYP2D)-linked monooxygenase systems in vivo were found to be due to a decrease of the content of the P450IID (CYP2D) protein.

Interleukin 2 induction of cytochrome P450-linked monooxygenase systems of rat liver microsomes

The effects of interleukin 2 (IL-2), a pivotal cytokine for generating an effective immune response, on rat liver microsomal cytochrome P450-linked monooxygenase systems were investigated by measuring the contents of cytochromes b 5 and P450, and the activities of various xenobiotic-metabolizing enzymes [debrisoquine and bufuralol monooxygenases (CYP2D), 7-ethoxycoumarin O-deethylase, benzphetamine N-demethylase, aniline hydroxylase and p-nitroanisole N-demethylase]. The enzymatic activities except for p-nitroanisole N-demethylase and aniline hydroxylase were increased approximately to 1.3-fold of those of untreated liver microsomes following intraperitoneal infection of IL-2 (15 U/rat). However, the amount of immunoreactive b 5 protein, and the activities of aniline hydroxylase and p-nitroanisole N-demethylase were not changed by injection of IL-2. To elucidate further the mechanism of the induction of CYP2D by IL-2, quantitative analyses of immunoreactive CYP2D protein and its mRNA were conducted by western blot and slot blot hybridization analyses. The results indicated that IL-2 induced an increase in the amounts of immunoreactive CYP2D protein and its mRNA. These enzyatic activities were thus up-regulated at the mRNA level.

Effects of interleukin 1α on the activities and gene expressions of the cytochrome p450iid subfamily

The mechanism by which recombinant human interleukin 1α-(rhIL-1α) inhibits the activities of drug-metabolizing enzymes of rat liver microsomes, especially debrisoquine monooxygenase and bufuralol monooxygenase (both cytochrome P450IID supported reactions), as well as other enzymes, was investigated by injecting IL-1α into rats. rhIL- 1α suppressed the activities of various P450-linked monooxygenase systems such as aminopyrine N-demethylase, benzphetamine N-demethylase, and 7-ethoxycoumarin O-deethylase. It also suppressed the activities of debrisoquine monooxygenase and bufuralol monooxygenase. On the other hand, IL-1α had little effect on the activity of p-nitroanisole N-demethylase. The suppression of debrisoquine monooxygenase and bufuralol monooxygenase activities was caused by a decrease in the amounts of immunoreactive P450IID protein and its mRNA. The reduction rates in the level of immunoreactive P450IID protein and its mRNA were comparable. These results suggest that at the mRNA level, the enzymatic activities of debrisoquine monooxygenase and bufuralol monooxygenase are down-regulated by IL-1α.

Metabolic deactivation of furylfuramide by cytothrome P450 in human and liver microsomes

Metabolic deactivation of furylfuramide by human and rat liver microsomal cytochrome P450 enzymes has been investigated in a system measuring induction of umu gene expression response in Salmonella typhimurium TA1535/pSK1002. Both human and rat liver microsomes catalyzed the metabolism of furylfuramide to inactive form(s) that are incapable of inducing umu gene expression in the tester strain. The reaction required an NADPH-generating system and molecular oxygen and was inhibited by carbon monoxide, suggesting that a cytochrome P450-linked mono-oxygenase system is prerequisite for the deactivation reaction. With liver microsomes from variously pretreated rats, 3-methylcholanthrene was found to be a powerful inducer for the furylfuramide-metabolizing activity, and antibodies raised against rat P450IA1(BNF-B, c) and P450IA2(ISF-G, d) inhibited the microsomal activity. Human liver microsomal furylfuramide-metabolizing activity was also inhibited significantly by anti-P450IA2 IgG but weakly by anti-P450IA1 IgG. In liver microsomes prepared from seven different human samples, the activities of deactivation of furylfuramide were found to correlate with the amounts of immunoreactive protein related to rat P450IA2 and with the monooxygenase activities of metabolic activation of 2-amino-3,4-dimethyl-imidazo[4,5-f]quinoline (MeIQ) and of ethoxyresorufin O-deethylation. These results suggest that P450IA1 and P450IA2 in rats, and P450PA (IA2, the phenacetin O-deethylase and ortholog of rat P450IA2) in humans are the major enzymes involved in the deactivation of furylfuramide in liver microsomes. The metabolic studies involving HPLC analysis of products followed by spectrophotometric examination have also suggested that furylfuramide can be degraded very rapidly through the aerobic metabolism by liver microsomes.

Eicosanoids and the modulation of glomerular immune injury

For the purposes of this review, glomerular immune injury is viewed as a process initiated at the level of glomerular structures following deposition and interaction of defined immunologic reactants which may activate the synthesis of proinflammatory mediators. Renal hemodynamic perturbations and glomerular capillary wall injury resulting in proteinuria are the most frequent sequelae. When glomerular injury is initiated by deposition of antibody with specificity against glomerular antigens (structural or trapped) or against non-renal soluble antigens, the ensuing reaction can be of an inflammatory or noninflammatory type. The former is frequently associated with intraglomerular complement activation, recruitment of neutrophils and platelets and recruitment or enhanced expression of sensitized monocytes-macrophages. In noninflammatory forms, capillary wall injury can be mediated by antibody deposition alone or it may require complement activation. Regardless the type of injury, activation of a number of proinflammatory mediator systems has been documented following glomerular deposition of immunologic reactants. The metabolic products formed following activation of these systems may mediate perturbations in renal hemodynamics and in the permeability of the glomerular capillary wall. Moreover, they may play a regulatory role on the function of endogenous glomerular or blood borne cells recruited in glomerular structures following initiation of injury. Table 1 summarizes the mediator systems which have been studied in various forms of experimental glomerulonephritis. The use of powerful investigative tools such as tissue culture and analytical biochemistry has yielded an impressive amount of information regarding the biosynthetic capabilities of renal tissues. The renal glomerulus and its various cell types are now regarded as active sites of synthesis of lipid compounds with autacoid and proinflammatory effects. Of the compounds with proinflammatory effects, major emphasis has been placed on the biosynthesis and role of eicosanoids. Eicosanoids are oxygenated metabolites of arachidonic acid (eicosatetraenoic acid) and other polyunsaturated fatty acids which contain 20 carbons (eicosa indicates 20). These polyunsaturated fatty acids are derived from dietary essential fatty acids and are present in esterified form on membrane phosphoglycerides of most mammalian cells. The literature on eicosanoids has been expanding exponentially with approximately 10 publications now appearing daily, a substantial number of which pertain to the kidney. The reader is, therefore, referred to a number of key references regarding the distribution of arachidonate on renal cell phospholipids [11], the release of arachidonate in response to cell-surface perturbations or receptor-mediated mechanisms [12], the renal compartmentalization of enzymes involved in the sequential conversion of arachidonate to prostaglandins (PG), thromboxanes (Tx) [13], the metabolites originating via the cytochrome P450-linked monooxygenase system [14] and the biosynthesis and proinflammatory role of arachidonate lipoxygenation products (hydroxyeicosatetraenoic acids, leukotrienes and lipoxins) [15, 16]. Eicosanoids exhibit a bewildering and often widely differing spectrum of biological activities with frequently opposing biological effects which may further depend on the species studied. At the risk of oversimplifying, this review will regard the renal effects of arachidonate cyclooxy-genation products (dienoic prostaglandins and thromboxanes) as being primarily vasoactive while those of arachidonate lipoxygenation products (hydroxyeicosatetraenoic acids and leukotrienes) as being mainly proinflammatory. Of additional relevance to this review is the role of eicosanoids on immuno-regulation [17] and their potential role in regulating glomerular cell function, which has primarily been explored in mesangial cells with respect to their contractile properties [18, 19].

On the role of calcium in chemical toxicity.

It is now well established that the toxic effects of a variety of chemicals are me­diated by reactive products formed during their biotransformation in the organ­ism. It is equally clear that there exist protective systems which can trap or inac­tivate toxic metabolites and thereby prevent their accumulation within the tissues and subsequent toxicity. Although phase I reactions, in particular those mediated by the cytochrome P450-linked monooxygenase system, are most often responsi­ble for the production of toxic metabolites, there are now many examples of met­abolic activation via phase II reactions, despite the fact that the latter normally serve a protective function (Jakoby 1980). It therefore follows that the formation of toxic metabolites cannot be attributed to any single enzyme or enzyme system, and that the balance between metabolic activation and inactivation is critical in deciding whether exposure to a potentially toxic compound will result in toxicity or not.

Renal arachidonic acid metabolism. The third pathway.

Cells were isolated from the outer medulla of the rabbit kidney, primarily from the thick ascending limb of Henle's loop (mTALH). These mTALH cells are heavily invested with a cytochrome P450-linked monooxygenase that represents the third pathway by which arachidonic acid is metabolized. After cell separation, approximately 80% of the cells proved to be mTALH in origin, based on electron microscopic criteria and immunofluorescent localization of Tamm-Horsfall protein, a specific marker for mTALH cells. The specific activity of alkaline phosphatase, a marker for proximal tubular cells, decreased threefold after separation of mTALH cells from outer medullary cells, associated with a fourfold increase in the capacity of the separated mTALH cells to metabolize arachidonic acid. Incubation of mTALH cells with 14C-arachidonic acid resulted in formation of oxygenated metabolites, identified as two peaks (P1 and P2), which accounted for 30 to 40% of the recovered radioactivity. Formation of prostaglandin E2 and F2 alpha accounted for only 3 to 5%. The chromatographic retention times of P1 and P2 were different from products of lipoxygenases. An inhibitor of cytochrome P450-dependent enzymes, SKF-525A (50 microM), reduced product formation by mTALH cells by more than 70%, while induction of cytochrome P450 increased product formation. Formation of P1 and P2 by cell-free homogenates of mTALH was totally dependent on the presence of nicotinamide adenine dinucleotide phosphate, reduced form (NADPH), which suggests a NADPH-dependent cytochrome P450-linked monooxygenase pathway. Vasopressin and calcitonin (10(-10) M to 10(-7) M) stimulated release of arachidonic acid metabolites from mTALH cells.(ABSTRACT TRUNCATED AT 250 WORDS)