Pregnane X receptor activation induces liver enlargement and regeneration and simultaneously promotes the metabolic activity of CYP3A1/2 and CYP2C6/11 in rats.

Human pregnane X receptor (PXR) is critical for regulating the expression of key drug-metabolizing enzymes such as CYP3A and CYP2C. Our recent study revealed that treatment with rodent-specific PXR agonist pregnenolone-16α-carbonitrile (PCN) significantly induced hepatomegaly and promoted liver regeneration after two-thirds partial hepatectomy (PHx) in mice. However, it remains unclear whether PXR activation induces hepatomegaly and liver regeneration and simultaneously promotes metabolic function of the liver. Here, we investigated the metabolism activity of CYP1A2, CYP3A1/2 and CYP2C6/11 during PXR activation-induced liver enlargement and regeneration in rats after cocktail dosing of CYP probe drugs. For PCN-induced hepatomegaly, a notable increase in the metabolic activity of CYP3A1/2 and CYP2C6/11, as evidenced by the plasma exposure of probe substrates and the AUC ratios of the characteristic metabolites to its corresponding probe substrates. The metabolic activity of CYP1A2, CYP3A1/2 and CYP2C6/11 decreased significantly after PHx. However, PCN treatment obviously enhanced the metabolic activity of CYP2C6/11 and CYP3A1/2 in PHx rats. Furthermore, the protein expression levels of CYP3A1/2 and CYP2C6/11 in liver were up-regulated. Taken together, this study demonstrates that PXR activation not only induces hepatomegaly and liver regeneration in rats, but also promotes the protein expression and metabolic activity of the PXR downstream metabolizing enzymes such as CYP3A1/2 and CYP2C6/11 in the body.

Gender differences in plasma pharmacokinetics and hepatic metabolism of geissoschizine methyl ether from Uncaria hook in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Geissoschizine methyl ether (GM), an indole alkaloid from Uncaria hook, is an active ingredient in the traditional Japanese Kampo medicine yokukansan, which is used to treat neurosis, insomnia, irritability, and night crying in children. AIM OF THE STUDY: Recent our pharmacokinetic studies suggested that there may be gender differences in the plasma concentrations of GM in rats, but not in humans. However, the details of this difference remain unverified. The purpose of this study was to clarify the reasons for the gender differences in rats. MATERIALS AND METHODS: GM plasma pharmacokinetics was compared in male and female rats orally administered yokukansan (4 g/kg). To confirm the involvement of cytochrome P450 (CYP) in GM liver metabolism, GM was incubated with male and female rat liver S9 fraction in the absence or presence of 1-aminobenzotriazole (a nonspecific CYP inhibitor). CYP isoforms involved in GM metabolism were estimated using recombinant rat CYP isoforms and anti-rat CYP antibodies. RESULTS: The maximum GM plasma concentrations were significantly higher in female than in male rats. When GM was incubated with rat liver S9 fractions, GM reduction was more striking in male S9 (69.3%) than that in female S9 (10.0%) and was completely blocked with nonspecific CYP inhibitor 1-aminobenzotriazole. Screening experiments using recombinant rat cytochrome P450 (CYP) isoforms showed that CYP1A1, CYP2C6, CYP2C11, CYP2D1, and CYP3A2 were involved in GM metabolism. Of these CYP isoforms, the use of anti-rat CYP antibodies indicated that male-dependent CYP2C11 and CYP3A2 were predominantly involved in the liver microsomal GM metabolism with gender differences. CONCLUSIONS: These results suggest that the cause of gender differences in plasma GM pharmacokinetics in rats is most likely because of male-dependent CYP2C11 and CYP3A2, and provide also useful information to further evaluate the pharmacological and toxicological effects in future. This study is the first to demonstrate that the gender differences in plasma GM pharmacokinetics in rats are caused by the gender-dependent metabolism of GM.

Neuroinflammation is able to downregulate cytochrome P450 epoxygenases 2J3 and 2C11 in the rat brain.

Cytochrome P450 (CYP) epoxygenases have been considered the main producers of epoxyeicosatrienoic acids (EETs) through the oxidation of arachidonic acid (AA). EETs display various biological properties, notably their powerful anti-inflammatory activities. In the brain, EETs have proven to be neuroprotective and to improve neuroinflammation. However, it is known that inflammation could modify CYP expression. We have previously reported that an inflammatory process in astrocytes is able to down-regulate CYP2J3 and CYP2C11 mRNA, protein levels, and activity (Navarro-Mabarak et al., 2019). In this work, we evaluated the effect of neuroinflammation in protein expression of CYP epoxygenases in the brain. Neuroinflammation was induced by the intraperitoneal administration of LPS (1 mg/kg) to male Wistar rats and was corroborated by IL-6, GFAP, and Iba-1 protein levels in the cortex over time. CYP2J3 and CYP2C11 protein levels were also evaluated in the cortex after 6, 12, 24, 48, and 72 h of LPS treatment. Our results show for the first time that neuroinflammation is able to downregulate CYP2J3 and CYP2C11 protein expression in the brain cortex.

Exploring the biotransformation of N-(2-hydroxyphenyl)-2-propylpentanamide (an aryl valproic acid derivative) by CYP2C11, using in silico predictions and in vitro studies.

OBJECTIVES: N-(2-hydroxyphenyl)-2-propylpentanamide (HO-AAVPA), a derivative of valproic acid (VPA), has been proposed as a potential anticancer agent due to its improved antiproliferative effects in some cancer cell lines. Although there is evidence that VPA is metabolized by cytochrome P450 2C11 rat isoform, HO-AAVPA CYP-mediated metabolism has not yet been fully explored. Therefore, in this work, the biotransformation of HO-AAVPA by CYP2C11 was investigated. METHODS: Kinetic parameters and spectral interaction between HO-AAVPA and CYP were evaluated using rat liver microsomes. The participation of CYP2C11 in metabolism of HO-AAVPA was confirmed by cimetidine (CIM) inhibition assay. Docking and molecular dynamics simulations coupled to MMGBSA methods were used in theoretical study. KEY FINDINGS: HO-AAVPA is metabolized by CYP enzymes (KM  = 38.94 µm), yielding a hydroxylated metabolite according to its HPLC retention time (5.4 min) and MS analysis (252.2 m/z). In addition, CIM inhibition in rat liver microsomes (Ki  = 59.23 µm) confirmed that CYP2C11 is mainly involved in HO-AAVPA metabolism. Furthermore, HO-AAVPA interacts with CYP2C11 as a type I ligand. HO-AAVPA is stabilized at the CYP2C11 ligand recognition site through a map of interactions similar to other typical CYP2C11 substrates. CONCLUSION: Therefore, rat liver CYP2C11 isoform is able to metabolize HO-AAVPA.

Pharmacokinetic interference of doxorubicin with tolbutamide due to reduced metabolic clearance with increased serum unbound fraction in rats.

The study examined the effect of doxorubicin (DOX) on the hepatic expression of CYP2C and its activity for metabolizing tolbutamide (TB), a specific CYP2C substrate, in rats and whether the pharmacokinetics of tolbutamide were altered by doxorubicin exposure. The expression level of hepatic CYP2C11 was depressed 1 day after doxorubicin administration (day 1), and this effect on CYP2C11 was augmented on day 4. However, the expression level of hepatic CYP2C6 remained unchanged. The activity of tolbutamide 4-hydroxylation in hepatic microsomes was decreased with time following doxorubicin administration. Regarding the enzyme kinetic parameters for tolbutamide 4-hydroxylation on day 4, the maximum velocity (Vmax ) was significantly lower in the DOX group than that in the control group, while the Michaelis constant (Km ) was unaffected. On pharmacokinetic examination, the total clearance (CLtot ) of tolbutamide on day 4 was increased, despite the decreased metabolic capacity. On the other hand, the serum unbound fraction (fu ) of tolbutamide was elevated with a reduced serum albumin concentration in the DOX group. Contrary to CLtot , CLtot /fu , a parameter approximated to the hepatic intrinsic clearance of unbound tolbutamide, was estimated to be significantly reduced in the DOX group. These findings indicate that the metabolic capacity of CYP2C11 in the liver is depressed time-dependently by down-regulation after doxorubicin exposure in rats, and that the decreased enzyme activity of TB 4-hydroxylation in hepatic microsomes reflects the pharmacokinetic change of unbound tolbutamide, not total tolbutamide, in serum.

Altered cytokine profile under control of the serotonergic system determines the regulation of CYP2C11 and CYP3A isoforms.

The aim of this study is to assess a potential mechanism by which the serotonergic system can control the expression and activity of cytochrome (CYP) 2C11 and CYP3A isoforms during liver insufficiency. A rat model of diethylnitrosamine (DEN)-induced liver insufficiency was developed by administering 50 mg/kg of DEN twice a week for 7 weeks. Dysfunction of the serotonergic system was evoked by feeding the rats with a tryptophan-free diet for three weeks. Dysfunction of the serotonergic system during liver insufficiency decreased the level of proinflammatory cytokines (TGF-ß and IL-1ß) and increased the level of an anti-inflammatory cytokine (IL-4). Simultaneously, activation of the repressive mechanism IL-4/JAK1/STAT6/SOCS1 of the JAK2/STAT5b-mediated signal transduction pathway and the pERK1/2/GR/STAT6 signal transduction pathway resulted in the suppression of the CYP2C11 and CYP3A isoforms. Moreover, dysfunction of the serotonergic system during liver insufficiency equalized the level of testosterone to the basal level, did not change the steady state of the corticosterone level and significantly enhanced the reduced level of growth hormone. An altered cytokine profile under control of the serotonergic system determines the regulation of CYP2C11 and CYP3A isoforms during liver insufficiency through mechanisms based on posttranscriptional and posttranslational processes.

Activation of 5-HT1A Receptors in the Hypothalamic Paraventricular Nuclei Negatively Regulates Cytochrome P450 Expression and Activity in Rat Liver.

Our recent work suggested a negative effect for the serotonergic innervation of the paraventricular nuclei (PVN) of the hypothalamus on growth hormone secretion and growth hormone-dependent expression of CYP2C11. The aim of our present research was to determine the effect of the activation of the 5-hydroxytryptamine [(5-HT) serotonin] 5-HT1 or 5-HT2 receptors in the PVN on the expression and activity of cytochrome P450 in male rat liver. The serotonergic agonists 5-carboxyamidotryptamine [(5-CT), a 5-HT1 receptor-type agonist], 8-hydroxy-2-(di-n-propyloamino)-tetralin [(8-OH-DPAT), a 5-HT1A receptor agonist], sumatriptan (a 5-HT1B/D receptor agonist), and 2,5-dimethoxy-4-iodoamphetamine [(DOI), a 5-HT2A/C receptor agonist] were individually injected into the PVN. The liver cytochrome P450 activity and expression and the levels of serum and pituitary and hypothalamic hormones were measured. 5-CT and 8-OH-DPAT significantly decreased the activity and expression of CYP2C11 at both the mRNA and protein levels, which was accompanied by an increase in pituitary and hypothalamic somatostatin levels and a decrease in the serum growth hormone concentration. The expression of CYP3A1/23 also decreased. The serum corticosterone concentration declined after the injection of 8-OH-DPAT. The obtained results indicated that 5-HT1A but not the 5-HT1B/D or 5-HT2 receptors in the PVN are engaged in the negative neuroendocrine regulation of cytochrome P450 via the stimulation of hypothalamic somatostatin secretion and in the decreases in the serum growth hormone and corticosterone concentrations. Since the affected enzymes metabolize steroids and drugs and 5-HT1A receptors are engaged in the action of psychotropic drugs, the results obtained may be of both physiologic and pharmacological meaning.

Altered tolbutamide pharmacokinetics by a decrease in hepatic expression of CYP2C6/11 in rats pretreated with 5-fluorouracil.

1. We investigated the change in the pharmacokinetic profile of tolbutamide (TB), a substrate for CYP2C6/11, 4 days after single administration of 5-fluorouracil (5-FU), and the hepatic gene expression and activity of CYP2C6/11 were also examined in 5-FU-pretreated rats. 2. Regarding the pharmacokinetic parameters of the 5-FU group, the area under the curve (AUC) was significantly increased, and correspondingly, the elimination rate constant at the terminal phase (ke) was significantly decreased without significant change in the volume of distribution at the steady state (Vdss). 3. The metabolic production of 4-hydroxylated TB in hepatic microsomes was significantly reduced by the administration of 5-FU. 4. The expression level of mRNAs for hepatic CYP2C6 and CYP2C11 was significantly lower than in the control group when the rats were pretreated with 5-FU. 5. These results demonstrated that the pharmacokinetic profile of TB was altered by the treatment with 5-FU through a metabolic process, which may be responsible for the decreased CYP2C6/11 expression at mRNA levels.

Impaired liver cytochrome P450 2C11 activity after dual antiplatelet therapy with aspirin and clopidogrel in rats.

1. Aspirin (ASA) and clopidogrel (CLP) are used in combination as dual antiplatelet therapy (DAPT) for acute coronary syndrome based on their complementary mechanisms for platelet aggregation inhibition. However, the pharmacokinetics of such drug combination usage has not been thoroughly investigated. 2. In the current study, an LC-MS/MS method was developed to simultaneously determine the plasma concentrations of ASA and its metabolite salicylic acid (SA) with CLP and its metabolites, clopidogrel carboxylic acid (CLPM) and clopidogrel active metabolite derivative (CAMD). The pharmacokinetics of ASA, SA, CLP, CLPM and CAMD in rats receiving two-week DAPT with ASA and CLP were then determined. 3. After two-week DAPT with ASA and CLP in rats, the activities of aspirin esterase and rCyp2c11, enzymes mediating rat metabolism of ASA and CLP, respectively, in prepared rat liver microsomes were measured followed by further determination of rCyp2c11 mRNA expressions. The results demonstrated that DAPT led to minimal impact on aspirin esterase activity but significant decrease in rCyp2c11 activity and mRNA expression. 4. In conclusion, our findings on impairment in rCyp2C11 activity and mRNA expression by DAPT in rats could provide guidance on its safe clinical use with other CYP 2C19 substrates.

Growth hormone: a newly identified developmental organizer.

The sexually dimorphic expression of cytochromes P450 (CYP) drug-metabolizing enzymes has been reported in all species examined. These sex differences are only expressed during adulthood and are solely regulated by sex differences in circulating growth hormone (GH) profiles. Once established, however, the different male- and female-dependent CYP isoform profiles are permanent and immutable, suggesting that adult CYP expression requires imprinting. As the hormone that regulates an adult function is likely the same hormone that imprints the function, we selectively blocked GH secretion in some newborn male rats, whereas others received concurrent physiologic replacement of rat GH. The results demonstrate that adult male GH activation of the signal transduction pathway regulating expression of the principal CYP2C11 isoform is obligatorily dependent on perinatal GH imprinting, without which CYP2C11 and drug metabolism would be permanently and profoundly suppressed. As there are other adult metabolic functions also regulated by GH, pediatric drug therapy known to disrupt GH secretion could unintentionally impair adult health.

A new in vivo analysis model to detect sexually dimorphic rat liver cytochrome P450 gene expression dependent on growth hormone secretory patterns.

Several drug-metabolizing cytochrome P450 (CYP) enzymes exhibit sexual dimorphism depending on the pituitary growth hormone (GH) secretory patterns. However, the mechanism underlying CYP sexual dimorphism remains unclear. We previously established a transgenic (Alb-DsRed2 Tg) rat that expressed red fluorescent DsRed2 protein, particularly in hepatocytes, to visualize cell differentiation and multiplication and found that hepatic DsRed2 expression exhibited sexual dimorphism that was limited to adult males. In this study, we compared the expression patterns between sexual dimorphic Cyps and DsRed2 in Tg rats after experimentally reversing the GH secretory patterns in males and females. Postnatal day 1 male and female Tg rats were gonadectomized and then testosterone propionate (0.25 mg/rat) was subcutaneously administered to ovariectomized females immediately after surgery. Cyp mRNA and DsRed2 expression levels were quantified using RT-PCR and an in vivo imaging system, respectively. GH-dependent Cyps and hepatic DsRed2 expression patterns were reversed in males and females at 9 weeks after birth and were significantly correlated (P<0.05). This suggested that DsRed2 expression in these Tg rats depended on GH secretory patterns. Based on DsRed2 fluorescence, this Tg rat model could become a tool to readily and effectively evaluate changes in GH-dependent Cyp expression.

Proteomic analysis for the impact of hypercholesterolemia on expressions of hepatic drug transporters and metabolizing enzymes.

1. Our objective is to investigate the alterations of hepatic drug transporters and metabolizing enzymes in hypercholesterolemia. Male Sprague-Dawley rats were fed high-cholesterol chows for 8 weeks to induce hypercholesterolemia. Protein levels of hepatic drug transporters and metabolizing enzymes were analyzed by iTRAQ labeling coupled with LC TRIPLE-TOF. 2. Total 239 differentially expressed proteins were identified using proteomic analysis. Among those, protein levels of hepatic drug transporters (MRP2, ABCD3, OAT2, SLC25A12, SCL38A3, SLC2A2 and SLC25A5) and metabolizing enzymes (CYP2B3, CYP2C7, CYP2C11, CYP2C13, CYP4A2 and UGT2B) were markedly reduced, but the levels of CYP2C6 and CYP2E1 were increased in hypercholesterolemia group compared to control. Decreased expressions of drug transporters MRP2 and OAT2 were further confirmed by real time quantitative PCR (RT-qPCR) and western blot. 3. Ingenuity pathway analysis revealed that these differentially expressed proteins were regulated by various signaling pathways including nuclear receptors and inflammatory cytokines. One of the nuclear receptor candidates, liver X receptor alpha (LXRα), was further validated by RT-qPCR and western blot. Additionally, LXRα agonist T0901317 rescued the reduced expressions of MRP2 and OAT2 in HepG2 cells in hypercholesterolemic serum treatment. 4. Our present results indicated that hypercholesterolemia affected the expressions of various drug transporters and metabolizing enzymes in liver via nuclear receptors pathway. Especially, decreased function of LXRα contributes to the reduced expressions of MRP2 and OAT2.

Epoxyeicosatrienoic acid analogue mitigates kidney injury in a rat model of radiation nephropathy.

Arachidonic acid is metabolized to epoxyeicosatrienoic acids (EETs) by CYP epoxygenases, and EETs are kidney protective in multiple pathologies. We determined the ability of an EET analogue, EET-A, to mitigate experimental radiation nephropathy. The kidney expression of the EET producing enzyme CYP2C11 was lower in rats that received total body irradiation (TBI rat) compared with non-irradiated control. At 12 weeks after TBI, the rats had higher systolic blood pressure and impaired renal afferent arteriolar function compared with control, and EET-A or captopril mitigated these abnormalities. The TBI rats had 3-fold higher blood urea nitrogen (BUN) compared with control, and EET-A or captopril decreased BUN by 40-60%. The urine albumin/creatinine ratio was increased 94-fold in TBI rats, and EET-A or captopril attenuated that increase by 60-90%. In TBI rats, nephrinuria was elevated 30-fold and EET-A or captopril decreased it by 50-90%. Renal interstitial fibrosis, tubular and glomerular injury were present in the TBI rats, and each was decreased by EET-A or captopril. We further demonstrated elevated renal parenchymal apoptosis in TBI rats, which was mitigated by EET-A or captopril. Additional studies revealed that captopril or EET-A mitigated renal apoptosis by acting on the p53/Fas/FasL (Fas ligand) apoptotic pathway. The present study demonstrates a novel EET analogue-based strategy for mitigation of experimental radiation nephropathy by improving renal afferent arteriolar function and by decreasing renal apoptosis.

Differential Effects of Sunitinib on the Expression Profiles of Xenobiotic-Metabolizing Enzymes and Transporters in Rat Liver and Kidneys.

Sunitinib (SUN) is a multi-targeted tyrosine kinase inhibitor that was recently approved for the treatment of gastrointestinal tract and renal cancers. To date, very little is known about the effects of SUN on the expression of hepatic and renal xenobiotic-metabolizing enzymes (XMEs) and transporters. The present study was designed to investigate the capacity of chronic SUN treatment to modulate the mRNA and protein expression levels of phase I cytochrome P450 (CYP), phase II conjugating enzymes, and phase III transporters in rat liver and kidneys. For this purpose, SUN (25, 50 and 100 mg/kg) was injected IP into Wistar albino rats for 4 weeks; thereafter, the mRNA and protein expression levels of several XMEs and transporters were determined by RT-PCR and Western blot analysis, respectively. Real-time PCR analysis showed that SUN significantly induced the hepatic and renal CYP1A1, 1A2, 1B1, 2E1 and 4F4, whereas it inhibited CYP2C11 and 4A2. Furthermore, SUN specifically induced renal, but not hepatic, CYP2J3 and 3A2, while it induced only hepatic CYP4A1. With regard to phase II, SUN induced hepatic GSTA1 and UGT1A and renal NQO1 and UGT1A mRNA levels, whereas it inhibited renal GST1A expression. On the other hand, both renal and hepatic P-gp, MRP2 and BCRP transporters were significantly induced by SUN at the mRNA and protein expression levels. Importantly, these differential effects were associated with changes in oxidative stress genes and lipid peroxidation levels. In conclusion, SUN can serve as XME and transporters modulator, which potentially may counteract the efficacy of the treatment, adverse reactions and drug interactions in SUN treatment.

Inhibitory effects of curcumin on activity of cytochrome P450 2C9 enzyme in human and 2C11 in rat liver microsomes.

Cytochrome P450 2C9 (CYP2C9), one of the most important phase I drug metabolizing enzymes, could catalyze the reactions that convert diclofenanc into diclofenac 4'-hydroxylation. Evaluation of the inhibitory effects of compounds on CYP2C9 is clinically important because inhibition of CYP2C9 could result in serious drug-drug interactions. The objective of this work was to investigate the effects of curcumin on CYP2C9 in human and cytochrome P450 2C11 (CYP2C11) in rat liver microsomes. The results showed that curcumin inhibited CYP2C9 activity (10 µmol L(-1) diclofenac) with half-maximal inhibition or a half-maximal inhibitory concentration (IC50) of 15.25 µmol L(-1) and Ki = 4.473 µmol L(-1) in human liver microsomes. Curcumin's mode of action on CYP2C9 activity was noncompetitive for the substrate diclofenanc and uncompetitive for the cofactor NADPH. In contrast to its potent inhibition of CYP2C9 in human, diclofenanc had lesser effects on CYP2C11 in rat, with an IC50 ≥100 µmol L(-1). The observations imply that curcumin has the inhibitory effects on CYP2C9 activity in human. These in vitro findings suggest that more attention should be paid to special clinical caution when intake of curcumin combined with other drugs in treatment.

Changes of hepatic lipid mediators associated with intake of high-fat diet for 12 weeks in endotoxemic rats using LC-ESI-MS/MS.

BACKGROUND & AIMS: It has recently been reported that anti-inflammatory lipid mediators are increased in the late phase of acute inflammation, whereas proinflammatory lipid mediators are regulated at the initiation of inflammation. The purpose of this study was to evaluate changes of hepatic lipid mediators due to high-fat diet (HFD) feeding in endotoxemic rats. METHODS: Male Wistar rats were fed either HFD or control diet for 12 weeks, and were then killed 0, 1.5, and 6 h after lipopolysaccharide (LPS) injection. Analyses included lipidomics assessment of mediators using liquid chromatography-electrospray ionization/multi-stage mass spectrometry; measuring expression of hepatic polyunsaturated fatty acid (PUFA)-oxidizing enzyme, tumor necrosis factor (TNF)-α, interleukin (IL)-6, and inducible nitric oxide synthase mRNA levels; blood biochemical tests; and liver histology. RESULTS: HFD feeding worsened liver injury, increased expression of TNF-α and IL-6 mRNA, and increased oxidative stress after LPS injection. PUFA-oxidizing enzymes were higher in HFD-fed rats after LPS injection. The proinflammatory prostaglandin (PG)E2 and thromboxane B2 were increased 1.5 h after LPS injection, and had decreased by 6 h in HFD-fed rats. In contrast, potent pro-resolving resolvins derived from eicosapentaenoic acid and docosahexaenoic acid were not detected, but anti-inflammatory epoxyeicosatrienoic acids, lipoxin A4, and 15-deoxy-PGJ2 were increased after LPS injection in HFD-fed rats. CONCLUSIONS: HFD feeding for 12 weeks enhanced proinflammatory lipid mediators 1.5 h after LPS injection suggesting relation to liver injury.

Rhythmic expression of cytochrome P450 epoxygenases CYP4x1 and CYP2c11 in the rat brain and vasculature.

Mammals have circadian variation in blood pressure, heart rate, vascular tone, thrombotic tendency, and cerebral blood flow (CBF). These changes may be in part orchestrated by circadian variation in clock gene expression within cells comprising the vasculature that modulate blood flow (e.g., fibroblasts, cerebral vascular smooth muscle cells, astrocytes, and endothelial cells). However, the downstream mechanisms that underlie circadian changes in blood flow are unknown. Cytochrome P450 epoxygenases (Cyp4x1 and Cyp2c11) are expressed in the brain and vasculature and metabolize arachidonic acid (AA) to form epoxyeicosatrienoic acids (EETs). EETs are released from astrocytes, neurons, and vascular endothelial cells and act as potent vasodilators, increasing blood flow. EETs released in response to increases in neural activity evoke a corresponding increase in blood flow known as the functional hyperemic response. We examine the hypothesis that Cyp2c11 and Cyp4x1 expression and EETs production vary in a circadian manner in the rat brain and cerebral vasculature. RT-PCR revealed circadian/diurnal expression of clock and clock-controlled genes as well as Cyp4x1 and Cyp2c11, within the rat hippocampus, middle cerebral artery, inferior vena cava, hippocampal astrocytes and rat brain microvascular endothelial cells. Astrocyte and endothelial cell culture experiments revealed rhythmic variation in Cyp4x1 and Cyp2c11 gene and protein expression with a 12-h period and parallel rhythmic production of EETs. Our data suggest there is circadian regulation of Cyp4x1 and Cyp2c11 gene expression. Such rhythmic EETs production may contribute to circadian changes in blood flow and alter risk of adverse cardiovascular events throughout the day.

Irreversible perinatal imprinting of adult expression of the principal sex-dependent drug-metabolizing enzyme CYP2C11.

We proposed to determine whether, like other sexual dimorphisms, drug metabolism is permanently imprinted by perinatal hormones, resulting in its irreversible sex-dependent expression. We treated newborn male rats with monosodium glutamate (MSG), a total growth hormone (GH) blocker, and, using cultured hepatocytes, examined expression of adult CYP2C11, the predominant cytochrome-P450 expressed only in males, as well as the signal transduction pathway by which episodic GH solely regulates the isoform's expression. In addition, adolescent hypophysectomized (hypox) male rats served as controls in which GH was eliminated after the critical imprinting period. Whereas renaturalization of the masculine episodic GH profile restored normal male-like levels of CYP2C11, as well as CYP2C12, in hepatocytes from hypox rats, the cells derived from the MSG-treated rats were completely unresponsive. Moreover, GH exposure of hepatocytes from hypox rats resulted in normal induction, activation, nuclear translocation, and binding to the CYP2C11 promoter of the signal transducers mediating GH regulation of CYP2C11 expression, which dramatically contrasted with the complete unresponsiveness of the MSG-derived hepatocytes, also associated with hypermethylation of GH-response elements in the CYP2C11 promoter. Lastly, neonatal MSG treatment had no adverse effect on postnatal and adult testosterone levels. The results demonstrate that the sexually dimorphic expression of CYP2C11 is irreversibly imprinted shortly after birth by a hormone other than the customary testosterone, but likely by GH.

Protein restoration in low-birth-weight rat offspring derived from maternal low-protein diet leads to elevated hepatic CYP3A and CYP2C11 activity in adulthood.

The World Health Organization has identified hypercholesterolemia to be one of the major symptoms encompassing the metabolic syndrome. Moreover, epidemiologic evidence indicates that low-birth-weight offspring are at greater risk of developing the metabolic syndrome. Previous work in our laboratory demonstrated that maternal protein restriction (MPR) results in impaired fetal growth and hypercholesterolemia in adulthood. This was attributed to repression of hepatic CYP7A1, a rate-limiting enzyme that catabolizes cholesterol to bile acids. Another important function of hepatic cytochrome P450 enzymes is the phase I oxidative metabolism of drugs (i.e., statins for hypercholesterolemia), which can significantly impact pharmacokinetics. We hypothesized that MPR offspring may have altered ability to metabolize drugs in adulthood. To address this hypothesis, we maintained Wistar rats on a 20% protein diet (control) or a low 8% protein diet throughout prenatal and postnatal life (LP1) or exclusively during prenatal life and weaning (LP2). Intriguingly CYP3A and CYP2C11 intrinsic clearance (Vmax/Km) was significantly increased exclusively in LP2 offspring at postnatal day 130 compared with control or LP1 offspring, as evaluated by testosterone enzyme kinetics in liver microsomes. The increase in activity was secondary to an increase in CYP3A23 and CYP2C11 mRNA. Collectively, these findings suggest that a low-birth-weight offspring with postnatal catch-up growth may have a diminished response to xenobiotics metabolized by CYP3A and CYP2C11 enzymes.

Disturbed balance between phase I and II metabolizing enzymes in ovarian endometriosis: a source of excessive hydroxy-estrogens and ROS?

Oxidative metabolism of estrogens was studied in 31 ovarian endometriosis and 29 normal endometrium samples, by qPCR. Expression was monitored for genes encoding five estrogen hydroxylating, five hydroxy (OH)-estrogen conjugating, and three estrogen quinone detoxifying enzymes. CYP1B1, COMT, NQO1, and GSTP1 protein levels were determined using Western blotting and immunohistochemistry staining. Increased expression of CYP1A1, CYP3A7 and COMT, and higher levels of MB-COMT were seen in endometriosis, as compared to normal endometrium. Expression of CYP1B1, CYP3A5, SULT1A1 and NQO2 was unchanged, with comparable CYP1B1 protein levels. Expression of SULT1E1, SULT2B1, UGT2B7, NQO1, and GSTP1 was decreased. Three NQO1 isoforms were detected; NQO1c appears to be endometriosis-specific. Our data indicate a disturbed balance between phase I and II metabolizing enzymes in endometriosis, potentially leading to excessive OH-estrogen and altered ROS formation, and stimulation of proliferation of ectopic endometrium. This is the first report on disturbed expression of estrogen oxidative metabolism genes in ovarian endometriosis.