Thiomethylstilbenes as inhibitors of CYP1A1, CYP1A2 and CYP1B1 activities

Resveratrol (3,5,4'-trihydroxy-trans-stilbene) is a natural stilbene derivative occurring in grapes, peanuts and red wine. Its chemopreventive action has been established in studies on animal models. Recently, numerous classes of compounds with stilbene backbone have been investigated for their biological activity concerning cancer prevention; e. g. resveratrol methyl ethers appeared to be specific and potent inhibitors of cytochromes P450 (CYP) family 1 involved in the activation of procarcinogens. Since the replacement of the 4'-hydroxyl with a thiomethyl group is supposed to reduce toxicity of stilbene derivatives, the purpose of this study was the synthesis and evaluation of a series of 4-thiomethyl-trans-stilbene derivatives differing in a number and position of additional methoxy groups. Their inhibitory potency toward human recombinant CYPs: CYP1A1, CYP1A2 and CYP1B1 have been studied and compared with the effect of resveratrol and its analogues. Among compounds tested, 2-methoxy-4'-thiomethyl-trans-stilbene and 3-methoxy-4'-thiomethyl-trans-stilbene demonstrated the most potent and selective inhibitory effect on CYP1A1 and CYP1B1 activities. The results of our study indicate that modification of stilbene derivatives with thiomethyl group may influence the selectivity and inhibitory potency of these compounds toward P450 isozymes. Thus, it should be considered in developing new chemopreventive agents based on their mechanism of action.     

Effects of various ginkgo biloba extracts and proanthocyanidin on hepatic cytochrome P450 activity in rats

In previous papers, we showed that Ginkgo biloba extract (GBE) induced hepatic cytochrome P450 (CYP) activity, in particular pentoxyresorufin O-dealkylase (PROD; corresponding to CYP2B type) in rats, and that GBE influenced the efficacy of co-administered drugs. In this study, to clarify the nature of the induction, we examined the effects of GBE samples from different sources and some major constituents of GBE on rat hepatic CYP in vitro and in vivo. In the study in vitro, eight GBE samples dose-dependently inhibited PROD activity in microsomes prepared from GBE-treated rats, and the inhibitory ratio correlated well with the content of proanthocyanidin in the GBE samples. Moreover, among six GBE constituents examined, proanthocyanidin markedly inhibited the PROD activity. However, administration of two GBE extracts with different proanthocyanidin contents to rats induced hepatic CYP activity, including PROD, to similar extents, and proanthocyanidin alone did not induce PROD activity. Furthermore, GBE samples extracted with both acetone-water and ethanol-water showed similar induction of CYPs in rats in vivo. These results suggest that most GBE samples available in Japan induce CYPs in rats regardless of the preparation method of the GBE, and that proanthocyanidin is not responsible for the induction. Further studies will be necessary to identify the constituent(s) of GBE involved in the induction of CYPs in vivo.     

Effects of green tea extract and its components on antioxidant status and activities of xenobiotic metabolizing enzymes of rats

Dietary administration of green tea extract (GTE) or epigallocatechin gallate (EGCG), quercetin (Qu) or caffeine (Cf) in doses equal to their concentration in GTE led to an increase of serum and liver antioxidant capacity and strengthening stability of microsomal and lysosomal membranes in rats. The antioxidant efficiency of EGCG and Qu was considerably higher than that of GTE. There were significant differences in the effects of EGCG, Qu and GTE on the activities and expression of mRNA for CYP1A1, CYP1A2 and CYP3A1. But feeding both GTE and Cf to rats results in similar elevated activities of CYP1A1, CYP1A2, UDP-glucuronosyl transferase and glutathion transferase. Our results suggest that Cf is the main contributor to GTE effects on activities of xenobiotic metabolizing enzymes.     

In vitro transactivation potencies of black-footed albatross (Phoebastria nigripes) AHR1 and AHR2 by dioxins to predict CYP1A expression in the wild population

Our previous studies have detected high levels of dioxins and related compounds (DRCs) including polychlorinated dibenzo-p-dioxins (PCDDs), furans (PCDFs), and coplanar PCBs (Co-PCBs) in the black-footed albatross (BFA), Phoebastria nigripes, from the North Pacific region. We have also cloned two aryl hydrocarbon receptors, AHR1 and AHR2, of the BFA. To evaluate the sensitivity to DRCs in the BFA and to assess the status of cytochrome P450 1A (CYP1A) induction in the wild population, this study investigated the mRNA expression levels of BFA AHR1 and AHR2 and also the transactivation potencies of each AHR by 15 selected DRC congeners. Quantitative real-time PCR of BFA AHR mRNAs showed that hepatic AHR1 is more highly expressed than AHR2. Transactivation by graded concentrations of individual DRCs was measured in COS-7 cells, where BFA AHR1 or AHR2 was transiently transfected. For congeners that exhibited AHR-mediated dose-dependent activities, 50% effective concentration (EC(50)) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) relative potencies (REPs) were estimated. Based on the estimates of the REPs, TCDD induction equivalency factors (IEFs) were determined. For BFA AHR1, PeCDF was equipotent to TCDD, but other congeners exhibited lower IEFs. For BFA AHR2, PCDD/F congeners except OCDD/F showed IEFs ≥ 1.0. Using BFA AHR1- or AHR2-IEFs and hepatic concentrations of DRCs in North Pacific BFAs, TCDD induction equivalents (IEQs) were calculated. We further constructed nonlinear regression models on the relationships between BFA AHR1- or AHR2-IEF derived total IEQ or WHO-TEF derived total TEQ and ethoxyresorufin-O-deethylase activity (EROD) in the liver of wild BFAs. The results indicated that the relationships of BFA AHR1- and AHR2-based IEQs and EROD were predictable from BFA AHR1- and AHR2-mediated transactivation by TCDD, respectively. Collectively, these results suggest that the in vitro assay incorporating the AHR of species of concern would be a useful tool to predict the sensitivity to DRCs in the species and CYP1A induction in the wild population.     

Effects of flavonoids on CYP1 expression in RL95-2 endometrial carcinoma cells

RL95-2 endometrial cancer cells were used to study cytochrome P450-mediated chemopreventative mechanisms of four flavonoids found in foods. To investigate enzymatic CYP1 inhibition, intact cells were induced with benzo(a)pyrene or 2,3,7,8-tetrachlorodibenzo-p-dioxin. Quercetin, kaempferol and myricetin inhibited CYP1 activity dose-dependently with IC₅₀s ranging from 2.2 to 4 μM; while amentoflavone was inactive. Further experiments were designed to determine if flavonoids also interacted with the AhR or caused a decrease in CYP1 protein or mRNA expression. CYP1A1 protein expression was inhibited in cells co-treated with TCDD and quercetin, kaempferol or myricetin compared with TCDD alone, but amentoflavone was ineffective. Relatively higher (∼7-fold) basal levels of CYP1B1 protein were not significantly affected by flavonoid treatments. In general, at the message level significant inhibition of induced CYP1A1 or CYP1B1 was not detected following flavonoid cotreatment. Despite the common inhibitory effects of quercetin, kaempferol, and myricetin on induced CYP1A1-dependent activity and protein expression, the mechanisms of CYP1 inhibition in this cell line are complex and dependent on the CYP gene, AhR inducer and the flavonoid.     

Hepatic CYP1A induction by dioxin-like compounds, and congener-specific metabolism and sequestration in wild common cormorants from Lake Biwa, Japan

The present study examines the effects of polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), and coplanar polychlorinated biphenyls (Co-PCBs) on hepatic cytochromes P450 (CYP) in the wild population of common cormorants from Lake Biwa, Japan, and discusses functional roles of CYP1A in terms of correlation analysis between tissue concentrations of individual congeners and expression levels of CYP1A. Levels of alkoxyresorufin (methoxy-, ethoxy-, pentoxy-, and benzyloxyresorufin) O-dealkylase activities and a protein cross-reacted with anti-rat CYP1A1 polyclonal antibodies showed significant positive correlations with total 2,3,7,8-tetrachlorodibenzo-p-dioxin toxic equivalents (TEQs) or TEQs for most individual congeners in the liver of cormorants, suggesting induction of CYP1A-like protein by these chemicals. In contrast, TEQs for lower chlorinated congeners, 2,3,7,8-T4CDF and PCB77, showed relatively low correlations with the expression level of CYP1A-like protein. Concentrations of 2,3,7,8-T4CDF and PCB77 normalized to a relatively recalcitrant congener, PCB169, were negatively correlated with the CYP1A-like protein level. These results indicate preferential metabolism of those congeners by CYP1A-like protein that was induced by TEQs. Concentration ratios of liverto pectoral muscle for certain congeners significantly increased with an elevation of the CYP1A-like protein level. Comparing the results in the present study with those of previous studies using rodents treated with certain dioxin-like congeners, these congeners in the liver may be sequestered by CYP1A. Levels of cross-reactive proteins with anti-rat CYP2B1, CYP2C6, and CYP3A2 polyclonal antibodies correlated with neither TEQs nor liver/muscle concentration ratios of congeners. We conclude that the potential for CYP1A induction, and metabolism and sequestration of dioxin-like compounds by CYP1A, may be a critical factor for assessing the ecological risk in wild avian species.     

Quercetin attenuates ethanol-derived microsomal oxidative stress: Implication of haem oxygenase-1 induction

As the main source of reactive oxygen species (ROS), hepatic microsomes are susceptible to ROS attack, especially upon CYP2E1 activation by ethanol. The objective of this study was to evaluate the hepatoprotective effect of quercetin, by inducing haem oxygenase-1 (HO-1), on ethanol-induced microsomal oxidative stress. Chronic alcohol administration to adult rats (4.0 g/kg for 90 days) resulted in microsomal redox disturbance and liver dysfunction, accompanying CYP2E1 upregulation and HO-1 downregulation of both protein expression and enzymatic activity. Quercetin (100 mg/kg) induced HO-1, which was not completely suppressed by ethanol. Moreover, quercetin pretreatment to ethanol-fed rats lowered CYP2E1 induction, partially normalised ethanol-overwhelmed microsomal antioxidative system, decreased ROS level and lipid peroxidation, and alleviated the leakage of transaminases. Given the beneficial effect of HO-1, its induction by quercetin may contribute to the protective role against CYP2E1-mediated oxidative stress on hepatic microsomes.     

Liquorice reduced cyclosporine bioavailability by activating P-glycoprotein and CYP 3A

Liquorice (root of Glycyrrhiza uralensis FISCH) is an ingredient of candies and used as a popular medicine in Europe and oriental countries. Cyclosporine (CsA), an immunosuppressant with narrow therapeutic window, is widely used in transplant patients. The absorption and disposition of CsA were associated with P-glycoprotein (P-gp) and cytochrome P450 3A4 (CYP3A4). This study investigated the effects of liquorice extract (LE) and its major ingredient, glycyrrhizin (GZ), on CsA pharmacokinetics in rats. The results indicated that LE and GZ significantly decreased the peak blood concentration and the areas under the curves of CsA in rats. Mechanism studies revealed that glycyrrhetic acid (GA), the major metabolite of GZ, significantly activated the functions of P-gp and CYP3A4. In conclusion, liquorice significantly reduced the oral bioavailability of CsA through activating P-gp and CYP3A4.     

In silico metabolism studies of dietary flavonoids by CYP1A2 and CYP2C9

Dietary flavonoids are important contributors to cancer prevention, due to their interactions with CYP family enzymes. This study describes the application of in silico tools to study the metabolism of dietary flavonoid compounds such as quercetin, rutin, naringenin and naringin, aiming to propose their potential metabolites and chemical structures and also to investigate the abilities of these compounds to interact with cytochrome P450 CYP1A2 and CYP2C9 isoforms. The reactions founded suggest that the presence of the sugar chain may account for significant change in the position of the flavonoid in the active site, due to the steric contacts. Another important finding is the participation of the amino acid residues in the metabolism. In CYP2C9, the Asn474, Ser209, Thr304 and Arg108 residues participated almost in all of the metabolically active conformations, suggesting that these residues are important for the regioselectivity and positioning of the substrate. The same was observed for the Asp313, Phe226, Phe256 and Phe260 residues of the CYP1A2 isoform. The results presented here afford new opportunities for improving the application of dietary flavonoids for the prevention of chronic disorders, as they're capable to be readily metabolized by CYP450 family.     

Inhibition of human recombinant cytochromes P450 CYP1A1 and CYP1B1 by trans-resveratrol methyl ethers

CYP1A1 and CYP1B1 are the inducible forms of cytochrome P450 expressed in extrahepatic tissues, which are responsible for the biotransformation of polycyclic aromatic hydrocarbons, heterocyclic amines and estradiol to the carcinogenic intermediates. The aim of our research was to determine and compare the inhibitory effect of naturally occurring analogues of trans-resveratrol on the catalytic activities of human recombinant CYP1A1 and CYP1B1. Pinostilbene (3,4'-dihydroxy-5-methoxystilbene), desoxyrhapontigenin (3,5-dihydroxy-4'-methoxystilbene), and pterostilbene (3,5-dimethoxy-4'-hydroxystilbene) appeared to be very potent inhibitors of CYP1A1 catalytic activity with Ki values of 0.13, 0.16 and 0.57 microM, respectively. Results from this study indicate that trans-resveratrol analogues in which the hydroxy groups are substituted by methoxy groups exhibit a remarkably stronger inhibitory effect towards CYP1A1 in comparison to the parent compound. On the contrary, the potency of pinostilbene, desoxyrhapontigenin and pterostilbene towards CYP1B1 with Ki values of 0.90, 2.06 and 0.91 microM, respectively, was comparable to that of resveratrol. It appears that between these analogues, inhibition of CYP1A1 and CYP1B1 catalytic activities does not vary much regardless of the number and position of methylether substitution. The results suggest that the trans-resveratrol analogues: pinostilbene, desoxyrhapontigenin and pterostilbene, which occur in some food plants, might be considered as promising chemopreventive agents.     

Induced CYP1A2 activity as a phenotypic biomarker in humans highly exposed to certain PCBs/PCDFs

Polychlorinated biphenyls (PCBs), dibenzo-p-dioxins (PCDDs), and dibenzofurans (PCDFs) continue to be a worldwide public health concern due to their levels in the environment and humans, and associated adverse health effects. In animals, one of the most sensitive effects of physiologically significant body burdens has been the induction of cytochrome P450 1 (CYP1) family of enzymes. This study examined the capacity of CYP1 enzyme induction to be a biomarker of exposure to a mixture of PCBs and PCDFs and of adverse human health effects. We followed a group of people highly exposed to PCBs and PCDFs due to accidental ingestion of contaminated rice oil, the Yucheng cohort. A total of 174 Yucheng and 134 control subjects were studied. The caffeine breath test, a monitor of CYP1A2 activity, was conducted, and its results were compared to serum levels of chemicals and the subjects' medical history. Total dioxin serum toxic equivalency (TEQ) in the Yucheng cohort and their controls were 577 +/- 393 ppt lipid and 21 ppt lipid, respectively. CYP1A2 activity was elevated in Yucheng subjects more than 2-fold and correlated with serum TEQ (R2= 0.62). Manifestations like chloracne, fingernail abnormalities, and headaches were well predicted by P4501A2 activity. It is concluded that CYP1A2 induction seen in the Yucheng cohort is an excellent biomarker of exposure and human health effects in individual subjects and cohort.     

Lipid peroxidation in the rat liver in mild forms of vitamin A deficiency

The content of malonic dialdehyde (MDA) and diene conjugates (DC) in homogenates and microsomes of the liver, as well as the rate of spontaneous NADPH- and ascorbate-dependent lipid peroxidation (LPO) were studied in hepatic microsomes of rats with nonsevere types of experimental vitamin A deficiency: a) rapid and synchronously induced vitamin A deficiency developing in the animals with a pre-exhausted hepatic retinol reserve, which received retinoic acid, after the retinoic acid administration was stopped; b) subnormal provision with retinol by the injection to rats of minimal vitamin doses (7 IU/day). Both experimental models were characterized by a drastic exhaustion of vitamin A reserve in the liver, manifest disorders in the growth and appetite of the animals being absent. In both models the DC content and NADPH-dependent LPO rate in the liver of rats with vitamin A deficiency proved to be decreased as compared to the control, while the rate of ascorbate-dependent LPO was little changed. The study of the fatty-acid composition of microsomal lipids in synchronous vitamin A deficiency revealed decreased arachidonic acid and increased palmitic acid levels as compared to the controls. A conclusion has been made that the results obtained confirm the earlier data on the lowering of LPO intensity in severe types of retinol deficiency, and evidence specificity of shifts associated with deficiency of retinol proper, but not with secondary metabolic disorders due to the changes in nutrition and growth of animals.     

In vitro and in vivo assessment of cytochrome P450-mediated herb–drug interaction of Ssang-hwa-tang

We have evaluated the herb–drug interaction potential of Ssang-hwa-tang (SHT) mediated by cytochrome P450 (CYP) inhibition/induction. Further, the effects of fermentation on the CYP-mediated herb–drug interaction potential were determined. SHT showed inhibitory activity toward CYP1A2, but not 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, and 3A4 in human liver microsomes. The results of the enzyme kinetic study suggested that the SHT-induced CYP1A2 inhibition is mixed reversible inhibition. The hepatic CYP expression and activity in rats treated with SHT were examined. The expression/activity of CYP2E1 increased as a result of SHT extract treatment (P < 0.005 or P < 0.001, respectively), which raises the possibility that SHT may increase the toxicity of environmental toxicants through the elevation of CYP2E1-mediated metabolic activation. SHT fermentation using Lactobacillus fermentum or Lactobacillus gasseri resulted in attenuation of the SHT-induced CYP1A2 inhibition, but not CYP2E1 induction, suggesting that changes in the chemical composition of SHT through fermentation can affect the inhibition of CYP1A2 activity.     

The use of Yarrowia lipolytica for the expression of human cytochrome P450 CYP1A1

Cytochromes P450 constitute a superfamily of haem-thiolate mono-oxygenases that are involved in the oxidative metabolism of lipophilic subtrates. These enzymes require association with cytochrome P450 reductase (CPR) to achieve optimal activities. We have expressed human cytochrome P450 CYP1A1 under the POX2 promoter (pPOX2-CYP1A1) in Y. lipolytica, with or without overproduction of Y. lipolytica CPR expressed under the ICL promoter (pICL-CPR) or the POX2 promoter (pPOX2-CPR). Activity of cytochrome CYP1A1 was analysed by conversion of hydroxyresorufin to resorufin. Strain JMY330 and JMY330-CPR present no activity, the monocopy cytochrome CYP1A1 integrant JMY331 and JMY331-CPR derivatives present an average activity of 32.0 pM/min/dw and 48.3 and 64.6 pM/min/dw for pICL-CPR and pPOX2-CPR, respectively. Increase of CPR expression resulted in about two-fold higher activity. The multicopy 1A1 integrant JMY339 and JMY339-CPR derivatives present an activity of 129 pM/min/dw and 815-1845 pM/min/dw, respectively. Increase of CPR expression resulted in 6.3-12.8-fold higher activity, depending on the CPR transformant. We observed a 50-fold increase of activity between the monocopy integrant JMY331 as compared to the multicopies integrant JMY339-CPR in which CPR was overexpressed.     

丹参素上调LCAT和CYP7A1的表达对高血脂大鼠脂质紊乱的调节作用

为探究丹参素(Salvianic acid,SA)对高脂血症大鼠的调节作用及机制,将40只SD大鼠随机分为正常对照组(S,n=10)、高血脂模型组(M,n=10)、丹参素高剂量组(SAH,n=10)和低剂量组(SAL,n=10)。高脂饲料喂养建立高脂血症模型,边造模边灌胃给药。8周后,采用ELISA法测定各组大鼠血清中总胆固醇(TC)、甘油三脂(TG)、低密度脂蛋白胆固醇(LDL-C)、高密度脂蛋白胆固醇(HDL-C)、载脂蛋白AI(apo AI)和载脂蛋白B(apo B)的含量;RT-PCR和Western-blot检测卵磷脂胆固醇脂酰基转移酶(LCAT)和胆固醇7α-羟化酶(CYP7A1)的表达;HE染色检测肝脏组织病理学形态。结果发现丹参素能明显降低模型大鼠TC、TG、LDL-C和apo B含量(p0.05),上调HDL-C、apo AI、LCAT和CYP7A1的表达,同时减轻肝脏脂肪病变,高剂量组效果更显著。因此,丹参素对高血脂大鼠血脂紊乱的调节作用机制可能与调控脂质代谢载脂蛋白、LCAT和CYP7A1的表达有关。     

Modulation of biotransformation and elimination systems by BM-21, an aqueous ethanolic extract from Thalassia testudinum,and thalassiolin B on human hepatocytes

BM-21 is an extract obtained from Thalassia testudinum marine plant with pharmacological properties. The effects of BM-21 and thalassiolin B (TB), its main component, on enzyme and transport proteins involved in drug metabolism and excretion in human cultured hepatocytes were evaluated. Cells were exposed for 48 h to sub-cytotoxic concentrations of BM-21 or TB. Effects on P450 isoforms revealed significant reductions of CYP1A2, 3A4 and 2D6 activities (up to 56%, 66% and 44% inhibition, respectively) after exposition to BM-21, no changes on CYP2A6 and 2C9 activities. TB produced a concentration-dependent reduction of all P450 activities. In addition, a decrease in total UGT and UGT2B7 activities was found at 250 μg/mL BM-21, while UGT1A1 and 1A9 were significantly reduced (50 μg/mL). TB only inhibited significantly UGT1A9 activity. Both products were able to reduce P-gp activity in treated hepatocytes. Quantification of specific mRNAs revealed a reduction in CYP3A4 and 3A5 mRNAs content and an increase in CYP1A1 and 1A2 mRNAs. No appreciable effects in the levels of CYP2A6, 2B6, 2C9, 2C19, 2D6, 2E1, UGT1A1, UGT1A9 and ABCB1 (P-gp) were found. BM-21 inhibited P450, UGTs and P-gp activities in human hepatocytes; therefore, it should be examined for potential pharmacokinetic drug interactions in vivo.     

Short communication: insulin alters hepatic progesterone catabolic enzymes cytochrome P450 2C and 3A in dairy cows

High proportions of embryonic and early fetal losses in dairy cattle are associated with low peripheral concentrations of progesterone, which could result from increased catabolism, decreased production, or both. Progesterone catabolism occurs primarily in the liver via the cytochrome P450 2C (CYP2C) and cytochrome P450 3A (CYP3A) subfamilies (EC 1.14.14.1; unspecific monooxygenases). Recent observations from our laboratory have shown that the fractional rate constant of progesterone decay can be dramatically reduced by insulin because of a decrease in hepatic CYP2C and CYP3A activity. Little information exists on the regulation of progesterone catabolic enzymes in dairy cows. We hypothesized that elevated insulin concentrations would down-regulate hepatic CYP2C and CYP3A mRNA; therefore, our objectives were to determine the relative abundance of hepatic CYP2C and CYP3A mRNA in dairy cows in response to elevated concentrations of insulin. In the first experiment, 17 mature Holstein cows were drenched daily with 500 mL of water (n = 10) or propylene glycol (a gluconeogenic substrate; n = 7) from 10 d before their expected calving date until d 25 postpartum. Cows drenched with propylene glycol had a 30% increase in peripheral concentrations of insulin. Liver biopsies were collected on d 25 postpartum to determine the relative abundance of CYP2C and CYP3A mRNA. In the second experiment, 19 mature, lactating Holstein cows were randomly assigned to a hyperinsulinemic-euglycemic clamp (0.3 or 1.0 μg of insulin/kg of BW per h; n = 6 each) or remained as controls (saline infused; n = 7) for 96 h beginning on d 10 postpartum. Insulin infusion resulted in a 2.6- or 8- fold increase in peripheral concentrations of insulin, respectively. On d 14 postpartum, a liver biopsy was collected to determine CYP2C and CYP3A mRNA abundance. In experiment 1, the relative abundance of CYP2C mRNA in cows treated with propylene glycol did not differ from controls; however, the relative abundance of CYP3A mRNA in the propylene glycol group was 63% that of controls. For experiment 2, there was a dose-dependent decrease in the relative abundance of both CYP2C and CYP3A mRNA with increasing dosage of insulin. In conclusion, this study demonstrated that, in the cow, either providing a gluconeogenic feed-stuff or treatment with insulin decreased the abundance of mRNA for enzymes responsible for hepatic progesterone catabolism.     

Effects of diet enriched with restructured meats,containing Himanthalia elongata, on hypercholesterolaemic induction,CYP7A1 expression and antioxidant enzyme activity and expression in growing rats

Meat and pork consumptions are very high in Spain. Seaweeds are rich in fibre, minerals, and bioactive substances. Due to the growing demand for healthier meats, this work studied the effect of diets containing restructured pork (RP) enriched with Himanthalia elongata (Sea Spaghetti) on: (1) cholesterolaemia; (2) liver cytochrome P450 7A1 (CYP7A1) expression; (3) liver antioxidant enzyme activities and gene expression; (4) the liver antioxidant substrate concentrations. Four groups of 10 Wistar rats each were fed a mix of 85% AIN-93 M rodent diet and 15% freeze-dried RM for 35 days. The control group (C) consumed control RP; the Sea Spaghetti (SS) group, RP with 5% Sea Spaghetti. Animals on added cholesterol diets (CholC and CholSS) consumed their basal C and SS diets enriched with cholesterol and cholic acid as hypercholesterolaemic agent. Food intake was significantly affected by the alga × cholesterol interaction and by dietary cholesterol (both p < 0.001). Plasma cholesterol was significantly affected by the cholesterol × alga interaction (p < 0.05). CholC rats showed significantly higher plasma cholesterol (p < 0.001) than did their C counterparts, whilst serum cholesterol of CholSS was significantly lower (p < 0.05) than that of CholC. The glutathione peroxide (GSSG) concentrations and all mRNA expressions were significantly affected by the cholesterol × alga interaction (at least p < 0.05). SS vs C group showed significant (at least p < 0.05) increases in superoxide dismutase (Mn-SOD and Cu,Zn-SOD), glutathione peroxidase (GPx) and decrease of glutathione reductase (GR) expressions, and increased GR activity, GSSG and the redox index. CholSS vs CholC showed significant (at least p < 0.05) increases of CYP7A1, GR and Cu,Zn-SOD expression but decreases in catalase, Mn-SOD and GPx expression, and increase of GR activity. In conclusion, Sea Spaghetti could be widely used in RP design. Its addition to non-cholesterol enriched RP diet reduced oxidation mechanisms. SS-RP partially blocked the effect of the hypercholesterolaemic agent, giving rise to a new balance of the antioxidant enzyme expression.     

无斑鹞鲼蛋白质对大鼠胆固醇代谢的影响

目的：探讨无斑鹞鲼蛋白（Aetobatus flagelum protein,AFP）对高胆固醇模型大鼠胆固醇代谢的影响,并对其可能的作用机制加以研究。方法：5 周龄雄性Sprague-Dawley（SD）大鼠,适应性饲养1 周后,按体质量随机分为酪蛋白对照组、5% AFP组、10% AFP组,分别给予高胆固醇饲料及分别添加5%和10% AFP的高胆固醇饲料。28 d后测定大鼠血清总胆固醇（total cholesterol,TC）、高密度脂蛋白胆固醇（high density lipoprotein cholesterol,HDL-C）水平,肝脏TC、游离胆固醇水平,粪便中胆汁酸和中性固醇含量以及肝脏3-羟基-3-甲基戊二酸单酰辅酶A（3-hydroxy-3-methyl glutaryl coenzyme A reductase,HMG-CoA）、胆固醇酰基转移酶2（acyl coenzyme Acholesterolacyltransferase 2,ACAT2）、胆固醇7α-羟化酶1（cholesterol 7α-hydroxylase 1,CYP7A1）的mRNA表达量。结果：与酪蛋白对照组相比,无斑鹞鲼蛋白可显著降低大鼠血清和肝脏中的TC含量（P＜0.05）,明显增加血清HDL-C含量（P＜0.05）,极显著降低大鼠动脉粥样硬化指数（P＜0.01）。此外,无斑鹞鲼蛋白可明显增加大鼠粪便中胆汁酸和中性固醇的排出量（P＜0.05）,并可降低大鼠肝脏中ACAT2 mRNA的表达量（P＜0.05）,增加CYP7A1 mRNA的表达量（P＜0.05）,而对HMG-CoA mRNA的作用不显著。结论：无斑鹞鲼蛋白可明显降低大鼠血清和肝脏中的TC含量,降低大鼠血清动脉粥样硬化指数,其作用机制主要与增加肝脏内胆固醇的分解代谢以及促进粪便中性固醇和胆汁酸的排出有关。     

Potent inhibition of human cytochrome P450 3A4, 2D6, and 2C9 isoenzymes by grapefruit juice and its furocoumarins

ABSTRACT:  The cytochrome P450 enzyme family is the most abundant and responsible for the metabolism of more than 60% of currently marketed drugs and is considered central in many clinically important drug interactions. Seven different grapefruit and pummelo juices as well as 5 furocoumarins isolated from grapefruit juice were evaluated at different concentration on cytochrome P450 3A4 (CYP3A4), cytochrome P450 2C9 (CYP2C9), and cytochrome P450 2D6 (CYP2D6) isoenzyme activity. Grapefruit and pummelo juices were found to be potent inhibitors of cytochrome CYP3A4 and CYP2C9 isoenzymes at 25% concentration, while CYP2D6 is inhibited significantly low at all the tested concentration of juices ( P < 0.05). Among the 5 furocoumarins tested, the inhibitory potency was in the order of paradisin A > dihydroxybergamottin > bergamottin > bergaptol > geranylcoumarin at 0.1 μM to 0.1 mM concentrations. The IC₅₀ value was lowest for paradisin A for CYP3A4 with 0.11 μM followed by DHB for CYP2C9 with 1.58 μM.