Changes in uridine 5'-diphospho-glucuronosyltransferase 1A6 expression by histone deacetylase inhibitor valproic acid.

Valproic acid (VPA) is well-known as a histone deacetylase (HDAC) inhibitor. It has been reported that HDAC inhibitors enhance basal and aryl hydrocarbon receptor (AhR) ligand-induced aryl hydrocarbon receptor-responsive gene expression. Other studies suggested that HDAC inhibition might significantly activate the NF-E2-related factor-2 (Nrf2). Moreover, VPA activates mitogen-activated protein kinases (MAPKs). MAPK pathways regulate Nrf2 transactivation domain activity. Uridine 5'-diphospho-glucuronosyltransferase (UGT) 1A6 is one of the important isoforms to affect drug pharmacokinetics. UGT1A6 gene is regulated transcriptionally by AhR and Nrf2. The present study aimed to investigate whether UGT1A6 expression was changed by VPA and to elucidate the mechanism of the alteration. Following VPA treatment for 72 h in Caco-2 cells, UGT1A6 mRNA was increased by 7.9-fold. Moreover, UGT1A6 mRNA was increased by other HDAC inhibitors, suggesting that HDAC inhibition caused the UGT1A6 mRNA induction. AhR and Nrf2 proteins in the nucleus of Caco-2 cells were increased by 1.5- and 1.7-fold, respectively, following the VPA treatment. However, VPA treatment did not activate the extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) pathways in Caco-2 cells. In conclusion, we observed that VPA induced UGT1A6 mRNA expression via AhR and Nrf2 pathways, but not via the ERK or JNK pathways.

Biologic Response of Colorectal Cancer Xenograft Tumors to Sequential Treatment with Panitumumab and Bevacizumab.

Recent studies in RAS wild-type (WT) metastatic colorectal cancer (mCRC) suggest that the survival benefits of therapy using anti-epidermal growth factor receptor (anti-EGFR) and anti-vascular endothelial growth factor (anti-VEGF) antibodies combined with chemotherapy are maximized when the anti-EGFR antibody is given as first-line, followed by subsequent anti-VEGF antibody therapy. We report reverse-translational research using LIM1215 xenografts of RAS WT mCRC to elucidate the biologic mechanisms underlying this clinical observation. Sequential administration of panitumumab then bevacizumab (PB) demonstrated a stronger tendency to inhibit tumor growth than bevacizumab then panitumumab (BP). Cell proliferation was reduced significantly with PB (P < .01) but not with BP based on Ki-67 index. Phosphoproteomic analysis demonstrated reduced phosphorylation of EGFR and EPHA2 with PB and BP compared with control. Western blotting showed reduced EPHA2 expression and S897-phosphorylation with PB; RSK phosphorylation was largely unaffected by PB but increased significantly with BP. In quantitative real-time PCR analyses, PB significantly reduced the expression of both lipogenic (FASN, MVD) and hypoxia-related (CA9, TGFBI) genes versus control. These results suggest that numerous mechanisms at the levels of gene expression, protein expression, and protein phosphorylation may explain the improved clinical activity of PB over BP in patients with RAS WT mCRC.

Effect of single-walled carbon nanotubes on cytochrome P450 activity in human liver microsomes in vitro.

Single-walled carbon nanotubes (SWCNTs) are made from a rolled single sheet of graphene with a diameter in the nanometer range. SWCNTs are potential carriers for drug delivery systems because antibodies or drugs can be loaded on their surface; however, their effect on the activities of cytochrome P450 (CYP) remains unclear. The aim of this study was to investigate the effect of two kinds of SWCNTs with different lengths (FH-P- and SO-SWCNTs) on human CYP activity. In addition, other nano-sized carbon materials, such as carbon black, fullerene-C60 , and fullerene-C70 were also evaluated to compare their effects on CYP activities. Ten CYP substrates (phenacetin, coumarin, bupropion, paclitaxel, tolbutamide, S-mephenytoin, dextromethorphan, chlorzoxazone, midazolam, and testosterone) were used. Testosterone 6ß-hydroxylation and midazolam 1'-hydroxylation, which are catalysed by both CYP3A4 and CYP3A5 in liver microsomes, were decreased by 25% and 45%, respectively, in the presence of 0.1 mg/ml SO-SWCNT. Dextromethorphan O-demethylation, which is catalysed mainly by CYP2D6, was decreased by 40% in the presence of SO-SWCNT. Other CYP activities, however, were not attenuated by SO-SWCNT. FH-P-SWCNT, carbon black, fullerene-C60 , and fullerene-C70 at 0.1 mg/ml had no effect on CYP activities. The Ki values for testosterone 6ß-hydroxylation, midazolam 1'-hydroxylation, and dextromethorphan O-demethylation in liver microsomes were 136, 34, and 56 µg/ml, respectively. SO-SWCNT was determined to be a competitive inhibitor of CYP3A4, CYP3A5, and CYP2D6. These results suggest that the effect of SO-SWCNT differs among CYP isoforms, and that the inhibition potency depends on the physicochemical properties of the nanocarbons.

Effect of carbamazepine on expression of UDP-glucuronosyltransferase 1A6 and 1A7 in rat brain.

Because UDP-glucuronosyltransferase (Ugt) 1a6 and Ugt1a7 are highly expressed in the rat brain, changes in Ugt1a6 and Ugt1a7 expression may affect the pharmacokinetics of drugs and endogenous compounds in the brain. The present study aimed to elucidate the effect of carbamazepine (CBZ), a typical UGT inducer, on Ugt1a6 and Ugt1a7 expression in the rat brain. Sprague-Dawley rats were treated intraperitoneally for 7 d with CBZ (100 mg/kg/d). Ugt1a6 and Ugt1a7 mRNAs were induced by CBZ in the cerebellum, piriform cortex, and hippocampus (Ugt1a6: 3.1-, 2.4-, and 1.9-fold, respectively, Ugt1a7: 2.3-, 1.6-, and 3.1-fold, respectively); serotonin glucuronidation, which is catalyzed by Ugt1a6, was also increased by 2.8-, 1.7-, and 1.8-fold in these regions, respectively. The nuclear translocation of the constitutive androstane receptor was increased 1.4-fold in the cerebellum and piriform cortex, suggesting that brain Ugt1a6 and Ugt1a7 might be induced via the constitutive androstane receptor. However, the pregnane X receptor and nuclear factor erythroid 2-related factor 2 did not play decisive roles in the induction. Histone H3 lysine 9 acetylation, H3 lysine 4 pan-methylation, and H3 lysine 9 mono-methylation may not be required for the induction. This study clarified that CBZ affected Ugt1a6 and Ugt1a7 in the brain.

Species and Tissue Differences in ß-Estradiol 17-Glucuronidation.

Uridine 5'-diphosphate-glucuronosyltransferase (UGT) is expressed in the liver and extrahepatic tissues. One of the major metabolic pathways of ß-estradiol (E2) is glucuronidation at the 17-hydroxy position by UGTs. This study was performed to determine E2 17-glucuronidation kinetics in human and rodent liver, small intestine, and kidney microsomes and to clarify the species and tissue differences. In the human liver and small intestine, Eadie-Hofstee plots exhibited biphasic kinetics, suggesting that E2 17-glucuronide (E17G) formation was catalyzed by more than two UGT isoforms in both tissues. The Km values for E17G formation by the high-affinity enzymes in the human liver and small intestine were 1.79 and 1.12 µM, respectively, and corresponding values for the low-affinity enzymes were 3.72 and 11.36 µM, respectively. Meanwhile, E17G formation in the human kidney was fitted to the Hill equation (S50=1.73 µM, n=1.63), implying that the UGT isoform catalyzing E17G formation in the kidney differed from that in the liver and small intestine. The maximum clearance for E17G formation in the human kidney was higher than the intrinsic clearance in the liver. E17G formation in the rat liver and kidney exhibited biphasic kinetics, whereas that in the small intestine was fitted to the Hill equation. In mice, all 3 tissues exhibited biphasic kinetics. In conclusion, we reported species and tissue differences in E2 17-glucuronidation, which occurred not only in the human liver but also in the extrahepatic tissues particularly the kidney.

Characterization of ß-Estradiol 3-Glucuronidation in Rat Brain.

ß-Estradiol is conjugated by uridine 5'-diphosphate-glucuronosyltransferase (UGT) 1A to 3-glucuronide in the human liver. UGT1A has been found in the brain; therefore, UGT1A may be involved in ß-estradiol 3-glucuronidation in the brain. In the present study, we aimed to characterize the ß-estradiol 3-glucuronidation reaction in the rat brain. ß-Estradiol 3-glucuronidation was detected in eight rat brain regions (cerebellum, frontal cortex, parietal cortex, piriform cortex, hippocampus, medulla oblongata, striatum, and thalamus). ß-Estradiol 3-glucuronidation in the cerebellum was fitted to the Hill equation (S50=8.0 µM, n=1.1). In inhibition experiments, ß-estradiol 3-glucuronidation was inhibited to 73.6% in the cerebellum by 50 µM bilirubin, whereas it was reduced to 20.5% with 5 µM bilirubin in the liver. Unlike in the liver, Ugt1a1 may not be the main isoform catalyzing this glucuronidation in the brain. Serotonin and acetaminophen at 10 mM inhibited glucuronidation to 1.17 and 25.5%, respectively, in the cerebellum. In induction experiments, the administration of ß-naphthoflavone, carbamazepine, and phenobarbital did not increase ß-estradiol 3-glucuronidation in the brain except for phenobarbital in the striatum. In addition, ß-estradiol 3-glucuronidation was not correlated with serotonin or acetaminophen glucuronidation in the brain, suggesting that Ugt1a6 and Ugt1a7 are not major isoforms of ß-estradiol 3-glucuronidation in the rat brain. In the present study, although we were unable to identify the isoform responsible for ß-estradiol 3-glucuronidation, we confirmed that ß-estradiol could be metabolized to glucuronide in the brain under a different metabolic profile from that in the liver.

Effects of ß-Naphthoflavone on Ugt1a6 and Ugt1a7 Expression in Rat Brain.

Uridine 5'-diphosphate-glucuronosyltransferase (UGT) catalyzes a major phase II reaction in a drug-metabolizing enzyme system. Although the UGT1A subfamily is expressed mainly in the liver, it is also expressed in the brain. The purpose of the present study was to elucidate the effect of ß-naphthoflavone (BNF), one of the major inducers of drug-metabolizing enzymes, on Ugt1a6 and Ugt1a7 mRNA expression and their glucuronidation in the rat brain. Eight-week-old male Sprague-Dawley rats were treated intraperitoneally with BNF (80 mg/kg), once daily for 7 d. Ugt1a6 and Ugt1a7 mRNA expression increased in the cerebellum and hippocampus (Ugt1a6: 2.1- and 2.3-fold, respectively; Ugt1a7: 1.7- and 2.8-fold, respectively); acetaminophen glucuronidation also increased in the same regions by 4.1- and 2.7-fold, respectively. BNF induced Ugt1a6 and Ugt1a7 mRNA expression and their glucuronidation, and the degree of induction differed among 9 regions. BNF also upregulated CYP1A1, CYP1A2, and CYP1B1 mRNAs in the rat brain. Since the aryl hydrocarbon receptor signaling pathway was activated by BNF, it is indicated that Ugt1a6 and Ugt1a7 were induced via AhR in the rat brain. This study clarified that Ugt1a6 and Ugt1a7 mRNA expression and their enzyme activities were altered by BNF, suggesting that these changes may lead to alteration in the pharmacokinetics of UGT substrate in rat brain.

Effect of adrenalectomy on expression and induction of UDP-glucuronosyltransferase 1A6 and 1A7 in rats.

Uridine 5'-diphosphate (UDP)-glucuronosyltransferase 1A (UGT1A), which catalyzes major phase II reactions, is regulated by endogenous and exogenous factors via nuclear receptors such as the aryl hydrocarbon receptor (AhR). Glucocorticoid, one of the adrenocortical hormones, regulates AhR and UGT1A expression. We examined the effects of adrenalectomy on the expression and induction of UGT1A via AhR in the rat liver and small intestine. Rats were adrenalectomized bilaterally (ADX) or sham-operated (SHAM) and received intraperitoneal treatment with ß-naphthoflavone (BNF) for 4 d. Hepatic UGT1A6 and UGT1A7 mRNA levels were altered by ADX (0.1-fold and 1.6-fold, respectively). BNF treatment increased UGT1A6 and UGT1A7 mRNA expression and the intrinsic clearance of acetaminophen (APAP) glucuronidation, which is primarily catalyzed by UGT1A6 and UGT1A7, in both SHAM and ADX rats. Therefore, ADX rats maintained a functional AhR signaling pathway in the presence of BNF, expressed UGT1A6 and UGT1A7 mRNA, and showed APAP glucuronidation, namely induction by BNF via AhR was not abolished. Our results indicate that adrenal-dependent factors such as glucocorticoids are partially involved in the basal regulation of UGT1A6 and UGT1A7 transcription.

[Effect of tacrolimus on the pharmacokinetics and glucuronidation of SN-38, an active metabolite of irinotecan].

The present study has investigated the effect of tacrolimus on the pharmacokinetics of an active metabolite of irinotecan (CPT-11), 7-ethyl-10-hydroxy-camptothecin (SN-38) and SN-38 glucuronide (SN-38G) in rats. The effect of tacrolimus on SN-38 glucuronidation was also investigated in human and rat liver microsomes. When tacrolimus (0.5 mg/kg) was intravenously injected in rats 15 min before intravenous injection of CPT-11 (5 mg/kg), tacrolimus decreased the plasma concentration of SN-38G. Tacrolimus significantly decreased the area under plasma concentration-time curve (AUC) of SN-38G without change in the mean residence time. On the contrary, significant changes in the pharmacokinetic parameters of SN-38 were not observed. SN-38 glucuronidation in human and rat liver microsomes was inhibited dose-dependently by the presence of tacrolimus and the 50% inhibition concentration (IC50) values of tacrolimus in rat and human liver microsomes were 10.33 µM and 3.58 µM, respectively. When the inhibition type was determined by Lineweaver-Burk and Dixon plots, the inhibition was noncompetitive and the calculated inhibition constant (Ki) values for rat and human liver microsomes were 12.57 µM and 3.88 µM, respectively. These findings suggest that tacrolimus inhibits UGT1A1-mediated SN-38 glucuronidation. Considering the IC50 and Ki values for tacrolimus, it is likely that tacrolimus does not alter the pharmacokinetics of SN-38 and SN-38G at the clinically used dosages, suggesting the possibility that tacrolimus can use safely for cancer patients with irinotecan chemotherapy.

Electron transfer of site-specifically cross-linked complexes between ferredoxin and ferredoxin-NADP(+) reductase.

Ferredoxin (Fd) and Fd-NADP(+) reductase (FNR) are redox partners responsible for the conversion between NADP(+) and NADPH in the plastids of photosynthetic organisms. Introduction of specific disulfide bonds between Fd and FNR by engineering cysteines into the two proteins resulted in 13 different Fd-FNR cross-linked complexes displaying a broad range of activity to catalyze the NADPH-dependent cytochrome c reduction. This variability in activity was thought to be mainly due to different levels of intramolecular electron transfer activity between the FNR and Fd domains. Stopped-flow analysis revealed such differences in the rate of electron transfer from the FNR to Fd domains in some of the cross-linked complexes. A group of the cross-linked complexes with high cytochrome c reduction activity comparable to dissociable wild-type Fd/FNR was shown to assume a similar Fd-FNR interaction mode as in the native Fd:FNR complex by analyses of NMR chemical shift perturbation and absorption spectroscopy. However, the intermolecular electron transfer of these cross-linked complexes with two Fd-binding proteins, nitrite reductase and photosystem I, was largely inhibited, most probably due to steric hindrance by the FNR moiety linked near the redox center of the Fd domain. In contrast, another group of the cross-linked complexes with low cytochrome c reduction activity tends to mediate higher intermolecular electron transfer activity. Therefore, reciprocal relationship of intramolecular and intermolecular electron transfer abilities was conferred by the linkage of Fd and FNR, which may explain the physiological significance of the separate forms of Fd and FNR in chloroplasts.