Oxidative activities of heterologously expressed CYP107B1 and CYP105D1 in whole-cell biotransformation using Streptomyces lividans TK24

Cytochrome P450 enzymes are a major class of biocatalysts related to the oxidative metabolism of many drugs, assisted by electron transfer partners. The functional expression of the P450 gene in a heterologous host will lead to efficient biotransformation and biodegradation, which are useful in pharmaceutical improvement or environmental cleanup. The soluble cytochrome P450 monooxygenase systems CYP105D1 and CYP107B1 involved in the biotransformation of some xenobiotics, such as secondary metabolites or environmental pollutants, were expressed in Streptomyces lividans TK24 with the Streptomyces expression vector pIJ6021. In whole-cell biotransformation assay using these recombinant strains, the oxidative dealkylation of 7-ethoxycoumarin was detected without any foreign redox partners in the case of CYP107B1, while the activity of CYP105D1 was not monitored until this gene was coexpressed with the ferredoxin gene located downstream of the CYP105D1 gene, and the ferredoxin reductase gene SCF 15.02 from Streptomyces coelicolor A3(II). This result suggests that CYP107B1 is capable of utilizing an endogenous electron transfer partner from the host but not CYP105D1, and that CYP105D1 is complemented by some redox partner imported from closely related strains.     

罗布麻茶黄酮提取物对小鼠CYP2E1的影响

目的 CYP2E1是肝脏中主要的药物代谢酶,在肝脏疾病发生及发展中发挥重要的作用。本实验分别通过体内、体外实验,研究罗布麻茶黄酮提取物对小鼠CYP2E1活性和表达的影响,为评价罗布麻茶对肝脏的保护作用和药物的联合使用提供理论依据。方法将30只昆明小鼠随机分为空白对照组、黄酮提取物低剂量组和高剂量组(50和100 mg/kg),连续灌胃给药10天。采用探针药物法测定小鼠肝脏微粒体中CYP2E1催化对硝基苯酚代谢的活力。采用Western Blot法检测肝脏微粒体中CYP2E1蛋白的表达情况。体外抑制实验中,考察不同浓度的黄酮提取物对CYP2E1的抑制作用,计算半数抑制浓度(IC_(50))值。结果灌服高剂量黄酮提取物(100 mg/kg)后,小鼠体内CYP2E1酶活性和表达量均显著降低,黄酮提取物对CYP2E1的IC_(50)值为128.4μg/mL。结论罗布麻茶黄酮提取物对小鼠CYP2E1有抑制作用。     

The effect of solar radiation on the flavonol content in broccoli inflorescence

The effect of solar radiation on the quercetin and kaempferol contents in the inflorescence of three broccoli cultivars (‘Lord’, ‘Marathon’ and ‘Fiesta’) was investigated from 1999 to 2001. Great differences in the contents of both flavonols, dependent on growing time and cultivar, were found. Quercetin and kaempferol contents varied from 14.3 to 81.0 mg kg⁻¹ f.w. and from 35.9 to 213 mg kg⁻¹ f.w., respectively. Inflorescences of the cultivar ‘Lord’ were characterised by the highest mean content of quercetin and those of cultivar ‘Fiesta’ of kaempferol. The contents of both flavonols were highly positively correlated with total solar radiation in the period from planting to the harvest of broccoli inflorescences.     

Induction of cell apoptosis in 3T3-L1 pre-adipocytes by flavonoids is associated with their antioxidant activity

Obesity is biologically characterized at the cellular level by an increase in the number and size of adipocytes differentiated from fibroblastic pre-adipocytes in adipose tissue. In this study, we focused on the relationship between the influence of flavonoids on cell population growth and their antioxidant activity. The results showed that the inhibition of flavonoids (naringenin, rutin, hesperidin, resveratrol, naringin and quercetin) on 3T3-L1 pre-adipocytes was 28.3, 8.1, 11.1, 33.2, 5.6 and 71.5%, respectively. In oxygen radical absorbance capacity (ORAC) assay, quercetin had the highest ORAC(ROO) value among the six flavonoids tested. Apoptosis assays showed that quercetin increased apoptotic cells in time- and dose-dependent manner. Treatment of cells with quercetin decreased the mitochondrial membrane potential in the courses of time and dose. The cell apoptosis/necrosis assay showed that quercetin increased the number of apoptotic cells, but not necrotic cells. Quercetin treatment of cells caused a significant time- and dose-dependent increase in the caspase-3 activity. Western analysis indicated that treatment of quercetin markedly down-regulated PARP and Bcl-2 proteins, and activated caspase-3, Bax, and Bak proteins. These results indicate that quercetin efficiently inhibits cell population growth and induction of apoptosis in 3T3-L1 pre-adipocytes.     

不同生理状态下人参总皂苷对HepG2细胞CYP2B6 mRNA和蛋白表达的影响

本文研究人参总皂苷在正常和炎症状态下对HepG2细胞CYP2B6 mRNA和蛋白表达的影响。采用索式提取法提取人参总皂苷,Real-Time PCR及Western Blot技术检测细胞中CYP2B6 mRNA和蛋白表达。结果显示,正常状态下人参总皂苷低(15 g/mL)、中(60 g/mL)与高(240 g/mL)剂量均显著诱导CYP2B6 mRNA表达(p0.05),中、高剂量均显著诱导CYP2B6蛋白的表达(p0.05)。使用LPS孵育细胞制造炎症模型,LPS对CYP2B6 mRNA表达无影响,但显著诱导CYP2B6蛋白的表达(p0.05)。使用人参总皂苷和LPS共同孵育细胞后,炎症状态下人参总皂苷中、高剂量对CYP2B6 mRNA的表达仍有显著诱导效果(p0.05),低和中剂量则均能显著诱导CYP2B6蛋白的表达,但高剂量则对CYP2B6的蛋白表达则呈现抑制作用(p0.05)。提示炎症状态能够改变人参总皂苷对CYP2B6 mRNA和蛋白的表达,因此联合用药时,应考虑不同生理状态下人参对代谢性药物相互作用的影响。     

Genotypic and climatic influences on the concentration and composition of flavonoids in kale (Brassica oleracea var. sabellica)

The aim of this study was to determine the composition and concentration of flavonoid aglycones in kale, the dependence on genotype and their interaction with decreasing temperature and global radiation. Eight kale cultivars, comprising hybrid and traditional, old cultivars, were grown in a field experiment and harvested four times at 4-week intervals. The traditional, old cultivars in particular contained high concentrations of flavonoids. In all of the investigated cultivars, kaempferol was the main flavonoid aglycone, followed by quercetin and isorhamnetin, which was quantified in six of the eight cultivars. Furthermore, in six of the eight cultivars, the total concentration of flavonoids remained unchanged with decreasing temperature and global radiation. The quercetin concentration increased in five of these six cultivars, whereas the kaempferol concentration decreased. Interestingly, the quercetin-to-kaempferol ratio increased in all of the investigated cultivars, despite the fact that the radiation level decreased, suggesting that the impact of the decline in temperature could be responsible for this effect.     

Protective effects of protein hydrolysate from marine microalgae Navicula incerta on ethanol-induced toxicity in HepG2/CYP2E1 cells

Ethanol is independently known to cause tissue damage through various mechanisms. This study was designed to evaluate the protective effect of marine microalgae, Navicula incerta protein enzymatic hydrolysates (NEHs) against ethanol-induced hepatotoxicity in HepG2 cells transfected with human CYP2E1. Induction of CYP2E1 by ethanol is one of the central pathways by which ethanol generates a state of oxidative stress in hepatocytes. When the alcohol-induced cells were treated with NEHs at various concentrations, there was a dose-dependent decrease of gamma-glutamyl transpeptidase (GGT) activity in the culture media and loss of cell viability. Among the NEHs constituents the hydrolysates which were obtained by papain (P-NEH), pronase-E (PR-NEH) and α-chymotrypsin (A-NEH) activity attenuated the ethanol cytotoxicity effectively, respectively. The activity appeared to be a GGT inhibitor. Therefore, the cytoprotective effects at alcohol-induced HepG2/CYP2E1 cells could be attributed to the inhibition of GGT activity by NEHs. This study suggests that NEHs have enough potential to be considered as highly active compounds against ethanol toxicity which leads NEHs to be significant nutraceuticals.     

牛磺酸对HepG2细胞胆固醇水平的影响

目的：探讨牛磺酸对人肝癌细胞HepG2总胆固醇（total cholesterol,TC）、游离胆固醇（free cholesterol,FC）、胆固醇酯（cholesterol ester,EC）水平及对胆固醇代谢限速酶--胆固醇7α-羟化酶（cholesterol7α-hydroxylase,CYP7A1）蛋白表达的影响。方法：在培养液中添加终浓度为0.1、1、10、20 mmol/L的牛磺酸,分别在培养24、48 h后测定细胞内TC、FC及EC水平;在培养液中添加终浓度为20 mmol/L的牛磺酸,分别培养12、24、48、72 h后检测CYP7A1蛋白表达水平。结果：1、10、20 mmol/L的牛磺酸均可使HepG2细胞内TC、FC水平明显下降,且与牛磺酸浓度呈正比,但EC水平在牛磺酸浓度达1 mmol/L后即不随牛磺酸浓度的增加而进一步降低;培养48 h后HepG2细胞TC、FC水平的降低幅度大于24 h时;20 mmol/L牛磺酸使FC含量减少的作用强于对EC减少的作用;培养24 h后HepG2细胞中CYP7A1蛋白表达最强。结论：牛磺酸可增强CYP7A1蛋白表达,促进HepG2细胞内的胆固醇代谢,胆固醇降低程度与牛磺酸浓度及作用时间相关,作用48 h的效果明显优于24 h,且牛磺酸主要是降低了细胞内的FC水平。     

牛磺酸对肥胖大鼠甘油三酯和胆固醇代谢的影响

目的 探讨长期经饮水方式补充牛磺酸对高脂膳食所致肥胖大鼠降脂减重作用,对甘油三酯合成关键分子和胆固醇7α羟化酶(cholesterol 7α-hydroxylase, CYP7A1)表达调控关键分子的影响。方法 根据血清胆固醇水平和体重, SD大鼠被分为3组,为对照组(normal, N)饲喂基础维持饲料,肥胖组(high fat, HF)和牛磺酸组(high fat taurine, HFT)饲喂高脂饲料。N组和HF组自由饮水,而HFT组给予牛磺酸溶液代替水,持续12周。结果 与HF组相比,给予牛磺酸的HFT组大鼠体重、附睾周围脂肪重量、血清总胆固醇和低密度脂蛋白胆固醇、游离脂肪酸、固醇调节元件结合蛋白-1c (sterol regulatory element-binding protein-1c, SREBP-1c)和脂肪酸合成酶(fatty acid synthase, FAS)基因表达显著降低,粪便胆汁酸水平、脂蛋白脂肪酶(lipoprotein lipase, LPL)活性以及CYP7A1和Ser63p-c-Jun的蛋白表达显著增加,但肝细胞核因子4α(hepatocyte...     

Study of flavonoids in aqueous spinach extract using positive electrospray ionisation tandem quadrupole mass spectrometry

The presence of three flavonols (quercetin, kaempferol, myricetin) and two flavones (apigenin, luteolin) were investigated in the extract of fresh spinach leaves. Aqueous spinach extracts were prepared with a leaf/water ratio of 1:2 at 80 °C for 30 min stirring. Ferric ammonium sulphate method was used for measuring total polyphenols in the extracts and expressed as catechins and tannic acid equivalents. The flavonoids glycosides in the extract were hydrolysed to their aglycons with 1.2 M HCl in boiling 50% water methanol solution. The resulting aglycons were identified and quantified by a C18 reverse phase high-performance liquid chromatography (RP-HPLC). Furthermore, the results were confirmed by HPLC coupled to an electrospray ionisation tandem triple-quadrupole mass spectrometer ESI-MS performing low-energy collision induced dissociation (CID-MS/MS) in the collision cell (HPLC-ESI-MS/MS). Analyses were made in the multiple reaction monitory (MRM) mode. Results showed that total polyphenols contents in fresh spinach leaves were 270 mg kg⁻¹ and 390 mg kg⁻¹ as tannic acid and catechin equivalents, respectively, in which, major flavonoids aglycons were apigenin (170 mg kg⁻¹), quercetin (50 mg kg⁻¹) and kaempferol (30 mg kg⁻¹).     

Effects of sex, weight, diet and hCG administration on levels of skatole and indole in the liver and hepatic activities of cytochromes P4502E1 and P4502A6 in pigs

Cytochromes P4502E1 (CYP2E1) and P4502A6 (CYP2A6) catalyse metabolic reactions of skatole and indole metabolism. The objectives of this study were as follows: to evaluate whether activities of CYP2E1 and CYP2A6 in pigs of two live weights (LW) differ between males and females; to investigate whether activities of CYP2E1 and CYP2A6 are affected by hCG stimulation; and to investigate whether the levels of skatole and indole in the liver and the activities of CYP2E1 and CYP2A6 are affected by raw potato starch (RPS). Female pigs expressed higher CYP2A6 activity at 90kg LW, and higher CYP2E1 activity at 115kg LW compared to male pigs. Skatole levels in the liver were higher in male pigs than in female pigs at both LW, whereas indole levels were higher in males only at 115 kg LW. Neither levels of indolic compounds in the liver nor enzyme activities were affected by hCG stimulation. The inclusion of RPS in the diet reduced skatole levels in the liver in both sexes and increased CYP2A6 activity in female pigs. It was concluded that the incidence of boar taint may depend on both skatole amount, which reach the liver, and the activities of enzymes involved in skatole metabolism, which may vary depending on sex, live weight, and diet.     

Docosahexaenoic acid downregulates phenobarbital-induced cytochrome P450 2B1 gene expression in rat primary hepatocytes via the c-Jun NH2-terminal kinase mitogen-activated protein kinase pathway

Mitogen-activated protein kinase (MAPK) pathways play central roles in the transduction of extracellular stimuli into cells and the regulation of expression of numerous genes. Docosahexaenoic acid (DHA) was shown to be involved in the regulation of expression of drug metabolizing enzymes (DMEs) in rat primary hepatocytes in response to xenobiotics. Cytochrome P450 2B1 (CYP 2B1) is a DME that is dramatically induced by phenobarbital-type inducers. The constitutive androstane receptor (CAR) plays a critical role in regulating the expression of DMEs, and the phosphorylation/dephosphorylation of CAR is an important event in CYP 2B1 expression. In the present study, we determined the effect of DHA on MAPK transactivation and its role in CYP 2B1 expression induced by phenobarbital. c-Jun NH2-terminal kinase (JNK) JNK1/2 and ERK1/2 were activated by phenobarbital in a dose-dependent manner. DHA (100 muM) inhibited JNK1/2 and ERK2 activation induced by phenobarbital in a time-dependent manner. Both SP600125 (a JNK inhibitor) and SB203580 (a p38 MAPK inhibitor) inhibited CYP 2B1 protein and mRNA expression induced by phenobarbital. SB203580 significantly increased the intracellular 3'-5'-cyclic adenosine monophosphate (cAMP) concentration compared with a control group (p < 0.05). Our results suggest that inhibition of JNK activation by DHA is at least part of the mechanisms of DHA's downregulation of CYP 2B1 expression induced by phenobarbital.     

牛磺酸降低高胆固醇HepG2细胞内胆固醇的作用机制研究

目的:探讨牛磺酸对高胆固醇Hep G2细胞胆固醇降解作用及其机制。方法:以0.02 mmol/L胆固醇加入培养液中建立高胆固醇细胞模型后,分别加入1、10、20 mmol/L牛磺酸培养48 h,测定细胞内总胆固醇（TC）、游离胆固醇（FC）、胆固醇酯（EC）及细胞内总胆汁酸（TBAc）和培养液总胆汁酸（TBAm）水平;并检测20 mmol/L牛磺酸作用24 h后细胞CYP7A1及其调控分子的蛋白表达水平。结果:10 mmol/L和20 mmol/L牛磺酸作用48 h可显著降低细胞内TC、FC和EC（p<0.01）,同时升高了TBAc和TBAm（p<0.05或p<0.01）;20 mmol/L牛磺酸作用24 h时使CYP7A1显著增加（p<0.05）;20 mmol/L牛磺酸作用24 h后MAPK、ERK、JNK、c-jun和p-c-jun表达明显降低（p<0.05）,而对HNF4α则无显著影响。结论:牛磺酸可抑制高胆固醇Hep G2细胞MAPK/ERK和MAPK/JNK表达水平,降低c-jun蛋白表达及c-jun磷酸化水平,促进CYP7A1蛋白表达升高,从而加速胆固醇转化为胆汁酸。      

酒精对人原代培养肝细胞的氧化损伤与CYP 2E1关系的研究

目的 研究急性酒精暴露下对人原代培养肝细胞中CYP 2E1依赖的毒性作用和氧化损伤。方法 分离培养人原代肝细胞，以25-100mmol/L乙醇作用于人原代肝细胞9h及100mmol／L乙醇作用于人原代肝细胞0～24h后，检测人原代肝细胞中CYP 2E1的含量，并研100mmol/L乙醇作用于人原代肝细胞0～24h后，天冬氨酸转胺酶(aspartate transaminase，AST)的释放量及肝细胞中谷胱甘肽(Glutathione，GSH)、丙二醛(Malonclialdehyde，MDA)的含量。结果 急性酒精暴露导致人原代肝细胞中CYP 2E1的释放增加，并呈明显的剂量效应和时间效应关系；在100mmol/L乙醇作用下，AST和MDA明显升高，在0～24h内呈明显的时间效应关系，而GSH含量在6h后明显降低。结论 100mmol／L乙醇急性暴露可导致人原代培养肝细胞明显的氧化损伤，这种损伤与CYP 2E1活性的变化直接相关。     

Dietary calcium decreases plasma cholesterol by down‐regulation of intestinal Niemann–Pick C1 like 1 and microsomal triacylglycerol transport protein and up‐regulation of CYP7A1 and ABCG 5/8 in hamsters

Scope: It has been shown that calcium supplementation favorably modifies plasma lipoprotein profile in postmenopausal women. The present study investigated the interaction of dietary calcium with genes of transporters, receptors and enzymes involved in cholesterol metabolism. Methods and results: Forty‐eight ovariectomized hamsters were fed one of the four diets containing 0, 2, 6 and 8 g calcium per kg. Plasma total cholesterol (TC), triacylglycerols (TG), and non‐high density lipoprotein cholesterol were dose‐dependently decreased, whereas high‐density lipoprotein cholesterol (HDL‐C) was dose‐dependently increased with the increasing dietary calcium levels. Dietary calcium had no effect on protein mass of hepatic sterol regulatory element binding protein‐2 (SREBP), liver X receptor‐alpha (LXR), 3‐hydroxy‐3‐methylglutaryl‐CoA reductase (HMGR), LDL receptor (LDLR) and cholesterol‐7α‐hydroxylase (CYP7A1). However, dietary calcium up‐regulated the mRNA levels of hepatic CYP7A1 and intestinal ATP binding cassette transporters (ABCG5/8) whereas it down‐regulated the intestinal Niemann‐Pick C1 like 1 (NPC1L1) and microsomal triacylglycerol transport protein (MTP). In addition, dietary calcium increased the activity of intestinal acyl coenzyme A: cholesterol acyltransferase 2, while it decreased plasma cholesteryl ester transport protein (CETP). Conclusion: Beneficial modification of lipoprotein profile by dietary calcium was mediated by sequestering bile acid absorption and enhancing excretion of fecal cholesterol, via up‐regulation of mRNA CYP7A1 and intestinal ABCG 5/8 with down‐regulation of mRNA NPC1L1 and MTP.     

Protective effects of triterpenoids from Ganoderma resinaceum on H2O2-induced toxicity in HepG2 cells

Ganoderma resinaceum Boud. (Polyporeseae) has long been used for antioxidant, immunoregulation and liver protection. From the fruiting bodies of G. resinaceum, eight new lanostanoids, lucidones D–G (1–4), 7-oxo-ganoderic acid Z₂ (5), 7-oxo-ganoderic acid Z₃ (6), ganoderesin A (7), and ganoderesin B (8), together with six known lanostanoids (9–14) were isolated. The structures of new compounds were elucidated through extensive spectroscopic analysis. In an in vitro model, ganoderesin B (8), ganoderol B (10) and lucidone A (11) showed inhibitory effects against the increase of ALT and AST levels in HepG2 cells induced by H₂O₂ compared to a control group in the range of their maximum non-toxic concentration (MNTC). However, compounds 8, 10 and 11 displayed no anti-oxidant activities by DPPH assay. Meanwhile, activation for PXR (Pregnane X Receptor) of ganoderesin B (8), ganoderol B (10) and lucidone A (11) was evaluated; ganoderol (10) exhibited a vital activation for PXR-induced CYP3A4 expression. These results suggested that GTs (Ganoderma triterpenoids) exhibited hepatoprotective activities by lowering ALT and AST levels.