Tanshinone I increases CYP1A2 protein expression and enzyme activity in primary rat hepatocytes

This study investigated the effects of Danshen and its active ingredients on the protein expression and enzymatic activity of CYP1A2 in primary rat hepatocytes. The ethanolic extract of Danshen roots (containing mainly tanshinones) inhibited CYP1A2-catalyzed phenacetin O-deethylation (IC₅₀ = 24.6 μg/ml) in primary rat hepatocytes while the water extract containing mainly salvianolic acid B and danshenshu had no effect. Individual tanshinones such as cryptotanshinone, dihydrotanshinone, tanshinone IIA inhibited the CYP1A2-mediated metabolism with IC₅₀ values at 12.9, 17.4 and 31.9 μM, respectively. After 4-day treatment of the rat hepatocytes, the ethanolic extract of Danshen and tanshinone I increased rat CYP1A2 activity by 6.8- and 5.2-fold, respectively, with a concomitant up-regulation of CYP1A2 protein level by 13.5- and 6.5-fold, respectively. CYP1A2 induction correlated with the up-regulation of mRNA level of aryl hydrocarbon receptor (AhR), which suggested a positive feedback mechanism of tanshinone I-mediated CYP1A2 induction. A formulated Danshen pill (containing mainly danshensu and salvianolic acid B and the tanshinones) up-regulated CYP1A2 protein expression and enzyme activity, but danshensu and salvianolic acid B, when used individually, did not affect CYP1A2 activity. This study was the first report on the Janus action of the tanshinones on rat CYP1A2 activity.     

Potential involvement of serine/threonine protein phosphatases in apoptosis of HepG2 cells during selenite treatment

Selenium, an essential biological trace element present in both prokaryotic and eukaryotic cells, exerts its regulatory effect in a variety of cellular events, including cell growth, survival, and death. Selenium compunds have been shown in different cell lines to inhibit apoptosis by several mechanisms. Serine/threonine phosphatases (STPs) are potentially important in selenite-induced apoptosis because of their role in regulation of diverse set of cellular processes. In this study, the regulatory role of STPs in selenite-induced apoptosis has been implied by the use of two specific inhibitors: ocadaic acid and calyculin A. Our results show a decrease in cell density in HepG2 cells under selenite treatment. Resulting specific enzyme activities showed a concentration-dependent increase in all three phosphatase activities after 24 h in cells treated with 5 μM selenite and these activities decreased at 48 and 72 h. However, in cells treated with 10μM selenite, PP2A and PP2B decreased at 48 h, whereas PP2C activity did not change at this dose. In cells treated with 25μM, there was not a significant change in PP2C activity. These data suggest that the most specific response to selenite treatment was in PP2A and PP2B activities in a dose-dependent manner. Our results with OA and Cal-A further support the view that PP1 and PP2A might act as negative regulators of growth. With these data, we have first demonstrated the role of serine/threonine protein phosphatases in the signaling pathway of selenite-induced apoptosis and resulting cytotoxicity     

The role of protein tyrosine kinases in CYP1A1 induction by omeprazole and thiabendazole in rat hepatocytes

Benzimidazoles compounds like omeprazole (OME) and thiabendazole (TBZ) mediate CYP1A1 induction differently from classical aryl hydrocarbon receptor (AhR) ligands, 3-methylcholanthrene (3-MC) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). To clarify the involvement of an intracellular signal pathway in CYP1A1 induction by OME and TBZ, the TBZ, OME and 3-MC signal-transducing pathways were compared by using specific protein tyrosine kinase inhibitors in primary culture of rat hepatocytes. The effect of OME and TBZ (75-250 microM) on cytochrome P450 1A1 (CYP1A1) expression was therefore studied in primary cultures of rat hepatocytes after 24 h, 48 h and 72 h of exposure. Both compounds provoked a dose- and time-dependent increase in CYP1A1 (EROD activity, protein and mRNA levels), but OME was less effective at all the concentrations and times tested. The mechanism of benzimidazole-mediated induction of CYP1A1 was investigated by comparison with 3-MC, a prototypical AhR ligand. As expected, OME and TBZ were unable to displace [(3)H]-TCDD from its binding sites to the AhR in competitive binding studies. Moreover, classic tyrosine kinase inhibitor herbimycin A (HA) inhibited the two benzimidazoles-mediated CYP1A1 inductions, but only partially inhibited the 3-MC-mediated one. Another two tyrosine kinase inhibitors, Lavendustin A (LA) and genistein (GEN), had no effect on CYP1A1 induction by benzimidazoles and 3-MC. These results are consistent with the implication of a tyrosine kinase, most probably the Src tyrosine kinase, in the mechanism of CYP1A1 induction in rat hepatocytes.     

The involvement of protein kinases and protein phosphatases in signal transduction during neurotransmitter-induced stimulation of root water-pumping activity

In detached roots of etiolated maize (Zea mays L.) seedlings, neurotransmitters, adrenalin and noradrenalin, stimulated exudation by increasing the root pressure due to activation of its metabolic component. In these treatments, the osmotic pressure of the exudate was somewhat reduced. In contrast, a temperature coefficient Q₁₀ was increased, which as in accordance with the increase of the absolute value of the metabolic component and its proportion in the total root pressure. To obtain some information about transmitting the signals induced by adrenalin and noradrenalin action on water transport, we used two inhibitors of the most important and universal elements of signaling pathways, staurosporine (the inhibitor of protein kinases) and okadaic acid (the inhibitor of protein phosphatases). In control roots, staurosporine markedly slowed and okadaic acid accelerated exudation. In the presence of staurosporine in the incubation medium, a stimulatory effect of both neurotransmitters was completely abolished and the rate of exudation became even below the control value. Okadaic acid exerted an opposite action: it augmented markedly stimulatory effects of both neurotrasmitters. The data obtained indicated the involvement of protein kinases and protein phosphatases in transduction of signals induced by adrenalin and noradrenalin, which stimulated root water-pumping activity.     

Regulation and induction of CYP3A11, CYP3A13 and CYP3A25 in C57BL/6J mouse liver

This study reports that dexamethasone (DEX) significantly induces CYP3A11, CYP3A13 and CYP3A25 mRNA expression in male and female 4 days, 3 weeks and 18 weeks old C57BL/6J mice. Furthermore, CYP3A activity, as measured by erythromycin-N-demethylation, is also significantly increased. PXR, RXRalpha and CAR are known to be involved in the induction of CYP3As. Here we report nuclear receptors PXR and RXRalpha but not CAR demonstrate gender- and age-dependent expression. Also, treatment of C57BL/6J mice with DEX induces PXR but not RXRalpha or CAR. In summary, we demonstrate DEX is not only able to up-regulate CYP3A expression and activity, but also the nuclear receptor PXR through which it may exert this effect. Furthermore, the gender- and age-dependent pattern of basal PXR and RXRalpha expression is similar to the 3 CYP3As analysed.     

Mechanism of arsenite-mediated decreases in CYP3A23 in rat hepatocytes

In primary cultures of rat hepatocytes, exposure to arsenite causes a major decrease in dexamethasone (DEX)-mediated induction of CYP3A23 hemoprotein, with a minor decrease in CYP3A23 mRNA. Here we show that addition of heme did not prevent the arsenite-mediated decreases in CYP3A23 protein, and arsenite did not decrease intracellular glutathione levels, indicating that heme and glutathione were not limiting for formation of holoCYP3A23. We also investigated whether arsenite decreases CYP3A23 protein by increasing CYP3A23 degradation by the calpain pathway. The calpain inhibitor, calpeptin, caused greater than a 90% inhibition of calpain-mediated proteolysis, but had no effect on DEX-mediated induction of CYP3A23 protein following 24h treatments. However, calpeptin enhanced the effect of arsenite to decrease induction of CYP3A23 protein. In addition, in short-term studies, calpeptin appeared to be a suicidal inhibitor of CYP3A-catalyzed enzyme activity. Our findings suggest that CYP3A23 protein is not degraded by calpain-mediated proteolysis, even in the presence of arsenite.     

FMDV-2A sequence and protein arrangement contribute to functionality of CYP2B1-reporter fusion protein

Two optimized forms of green fluorescence proteins (GFP), enhanced GFP (EGFP) and humanized Renilla GFP (hrGFP), were used to track expression of cytochrome P450 2B1 (CYP2B1), an endoplasmic reticulum membrane-bound protein. In transiently expressing HEK293 cells we show that CYP2B1-GFP fusion proteins are stable and functional, whereas the vice-versa-arranged GFP-CYP2B1 fusions are not. The CYP2B1-hrGFP fusion protein is characterized by reduction in mean fluorescence intensity (MFI) to less than 20% of that of the hrGFP protein alone, accompanied by a 50% loss of CYP2B1 activity. Exchanging the linker for an alpha-helical peptide structure between CYP2B1 and hrGFP does not improve fusion protein activity. Insertion of a short linker (five amino acids) increases reporter protein fluorescence intensity twofold without improving CYP2B1 activity. Introduction of the foot and mouth disease virus 2A sequence providing cotranslational cleavage led to an unstable hrGFP-2A protein, whereas the corresponding EGFP-2A protein was stable and yielded an MFI superior to those of all other fusion constructs tested. CYP2B1 activity of the EGFP-2A-CYP2B1 protein was in the range of that of the unmodified CYP2B1. These data indicate that the protein arrangement EGFP-2A-CYP2B1 is superior to others, since it is most active and visible, which is essential for an effective tracking of the CYP2B1 enzyme.     

In vitro 3-D culture demonstrates incompetence in improving maintenance ability of primary hepatocytes

Primary hepatocytes (PHs) are considered the ‘gold standard’ in drug screening owing to their ability to express many drug-metabolizing enzymes and transporters. Culturing hepatocytes and maintaining their fate in vitro is a major issue since last decade. The main problem with in vitro hepatocytes culture is that they rapidly lose their hepatic morphology and liver-specific functions in culture. Herein, we isolated rat PHs, and cultured them in monolayers (2-D) and spheroids (3-D). The 2-D-cultured PHs exhibited elongated morphology, whereas the 3-D-cultured PHs exhibited spheroid morphology with gradual diameter decrease until 7 days. After 7 days of in vitro culture, PHs were analyzed for the expression of hepatic (Alb, Tf, and Afp) and apoptotic markers (Bax and Bcl2), and co-expression of CYP3A1 and Abumin after 2 and 7 days. Furthermore, in both cultures, PHs were induced with 3-methylcholanthrene (3-MC, Cyp1a-specific inducer) and dexamethasone (Cyp3a-specific inducer) for 48 and 72?h, respectively. The mRNA levels of Cyp1a and Cyp3a were analyzed in induced (3-MC, dexamethasone) and non-induced PHs. After 7 days of in vitro culture, PHs exhibited dramatic downregulation of hepatic marker expression in both cultures. Furthermore, apoptotic marker expression was higher in the 2-D-cultured PHs than 3-D-cultured PHs. The mRNA levels of Cyp1a and Cyp3a indicated higher RNA content in the 2-D-cultured PHs after 48?h of induction. Therefore, we concluded that there was no significant difference between the culture systems, and further studies are required to identify the essential components for in vitro PH culture rather than culture systems.     

Differential susceptibilities of serine/threonine phosphatases to oxidative and nitrosative stress

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are signal-transducing molecules that regulate the activities of a variety of proteins. In the present investigation, we have compared the effects of superoxide (O2-), nitric oxide (NO), and hydrogen peroxide (H2O2) on the activities of three highly homologous serine/threonine phosphatases, protein phosphatase type 1 (PP1), protein phosphatase type 2A (PP2A), and calcineurin (protein phosphatase type 2B). Although superoxide, generated from xanthine/xanthine oxidase or paraquat, and NO, generated from (+/-)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide or sodium nitroprusside, potently inhibited the phosphatase activity of calcineurin in neuroblastoma cell lysates, they had relatively little effect on the activities of PP1 or PP2A. In contrast, H2O2 inhibited the activities of all three phosphatases in lysates but was not a potent inhibitor for any of the enzymes. Calcineurin inactivated by O2-, NO, and H2O2 could be partially reactivated by the reducing agent ascorbate or by the thiol-specific reagent dithiothreitol (DTT). Maximal reactivation was achieved by the addition of both reagents, which suggests that ROS and RNS inhibit calcineurin by oxidizing both a catalytic metal(s) and a critical thiol(s). Reactivation of H2O2-treated PP1 also required the combination of both ascorbate and DTT, whereas PP2A required only DTT for reactivation. These results suggest that, despite their highly homologous structures, calcineurin is the only major Ser/Thr phosphatase that is a sensitive target for inhibition by superoxide and nitric oxide and that none of the phosphatases are sensitive to inhibition by hydrogen peroxide.     

氯吡格雷对大鼠肝药物酶CYP3A4和CYP1A2表达的影响

目的：氯吡格雷主要由CYP3A4催化使其激活,CYPlA2也参与氯吡格雷活化。关于氯吡格雷对肝微粒体酶的影响国内外文献报道不多,因此本实验通过检测肝细胞色素氧化酶CYP3A4和CYPlA2的表达,探讨氯吡格雷对大鼠肝药物酶的影响。方法：生理盐水为对照组,氯吡格雷设高、中、低三个剂量组（27,13．5,6．75mg／kg／d）,雄性健康大鼠连续灌胃给药7天,脱臼处死,取肝组织,通过westernblot法检测大鼠肝脏CYP3A4和CYPlA2蛋白表达情况。结果：1）、氯吡格雷抑制大鼠CYP3A4蛋白表达,氯吡格雷高中低剂量组分别比生理盐水组大鼠CYP3A4蛋白表达量降低（P〈0．05）;氯吡格雷低中高剂量组间进行比较,大鼠CYP3A4蛋白表达量呈梯度减少（P〈0．05）;2）、氯吡格雷抑制大鼠CYPlA2蛋白表达,氯吡格雷高中低剂量组分别比生理盐水组大鼠CYPlA2蛋白表达量降低（P〈0．05）,氯吡格雷低中高剂量组间进行比较,大鼠CYPlA2蛋白表达量呈梯度减少（P〈0．05）。结论：氯吡格雷使肝细胞色素氧化酶CYP3A4和CYPlA2的表达量减少,因此氯吡格雷高、中、低3个剂量组均不同程度的抑制大鼠肝脏CYP3A4和CYPlA2的表达,提示当氯吡格雷与某些主要经CYP3A4和CYPlA2代谢的药物合用时,发生代谢性相关作用的可能性大。     

Expression and induction of CYP3As in human fetal hepatocytes

CYP3A4 and CYP3A7 mRNA expression levels were markedly up-regulated by dexamethasone (DEX), but not by rifampicin (RIF). CYP3A5 mRNA level was not increased significantly by DEX, RIF, or phenobarbital. Testosterone 6beta-hydroxylase activity was induced to about 2-fold of control by DEX. However, concomitant treatment with RIF did not alter DEX-mediated induction of CYP3A mRNA expression and testosterone 6beta-hydroxylase activity. DEX-mediated induction of CYP3A mRNA was suppressed in a dose-dependent manner by RU486, a glucocorticoid receptor (GR) antagonist. At 5microM RU486, DEX-mediated induction of CYP3A4, CYP3A5, and CYP3A7 mRNA expression was inhibited almost completely. These results suggest that, in human fetal hepatocytes, PXR is not involved in DEX-mediated induction of CYP3A4 and CYP3A7, and that the induction is mediated directly by GR.     

Absence of enantioselectivity in the pharmacodynamics of P450 2B induction by 5-ethyl-5-phenylhydantoin in the male rat liver or in cultured rat hepatocytes

To explore the enantioselectivity of ligand interaction with the putative phenobarbital receptor, the pharmacodynamics of cytochrome P450 2B (CYP2B) induction by racemic 5-ethyl-5-phen-ylhydantoin and its two enantiomers were investigated in the male F344/NCr rat and in cultured adult male rat hepatocytes. Steady-state serum drug concentrations, measured following 14 days of administration of the compounds in the diet (0-1320 ppm, n = 3 rats per group), were used as an approximation of intrahepatocellular drug concentration. The serum xenobiotic concentrations associated with half-maximal hepatic CYP2B induction were 5-10 μM, based on measurement of pentoxy- or benzyloxyresorufin O-dealkylation activities, or immunoreactive CYP2B1 protein. The corresponding potency values in the hepatocyte culture experiments were 8-12 μM, based on measurement of total cellular RNA coding for CYP2B1. In both the in vivo and the hepatocyte culture experiments, the potencies for CYP2B induction were essentially equivalent for the racemate and the individual enantiomers of 5-ethyl-5-phenylhydantion. In the case of this compound, there would appear to be no enantioselectivity for CYP2B induction. This finding may be interpreted as evidence against receptor mediation in the induction of CYP2B activity, although it is also possible that a receptor is involved that does not exhibit enantioselectivity.     

Determination of serine/threonine protein phosphatase type 2B PP2B in lymphocytes by HPLC

Current cyclosporin (CsA) and tacrolimus (FK506) monitoring methods are based on blood concentrations determination, but the optimal sampling times are not clearly defined. An alternative pharmacodynamic approach has recently been introduced, based on assaying lymphocytes activity of calcineurin (PP2B), a phosphatase that is partially inhibited by CsA and FK506. However, the princeps method uses large amounts of radioactive [32P]substrate, raising a number of safety issues. Here we describe an alternative method for PP2B activity determination, based on HPLC coupled with spectrophotometric detection. A 19-amino-acid peptide is phosphorylated by a protein kinase, and further dephosphorylated by lymphocyte PP2B in the presence of okadaic acid. The two peptides are separated by using reverse-phase chromatography with a detection wavelength of 205 nm. After lymphocyte isolation by density-gradient centrifugation, PP2B activity is derived from the dephosphorylated peptide concentration measured during the first 6 min of the enzymatic reaction. This technique gives reproducible results and good analytical sensitivity with 10(6) lymphocytes. With an average isolation rate of 59.6%, only 7 ml of blood is required, making the method suitable for lymphopenic patients. Moreover, PP2B activity is stable in blood samples kept for 24h at room temperature and in isolated lymphocytes stored for 48 h at -20 degrees C. We validated the method by comparing median PP2B activity in 10 healthy volunteers (285.0+/-46.5 pmol/min/10(6)lymphocytes) and in 10 liver transplant patients (147.8+/-71.0 pmol/min/10(6)lymphocytes) (p<0.001). The relation between calcineurin activity and tacrolimus blood concentrations was also studied.     

Glucose-induced posttranslational activation of protein phosphatases PP2A and PP1 in yeast

The protein phosphatases PP2A and PP1 are major regulators of a variety of cellular processes in yeast and other eukaryotes. Here, we reveal that both enzymes are direct targets of glucose sensing. Addition of glucose to glucose-deprived yeast cells triggered rapid posttranslational activation of both PP2A and PP1. Glucose activation of PP2A is controlled by regulatory subunits Rts1, Cdc55, Rrd1 and Rrd2. It is associated with rapid carboxymethylation of the catalytic subunits, which is necessary but not sufficient for activation. Glucose activation of PP1 was fully dependent on regulatory subunits Reg1 and Shp1. Absence of Gac1, Glc8, Reg2 or Red1 partially reduced activation while Pig1 and Pig2 inhibited activation. Full activation of PP2A and PP1 was also dependent on subunits classically considered to belong to the other phosphatase. PP2A activation was dependent on PP1 subunits Reg1 and Shp1 while PP1 activation was dependent on PP2A subunit Rts1. Rts1 interacted with both Pph21 and Glc7 under different conditions and these interactions were Reg1 dependent. Reg1-Glc7 interaction is responsible for PP1 involvement in the main glucose repression pathway and we show that deletion of Shp1 also causes strong derepression of the invertase gene SUC2. Deletion of the PP2A subunits Pph21 and Pph22, Rrd1 and Rrd2, specifically enhanced the derepression level of SUC2, indicating that PP2A counteracts SUC2 derepression. Interestingly, the effect of the regulatory subunit Rts1 was consistent with its role as a subunit of both PP2A and PP1, affecting derepression and repression of SUC2, respectively. We also show that abolished phosphatase activation, except by reg1Δ, does not completely block Snf1 dephosphorylation after addition of glucose. Finally, we show that glucose activation of the cAMP-PKA (protein kinase A) pathway is required for glucose activation of both PP2A and PP1. Our results provide novel insight into the complex regulatory role of these two major protein phosphatases in glucose regulation.     

Prolongation of insulin-induced activation of mitogen-activated protein kinases ERK 1/2 and phosphatidylinositol 3-kinase by vanadyl sulfate, a protein tyrosine phosphatase inhibitor

Vanadium salts such as vanadyl sulfate (VS), potent inhibitors of protein tyrosine phosphatases, have been shown to mimic, augment, and prolong insulin's action. However, the molecular mechanism of responses to these salts is not clear. In the present studies, we examined if VS-induced effects on insulin action are associated with enhancement or augmentation in the activation state of key components of the insulin signaling pathway. Treatment of insulin receptor-overexpressing cells with insulin or VS resulted in a time-dependent transient increase in phosphorylation and activation of extracellular signal-regulated kinases 1 and 2 (ERK 1/2) that peaked at about 5 min, then declined rapidly to about baseline within 30 min. However, when the cells were treated with VS before stimulation with insulin, sustained ERK 1/2 phosphorylation and activation were observed well beyond 60 min. VS treatment also prolonged the insulin-stimulated activation of phosphatidylinositol 3-kinase (PI3-K), which was associated with sustained interaction between insulin receptor substrate-1 (IRS-1) and the p(85 alpha) subunit of phosphatidylinositol 3-kinase (PI3-K) in response to insulin. These data indicate that prolongation of insulin-stimulated ERK 1/2 and PI3-K activation by VS is due to a more stable complex formation of IRS-1 with the p(85 alpha) subunit which may, in turn, be responsible for its ability to enhance and extend the biological effects of insulin.     

Differential methotrexate hepatotoxicity on rat hepatocytes in 2-D monolayer culture and 3-D gel entrapment culture

It has been reported that 3-D cultures of hepatocytes or HepG2 cells were less susceptible to methotrexate (MTX) than their 2-D counterparts. Such a mechanism was addressed in this study by investigation of MTX hepatotoxicity in gel entrapped (3-D) rat hepatocytes vs. traditional monolayer culture (2-D). Similarly, gel entrapped hepatocytes showed higher drug resistance to MTX than hepatocyte monolayers in whatever culture medium with or without modification by hormone supplements (dexamethasone, glucagon and insulin). It was also found that medium modification by hormones greatly increased drug resistance of hepatocyte monolayers but has only a slight effect on 3-D cultured hepatocytes. These differential MTX toxicities regarding culture medium and culture models were assumed to correlate with multidrug resistance associated protein 2 (Mrp2). The involvement of Mrp2 was confirmed directly by the fact that MTX intracellularly accumulated less in gel entrapped hepatocytes than in hepatocyte monolayer but could be enhanced by Mrp2 inhibitors accompanied by reduced drug resistance. Furthermore, the expression of Mrp2 on gene level and transportation activity together with bile-duct-like structure were more significantly evidenced in 3-D gel entrapment culture than in 2-D monolayer culture. In conclusion, the highly preserved Mrp2 in 3-D gel entrapped hepatocytes determines its high drug resistance to MTX. Gel entrapped hepatocytes could be useful for investigation of hepatic transportation and hepatotoxicity.     

Long-term enhancement of cytochrome P450 2B1/2 expression in rat hepatocyte spheroids through adenovirus-mediated gene transfer

Tissue-like structures of cells organized in vitrohave a great potential for a number of clinical and biomedical applications. Cell functions may be modulated with gene delivery, improving the characteristics of these structures. Hepatocytes that self-assemble into spheroids can be transduced through adenovirus-mediated gene transfer. An adenoviral vector (AdGFP) was employed to deliver a gene encoding for green fluorescent protein (GFP) in rat hepatocyte spheroids. GFP fluorescence was detected for at least one month. Furthermore, the rat cytochrome P450 2B1 gene (CYP2B1) was transferred through infection with a recombinant adenovirus (AdCYP2B1) in hepatocyte spheroids cultured in suspension. The CYP2B1/2 mRNA and apoprotein levels were continuously higher for over 23 days compared to phenobarbital-induced and control cultures. P450-catalyzed pentoxyresorufin-O-dealkylation activity was also high in the AdCYP2B1-infected spheroids. In these spheroid cultures, albumin and urea levels were similar to those in uninfected spheroid cultures, indicating that expression of the CYP2B1transgene did not impair these liver-specific functions. Hepatocyte spheroids transduced by recombinant adenoviral vectors can be efficiently used for drug metabolism studies, in implantation, and in bioartificial liver devices. This revised version was published online in August 2006 with corrections to the Cover Date.