20-HETE and EETs in Diabetic Nephropathy: A Novel Mechanistic Pathway

Diabetic nephropathy (DN), a major complication of diabetes, is characterized by hypertrophy, extracellular matrix accumulation, fibrosis and proteinuria leading to loss of renal function. Hypertrophy is a major factor inducing proximal tubular epithelial cells injury. However, the mechanisms leading to tubular injury is not well defined. In our study, we show that exposure of rats proximal tubular epithelial cells to high glucose (HG) resulted in increased extracellular matrix accumulation and hypertrophy. HG treatment increased ROS production and was associated with alteration in CYPs 4A and 2C11 expression concomitant with alteration in 20-HETE and EETs formation. HG-induced tubular injury were blocked by HET0016, an inhibitor of CYPs 4A. In contrast, inhibition of EETs promoted the effects of HG on cultured proximal tubular cells. Our results also show that alteration in CYPs 4A and 2C expression and 20HETE and EETs formation regulates the activation of the mTOR/p70S6Kinase pathway, known to play a major role in the development of DN. In conclusion, we show that hyperglycemia in diabetes has a significant effect on the expression of Arachidonic Acid (AA)-metabolizing CYPs, manifested by increased AA metabolism, and might thus alter kidney function through alteration of type and amount of AA metabolites.     

Interaction of nitric oxide, 20-HETE, and EETs during functional hyperemia in whisker barrel cortex

Nitric oxide (NO) modulates vasodilation in cerebral cortex during sensory activation. NO is known to inhibit the synthesis of 20-HETE, which has been implicated in arteriolar constriction during astrocyte activation in brain slices. We tested the hypothesis that the attenuated cerebral blood flow (CBF) response to whisker stimulation seen after NO synthase (NOS) inhibition requires 20-HETE synthesis and that the ability of an epoxyeicosatrienoic acids (EETs) antagonist to reduce the CBF response is blunted after NOS inhibition but restored with simultaneous blockade of 20-HETE synthesis. In anesthetized rats, the increase in CBF during whisker stimulation was attenuated after the blockade of neuronal NOS with 7-nitroindazole. Subsequent administration of the 20-HETE synthesis inhibitor N-hydroxy-N'-(4-n-butyl-2-methylphenyl)formamidine (HET0016) restored the CBF response to control levels. After the administration of 7-nitroindazole, the inhibitory effect of an EETs antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE) on the CBF response was lost, whereas the simultaneous administration of 7-nitroindazole and HET0016 restored the inhibitory effect of 14,15-EEZE. The administration of HET0016 alone had only a small effect on the evoked CBF response in rats. Furthermore, in neuronal NOS(+/+) and NOS(-/-) mice, HET0016 administration did not increase the CBF response to whisker stimulation. In neuronal NOS(+/+) mice, HET0016 also blocked the reduction in the response seen with acute NOS inhibition. These results indicate that 20-HETE synthesis normally does not substantially restrict functional hyperemia. Increased NO production during functional activation may act dynamically to suppress 20-HETE synthesis or downstream signaling and permit EETs-dependent vasodilation. With the chronic loss of neuronal NOS in mice, other mechanisms apparently suppress 20-HETE synthesis or signaling.     

Disturbed ratio of renal 20-HETE/EETs is involved in androgen-induced hypertension in cytochrome P450 4F2 transgenic mice

We have previously established a cytochrome P450 4F2 (CYP4F2) transgenic mouse model. The present study elucidated the molecular foundation of hypertension by androgen-induction in this model. The renal expression of CYP4F2 in transgenic mice was highly expressed and strongly induced with 5α-dihydrotestosterone (DHT) treatment determined by Western blot. DHT also increased the renal arachidonic acid ω-hydroxylation and urinary 20-hydroxyeicosatetraenoic acid (20-HETE) excretion (P<0.01), and furthermore elevated the systolic blood pressure by 10 and 22 mm Hg (P<0.05) in female and castrated male transgenic mice, respectively. HET0016 completely eliminated the androgen-induced effects (P<0.01). Endogenous Cyp4a ω-hydroxylases, evaluated by real-time quantitative PCR, were significantly suppressed in transgenic mice (P<0.05). Importantly, transgenic mice with increased 20-HETE showed decreased epoxyeicosatrienoic acids (EETs) and increased dihydroxyeicosatetraenoic acids determined by liquid chromatography-tandem mass spectrometry, contributing to significantly raised ratio of 20-HETE/EETs in the urine and kidney homogenate (P<0.01). These data demonstrate that the androgen aggravated hypertension possibly through an altered ratio of 20-HETE/EETs in CYP4F2 transgenic mice.     

Inhibition of adrenal steroid metabolism by administration of 1-aminobenzotriazole to guinea pigs

Prior in vitro investigations demonstrated that the P450 suicide substrate, 1-aminobenzotriazole (ABT), was a potent inhibitor of xenobiotic metabolism but had no effect on steroidogenic enzymes in the guinea pig adrenal cortex. Studies were done to determine if ABT administration to guinea pigs in vivo also selectively inhibited adrenal xenobiotic metabolism. At single doses of 25 or 50 mg/kg, ABT effected rapid decreases in spectrally detectable adrenal P450 concentrations. The higher dose caused approx. 75% decreases in microsomal and mitochondrial P450 levels within 2 h. The decreases in P450 were sustained for 24 h but concentrations returned to control levels within 72 h. Accompanying the ABT-induced decreases in adrenal P450 content were proportionately similar decreases in P450-mediated xenobiotic and steroid metabolism. Microsomal benzo(a)pyrene hydroxylase, benzphetamine N-demethylase, 17-hydroxylase and 21-hydroxylase activities were decreased to 20–25% of control values by the higher dose of ABT. Mitochondrial 11β-hydroxylase and cholesterol sidechain cleavage activities were similarly diminished by ABT treatment. Adrenal 3β-hydroxysteroid dehydrogenase activity, by contrast, was not affected by ABT, indicating specificity for P450-catalyzed reactions. The results demonstrate that ABT in vivo is a non-selective inhibitor of adrenal steroid- and xenobiotic-metabolizing P450 isozymes. The absence of ABT effects on steroid metabolism in vitro suggests that an extra-adrenal metabolite may mediate the in vivo inhibition of steroidogenesis.     

Inhibition of 20-HETE abolishes the myogenic response during NOS antagonism in the ovine fetal pulmonary circulation

Mechanisms that maintain high pulmonary vascular resistance (PVR) and oppose vasodilation in the fetal lung are poorly understood. In fetal lambs, increased pulmonary artery pressure evokes a potent vasoconstriction, suggesting that a myogenic response contributes to high PVR in the fetus. In adult systemic circulations, the arachidonic acid metabolite 20-hydroxyeicosatetraenoic acid (20-HETE) has been shown to modulate the myogenic response, but its role in the fetal lung is unknown. We hypothesized that acute increases in pulmonary artery pressure release 20-HETE, which causes vasoconstriction, or a myogenic response, in the fetal lung. To address this hypothesis, we studied the hemodynamic effects of N-methylsufonyl-12,12-dibromododec-11-enamide (DDMS), a specific inhibitor of 20-HETE production, on the pulmonary vasoconstriction caused by acute compression of the ductus arteriosus (DA) in chronically prepared fetal sheep. An inflatable vascular occluder around the DA was used to increase pulmonary artery pressure under three study conditions: control, after pretreatment with nitro-L-arginine (L-NA; to inhibit shear-stress vasodilation), and after combined treatment with both L-NA and a specific 20-HETE inhibitor, DDMS. We found that DA compression after L-NA treatment increased PVR by 44 +/- 12%. Although intrapulmonary DDMS infusion did not affect basal PVR, DDMS completely abolished the vasoconstrictor response to DA compression in the presence of L-NA (44 +/- 12% vs. 2 +/- 4% change in PVR, L-NA vs. L-NA + DDMS, P < 0.05). We conclude that 20-HETE mediates the myogenic response in the fetal pulmonary circulation and speculate that pharmacological inhibition of 20-HETE might have a therapeutic role in neonatal conditions characterized by pulmonary hypertension.     

Angiotensin II attenuates the natriuresis of water immersion in humans

The hypothesis was tested that suppression of generation of ANG II is one of the mechanisms of the water immersion (WI)-induced natriuresis in humans. In one protocol, eight healthy young males were subjected to 3 h of 1) WI (WI + placebo), 2) WI combined with ANG II infusion of 0.5 ng. kg(-1). min(-1) (WI + ANG II-low), and 3) a seated time control (Con). In another almost identical protocol, 7-10 healthy young males were investigated to delineate the tubular site(s) of action of ANG II by the lithium clearance method (C(Li)) and were on an additional fourth study day subjected to infusion of ANG II at a rate of 1.5 ng. kg(-1). min(-1) (WI + ANG II-high). During WI + placebo, plasma concentration of ANG II decreased from 16 +/- 2 to 8 +/- 1 pg/ml (P < 0.05) and renal sodium excretion increased from 104 +/- 15 to 294 +/- 27 micromol/min (P < 0.05). During WI + ANG II-low, plasma ANG II was not suppressed by WI, and the natriuresis was blunted by 52 +/- 13% (P < 0.05). During WI + ANG II-low and WI + ANG II-high, an increase in C(Li) was prevented that was otherwise observed during WI, and fractional distal reabsorption of sodium was facilitated. In conclusion, maintaining plasma concentration of ANG II unchanged at the level of control attenuates the natriuresis of WI considerably in humans. Therefore, suppression of generation of ANG II is an important mechanism of the natriuresis of WI in humans. Furthermore, infusion of ANG II during WI prevents an otherwise induced increase in C(Li) and facilitates the fractional distal reabsorption of sodium, probably via an effect on aldosterone release.     

Inhibition of renal caveolin-1 reduces natriuresis and produces hypertension in sodium-loaded rats

Renal dopamine receptor function and ion transport inhibition are impaired in essential hypertension. We recently reported that caveolin-1 (CAV1) and lipid rafts are necessary for normal D(1)-like receptor-dependent internalization of Na-K-ATPase in human proximal tubule cells. We now hypothesize that CAV1 is necessary for the regulation of urine sodium (Na(+)) excretion (U(Na)V) and mean arterial blood pressure (MAP) in vivo. Acute renal interstitial (RI) infusion into Sprague-Dawley rats of 1 μg·kg?1·min?1 fenoldopam (FEN; D(1)-like receptor agonist) caused a 0.46 ± 0.15-μmol/min increase in U(Na)V (over baseline of 0.29 ± 0.04 μmol/min; P < 0.01). This increase was seen in Na(+)-loaded rats, but not in those under a normal-sodium load. Coinfusion with β-methyl cyclodextrin (βMCD; lipid raft disrupter, 200 μg·kg?1·min?1) completely blocked this FEN-induced natriuresis (P < 0.001). Long-term (3 day) lipid raft disruption via continuous RI infusion of 80 μg·kg?1·min?1 βMCD decreased renal cortical CAV1 expression (47.3 ± 6.4%; P < 0.01) and increased MAP (32.4 ± 6.6 mmHg; P < 0.001) compared with vehicle-infused animals. To determine whether the MAP rise was due to a CAV1-dependent lipid raft-mediated disruption, Na(+)-loaded rats were given a bolus RI infusion of CAV1 siRNA. Two days postinfusion, cortical CAV1 expression was decreased by 73.6 ± 8.2% (P < 0.001) and the animals showed an increase in MAP by 17.4 ± 2.9 mmHg (P < 0.01) compared with animals receiving scrambled control siRNA. In summary, acute kidney-specific lipid raft disruption decreases CAV1 expression and blocks D(1)-like receptor-induced natriuresis. Furthermore, chronic disruption of lipid rafts or CAV1 protein expression in the kidney induces hypertension.     

Inhibition of GATE-16 attenuates ATRA-induced neutrophil differentiation of APL cells and interferes with autophagosome formation

Autophagy is an intracellular bulk degradation process involved in cell survival upon stress induction, but also with a newly identified function in myeloid differentiation. The autophagy-related (ATG)8 protein family, including the GABARAP and LC3 subfamilies, is crucial for autophagosome biogenesis. In order to evaluate the significance of the GABARAPs in the pathogenesis of acute myeloid leukemia (AML), we compared their expression in primary AML patient samples, CD34⁺ progenitor cells and in granulocytes from healthy donors. GABARAPL1 and GABARAPL2/GATE-16, but not GABARAP, were significantly downregulated in particular AML subtypes compared to normal granulocytes. Moreover, the expression of GABARAPL1 and GATE-16 was significantly induced during ATRA-induced neutrophil differentiation of acute promyelocytic leukemia cells (APL). Lastly, knocking down GABARAPL2/GATE-16 in APL cells attenuatedneutrophil differentiation and decreased autophagic flux. In conclusion, low GABARAPL2/GATE-16 expression is associated with an immature myeloid leukemic phenotype and these proteins are necessary for neutrophil differentiation of APL cells.     

Regulation of arterial pressure: role of pressure natriuresis and diuresis

The importance of the renal pressure natriuresis and diuresis mechanisms in long-term control of body fluid volumes and arterial pressure has been controversial and difficult to quantitate experimentally. Recent studies, however, have demonstrated that in several forms of chronic hypertension caused by aldosterone, angiotensin II (AngII), vasopressin, or norepinephrine and adrenocorticotropin, increased renal arterial pressure is essential for maintaining normal excretion of sodium and water in the face of reduced renal excretory capability. When renal arterial pressure was servo-controlled in these models of hypertension, sodium and water retention continued unabated, causing ascites, pulmonary edema, or even complete circulatory collapse within a few days. Apparently, other mechanisms for volume homeostasis, such as the various natriuretic and diuretic factors that have been postulated, are not sufficiently powerful to maintain fluid balance in the absence of increased renal arterial pressure when renal excretory function is reduced in these forms of hypertension. The intrarenal mechanisms responsible for pressure natriuresis and diuresis are not entirely clear, but they seem to involve small increases in glomerular filtration rate and filtered load as well as reductions in fractional reabsorption in proximal and distal tubules. During chronic disturbances of arterial pressure additional factors, especially changes in AngII and aldosterone formation, act to amplify the effectiveness of the basic renal pressure natriuresis and diuresis mechanisms in regulating arterial pressure and body fluid volumes.     

20-Hydroxyeicosatetraenoic acid (20-HETE) is a novel activator of transient receptor potential vanilloid 1 (TRPV1) channel

TRPV1 is a member of the transient receptor potential ion channel family and is gated by capsaicin, the pungent component of chili pepper. It is expressed predominantly in small diameter peripheral nerve fibers and is activated by noxious temperatures >42 °C. 20-Hydroxyeicosatetraenoic acid (20-HETE) is a cytochrome P-450 4A/4F-derived metabolite of the membrane phospholipid arachidonic acid. It is a powerful vasoconstrictor and has structural similarities with other TRPV1 agonists, e.g. the hydroperoxyeicosatetraenoic acid 12-HPETE, and we hypothesized that it may be an endogenous ligand for TRPV1 in sensory neurons innervating the vasculature. Here, we demonstrate that 20-HETE both activates and sensitizes mouse and human TRPV1, in a kinase-dependent manner, involving the residue Ser(502) in heterologously expressed hTRPV1, at physiologically relevant concentrations.     

20-羟二十烷四烯酸在机体生理及病理调节中的作用

20多年前,国外学者就报道了机体内某些组织中存在着细胞色素P-450（cytochrome P-450,CYP）。已经证实它能催化花生四烯酸（arachidonic acid,AA）的ω-羟基生成20-羟二十烷四烯酸（20-hydroxyeicosatetraenoic acid,20-HETE）。随着研究的不断深入,人们逐渐发现作为第二信使,20-HETE在调节血管平滑肌紧张度、肾功能、脑血流量和肺血管舒张过程中发挥了重要作用;外源性的20-HETE对心脏冠脉循环也有明显的调节作用;并且它与高血压和妊娠毒血症等疾病的发生和发展关系密切。但国内对于20-HETE的相关研究还鲜有报道,本文将主要就20-HETE在机体生理及病理及病理调节中的作用作一综述。     

Inhibition of the NLRP3 inflammasome attenuates foam cell formation of THP-1 macrophages by suppressing ox-LDL uptake and promoting cholesterol efflux

The NOD-like receptor family, pyrin domain–containing protein 3 (NLRP3) inflammasome plays an important role in the development of atherosclerosis. The activated NLRP3 inflammasome has been reported to promote macrophage foam cell formation, but not all studies have obtained the same result, and how NLRP3 inflammasome is involved in the formation of foam cells remains elusive. We used selective NLRP3 inflammasome inhibitors and NLRP3-deficient THP-1 cells to assess the effect of NLRP3 inflammasome inhibition on macrophage foam cell formation, oxidized low-density lipoprotein (ox-LDL) uptake, esterification, and cholesterol efflux, as well as the expression of associated proteins. Inhibition of the NLRP3 inflammasome attenuated foam cell formation, diminished ox-LDL uptake, and promoted cholesterol efflux from THP-1 macrophages. Moreover, it downregulated CD36, acyl coenzyme A: cholesterol acyltransferase-1 and neutral cholesterol ester hydrolase expression; upregulated ATP-binding cassette transporter A1 (ABCA1) and scavenger receptor class B type I (SR-BI) expression; but had no effect on the expression of scavenger receptor class A and ATP-binding cassette transporter G1. Collectively, our findings show that inhibition of the NLRP3 inflammasome decreases foam cell formation of THP-1 macrophages via suppression of ox-LDL uptake and enhancement of cholesterol efflux, which may be due to downregulation of CD36 expression and upregulation of ABCA1 and SR-BI expression, respectively.     

Reversal of delayed vasospasm by an inhibitor of the synthesis of 20-HETE

This study characterized the time course of changes in cerebral blood flow (CBF) and vascular diameter in a dual-hemorrhage model of subarachnoid hemorrhage (SAH) in rats and examined whether acute blockade of the synthesis of 20-hydroxyeicosatetraenoic acid (20-HETE) with N-(3-chloro-4-morpholin-4-yl)phenyl-N'-hydroxyimido formamide (TS-011) can reverse delayed vasospasm in this model. Rats received an intracisternal injection of blood (0.4 ml) on day 0 and a second injection 2 days later. CBF was sequentially measured using laser-Doppler flowmetry, and the diameters of the cerebral arteries were determined after filling the cerebral vasculature with a casting compound. CBF fell to 67% of control after the first intracisternal injection of blood but returned to a value near control 24 h later. CBF again fell to 63% of control after a second intracisternal injection of blood and remained 30% below control for 5 days. The fall in CBF after the second intracisternal injection of blood was associated with a sustained 30% reduction in the diameters of the middle cerebral, posterior communicating, and basilar arteries. Acute blockade of the synthesis of 20-HETE with TS-011 (0.1 mg/kg i.v.), 5 days after the second SAH, increased the diameters of the cerebral arteries, and CBF returned to control. These results indicate that the rats develop delayed vasospasm after induction of the dual-hemorrhage model of SAH and that blockade of the synthesis of 20-HETE fully reverses cerebral vasospasm in this model. They also implicate 20-HETE in the development and maintenance of delayed cerebral vasospasm.     

Intrarenal mechanisms mediating pressure natriuresis: role of angiotensin and prostaglandins

The ability of the kidney to increase sodium and water excretion in response to increases in perfusion pressure has been recognized for more than 50 years. Because glomerular filtration rate is tightly autoregulated, pressure natriuresis occurs as the result of decreased tubular sodium reabsorption rather than increased filtered load. Micropuncture and microperfusion data support the contention that acute changes in arterial pressure can alter proximal tubule reabsorption; however, studies have failed to show a consistent association between changes in sodium excretion and peritubular, interstitial, or tubular pressures. Thus, the specific intrarenal mechanism for the change in tubular reabsorption in response to an acute change in arterial pressure does not appear to be related to the peritubular physical factors at the level of outer cortical nephrons. The possible roles of angiotensin and prostaglandins as humoral mediators of pressure natriuresis are considered in this report. Although angiotensin II is a powerful modulator of the slope of the pressure natriuresis relationship, the responsiveness of sodium excretion to arterial pressure is actually enhanced by angiotensin-converting enzyme inhibitors. These data suggest that angiotensin does not mediate the basic phenomenon. Recent experiments indicate that intrarenal prostaglandins also modulate the magnitude of the pressure natriuresis relationship, but these hormones do not appear to be essential for its basic manifestation.     

20-HETE and F2-isoprostanes in the metabolic syndrome: the effect of weight reduction

20-Hydroxyeicosatetraenoic acid (20-HETE) is a cytochrome P450 metabolite of arachidonic acid that regulates vascular function and sodium homeostasis. Studies showing an association between 20-HETE excretion, raised BMI, and oxidative stress suggest that 20-HETE may be important in the development of cardiovascular disease in the metabolic syndrome (MetS). We investigated whether 20-HETE and F₂-isoprostanes (markers of oxidative stress) were altered in the MetS before and after weight reduction. A case-controlled comparison of 30 participants with the MetS and matched controls showed that plasma and urinary 20-HETE and F₂-isoprostanes were significantly elevated in the MetS group. There was a significant gender × group interaction such that women with the MetS had higher urinary 20-HETE and F₂-isoprostanes compared to controls (p < 0.0001). In a randomized controlled trial, 42 participants with the MetS were assigned to 16 weeks of weight maintenance or a 12-week weight-loss program followed by 4 weeks weight stabilization. Relative to the weight-maintenance group, a 4-kg loss in weight resulted in a 2-mm Hg fall in blood pressure (BP) but did not alter urinary or plasma 20-HETE or F₂-isoprostanes. 20-HETE and oxidative stress may be important mediators of cardiovascular disease risk in the MetS. Although a 4% reduction in body weight reduced BP, there were no changes in plasma or urinary 20-HETE or F₂-isoprostanes.     

Inhibition of poly(ADP-ribose) polymerase-1 attenuates the toxicity of carbon tetrachloride

Carbon tetrachloride (CCl₄) is routinely used as a model compound for eliciting centrilobular hepatotoxicity. It can be bioactivated to the trichloromethyl radical, which causes extensive lipid peroxidation and ultimately cell death by necrosis. Overactivation of poly(ADP-ribose) polymerase-1 (PARP-1) can rapidly reduce the levels of β-nicotinamide adenine dinucleotide and adenosine triphosphate and ultimately promote necrosis. The aim of this study was to determine whether inhibition of PARP-1 could decrease CCl₄-induced hepatotoxicity, as measured by degree of poly(ADP-ribosyl)ation, serum levels of lactate dehydrogenase (LDH), lipid peroxidation, and oxidative DNA damage. For this purpose, male ICR mice were administered intraperitoneally a hepatotoxic dose of CCl₄ with or without 6(5H)-phenanthridinone, a potent inhibitor of PARP-1. Animals treated with CCl₄ exhibited extensive poly(ADP-ribosyl)ation in centrilobular hepatocytes, elevated serum levels of LDH, and increased lipid peroxidation. In contrast, animals treated concomitantly with CCl₄ and 6(5H)-phenanthridinone showed significantly lower levels of poly(ADP-ribosyl)ation, serum LDH, and lipid peroxidation. No changes were observed in the levels of oxidative DNA damage regardless of treatment. These results demonstrated that the hepatotoxicity of CCl₄ is dependent on the overactivation of PARP-1 and that inhibition of this enzyme attenuates the hepatotoxicity of CCl₄.     

Inhibition of poly(ADP-ribose) polymerase-1 attenuates the toxicity of carbon tetrachloride

Carbon tetrachloride (CCl(4)) is routinely used as a model compound for eliciting centrilobular hepatotoxicity. It can be bioactivated to the trichloromethyl radical, which causes extensive lipid peroxidation and ultimately cell death by necrosis. Overactivation of poly(ADP-ribose) polymerase-1 (PARP-1) can rapidly reduce the levels of β-nicotinamide adenine dinucleotide and adenosine triphosphate and ultimately promote necrosis. The aim of this study was to determine whether inhibition of PARP-1 could decrease CCl(4)-induced hepatotoxicity, as measured by degree of poly(ADP-ribosyl)ation, serum levels of lactate dehydrogenase (LDH), lipid peroxidation, and oxidative DNA damage. For this purpose, male ICR mice were administered intraperitoneally a hepatotoxic dose of CCl(4) with or without 6(5H)-phenanthridinone, a potent inhibitor of PARP-1. Animals treated with CCl(4) exhibited extensive poly(ADP-ribosyl)ation in centrilobular hepatocytes, elevated serum levels of LDH, and increased lipid peroxidation. In contrast, animals treated concomitantly with CCl(4) and 6(5H)-phenanthridinone showed significantly lower levels of poly(ADP-ribosyl)ation, serum LDH, and lipid peroxidation. No changes were observed in the levels of oxidative DNA damage regardless of treatment. These results demonstrated that the hepatotoxicity of CCl(4) is dependent on the overactivation of PARP-1 and that inhibition of this enzyme attenuates the hepatotoxicity of CCl(4).     

Determination of major UDP-glucuronosyltransferase enzymes and their genotypes responsible for 20-HETE glucuronidation

The compound 20-HETE is involved in numerous physiological functions, including blood pressure and platelet aggregation. Glucuronidation of 20-HETE by UDP-glucuronosyltransferases (UGTs) is thought to be a primary pathway of 20-HETE elimination in humans. The present study identified major UGT enzymes responsible for 20-HETE glucuronidation and investigated their genetic influence on the glucuronidation reaction using human livers (n = 44). Twelve recombinant UGTs were screened to identify major contributors to 20-HETE glucuronidation. Based on these results, UGT2B7, UGT1A9, and UGT1A3 exhibited as major contributors to 20-HETE glucuronidation. The K_m values of 20-HETE glucuronidation by UGT1A3, UGT1A9, and UGT2B7 were 78.4, 22.2, and 14.8 μM, respectively, while V_max values were 1.33, 1.78, and 1.62 nmol/min/mg protein, respectively. Protein expression levels and genetic variants of UGT1A3, UGT1A9, and UGT2B7 were analyzed in human livers using Western blotting and genotyping, respectively. Glucuronidation of 20-HETE was significantly correlated with the protein levels of UGT2B7 (r² = 0.33, P < 0.001) and UGT1A9 (r² = 0.31, P < 0.001), but not UGT1A3 (r² = 0.02, P > 0.05). A correlation between genotype and 20-HETE glucuronidation revealed that UGT2B7 802C>T, UGT1A9 −118T₉>T₁₀, and UGT1A9 1399T>C significantly altered 20-HETE glucuronide formation (P < 0.05–0.001). Increased levels of 20-HETE comprise a risk factor for cardiovascular diseases, and the present data may increase our understanding of 20-HETE metabolism and cardiovascular complications.