Published papers

Bafilomycin A1, a selective inhibitor of vacuolar H+-ATPase, induced neurite outgrowth of PC12 cells dose- and time-dependently: more than 50% of the cells extended neurite-like spikes after 24 h treatment with 100 nM bafilomycin A1. Its dose-response ran roughly parallel to that of a bafilomycin A1-induced lysosomal pH increase. It was inhibited by LiCl, an inhibitor of the phosphorylation of microtubule-associated proteins and, like nerve growth factor (NGF)-induced neurite outgrowth, it was also inhibited by cycloheximide and actinomycin D. But, unlike the NGF-effect, it was not associated with rapid induction of c-fos. © 1991.

Bafilomycin A1, a potent selective inhibitor of vacuolar H+-ATPase, inhibited the growth of a variety of cultured cells dose-dependently, including golden hamster embryo and NIH-3T3 fibroblasts, whether or not they were transformed, and PC12 and HeLa cells. The concentration of bafilomycin A1 for 50% inhibition of cell growth ranged from 10 to 50 n M. The dose response was nearly parallel with that of the bafilomycin A1-induced lysosomal pH increase. The degree of pH increase for growth inhibition produced by bafilomycin A1 was similar to that produced by NH4Cl in which little difference was recognized in effect among cell types. © 1993, Tissue Culture Association. All rights reserved.

Expression of two abundant mRNAs in the fat body of third instar larvae of Sarcophaga peregrina was studied using genomic clones of storage protein and 25K protein as probes. It was found that these two genes were expressed sequentially in the third instar with a 20-hr interval between their expressions. No amplification of genes was detected during larval development, suggesting that the high level of mRNA was due to efficient transcription. The contents of the two mRNAs in third instar larvae were about the same, although the synthesis of storage protein was much more than that of 25K protein. © 1983.

To explore the physiological roles of sulfotransferases (SULTs) in extra-hepatic tissues, we examined the expression of eight SULT genes by reverse transcription (RT)-PCR in human cell lines that were established from various tissues. Expression levels of SULTs were low in neural cell lines such as NB-1 and GI-1, and high in epithelial cell lines, such as Caco-2 and BeWo. SULT1C2 expression was abundant in all cell types, whereas that of SULT1E1, SULT1B1 or SULT2B1 was restricted to a specific cell type. SULT1C1, which can catalyze the sulfation of N-hydroxy-2-acetylaminofluorence, was expressed in Caco-2, BeWo and KB562. Induction of differentiation did not generally affect SULT expression, although that of SULT1C2 was reduced after differentiation of the neuroblastoma cell line, NB-1, was induced. The profile of SULT expression in the culture cells obtained here gives clues to understanding the physiological roles of SULT enzymes in extra-hepatic tissues or organs.

In order to explore the possible role of sulfation in the inactivation of environmental estrogens at gastrointestinal sites and their subsequent removal, we investigated the effects of phenolic environmental estrogens on sulfotransferase (ST) activity. The mouse intestine and a human colon carcinoma cell line, Caco-2, were studied. ST enzymes were found to have a high affinity for diethylstilbestrol (DES) and bisphenol A (BPA), whereas phenol ST (PST) activity was strongly inhibited by nonylphenol and genistein in both mice and humans. Kinetic analysis showed that this inhibition was competitive. These observations suggest that nonylphenol and genistein compounds might inhibit PST activity in the human intestine and that they might escape detoxification by sulfation.

We have previously reported that epigallocatechin gallate (EGCG) strongly inhibits the in vitro phenol sulfo-transferase (P-ST) activity of a human colon carcinoma cell line, Caco-2. In the present study, we examined the ability of EGCG to inhibit the sulfation of 1-naphthol in intact Caco-2 cells. Sulfation of 1-naphthol was detected in Caco-2 cells after 2 h of incubation, and was observed to continue for 24 h, resulting in an accumulation of sulfated 1-naphthol. Sulfation was strongly inhibited by the addition of EGCG to the culture medium. The IC50 of EGCG was calculated to be 20 μM; this value is similar to that obtained from in vitro assays (14 μM) [Ref. Tamura et al., Biol. Pharm. Bull., 23, 695, (2000)]. These results indicate that catechins are capable of inhibiting P-ST activity in intact cells as well as in vitro. We believe that the inhibitory activity of catechins might be the mechanism by which catechins (and green tea) exert anti-carcinogenic activity against procarcinogenic compounds that require P-ST activation in vivo.

In our current study, we investigated the expression profiles of the cytochromes P450 (CYPs) and transporters of the ocular tissues in Sprague-Dawley (SD) rats. Extensive expression of CYP1A1 in the cornea and CYP2E1 in the iris was observed whereas the expression of CYP2B1 and transporters was mostly ubiquitous throughout the ocular tissues. To further understand the regulation of these genes in ocular tissues, we investigated the effects of CYP inducers on the expression of the CYP and other xenobiotic-metabolizing enzyme genes in the cornea and lens. The administration of β-naphthoflavone (BNF), an agonist for aryl hydrocarbon receptor (AhR), induced CYP1A1 gene expression in the cornea, lens and liver of the rats, although the levels of induction were greatest in the liver. An AhR-sensitive UDP-glucuronosyl transferase (UGT) 1A6 gene was also induced in the cornea and the lens by BNF. Phenobarbital (PB) is a known inducer of the CYP2B genes, the expression of which is mediated by constitutive activated receptor (CAR), but did not induce CYP2B1 in the cornea or lens. This insensitivity to PB may be due to the lack of CAR expression in the ocular tissues as revealed in our present study. Pregnenolone-16α- carbonitrile (PCN) is known to induce CYP3A gene expression in the liver via the activation of pregnane X receptor (PXR). However, although PCN was found to induce the CYP3A1 gene in the rat cornea and liver, it failed to do so in the lens. In addition, another of the PXR-mediated genes, multidrug resistance-associated protein 3 (Mrp3), was not induced by PCN in either ocular region. Since the expression of the PXR gene was not detected in the rat ocular tissues, an unknown mechanism for the inducible regulation of CYP3A1 gene expression by PCN in the cornea is suggested. ©2009 The Pharmaceutical Society of Japan.

TR-TBT-18d-1 and -18d-2 are syncytiotrophoblast cell lines established from the placenta of a transgenic rat harboring a temperature-sensitive simian virus 40 large T-antigen (Tg-rat). To explore the functional roles of drug metabolizing enzymes in the syncytiotrophoblasts of the placenta, we analyzed the gene expression of both cytochrome P450 (CYP) and phase II conjugation enzymes in these TR-TBT cell lines. A relatively high level of expression of CYP1A1 and CYP2C11 was detectable in the 18d-1 cells whereas CYP2E1 was more strongly expressed in the 18d-2 cells. A moderate degree of expression of CYP2B2 and CYP3A, and low expression of CYP1A2, was observed in both cell lines, but no CYP3A2 was detectable in either. Relatively high levels of SULT2 family gene expression were also evident in these TR-TBT cells, although the SULT1 family genes were found to be either absent or present at only moderate levels. Strong expression of both the UGT1 and UGT2 subfamily, and also the glutathione S-transferase (GST) subfamily, was also observed in both cell lines. These results suggest that TR-TBT cells are a useful model system for the study of syncytiotrophoblasts, which act as a protective fetal barrier against harmful xenobiotics.

© Yosuke Nakazawa et al published by De Gruyter Open 2016. Background. Chaperone activity of α-crystallin in the lens works to prevent protein aggregation and is important to maintain the lens transparency. This study evaluated the effect of hesperetin on lens chaperone activity in selenite-induced cataracts. Methodology. Thirteen-day-old rats were divided into four groups. Animals were given hesperetin (groups G2 and G4) or vehicle (G1 and G3) on Days 0, 1, and 2. Rats in G3 and G4 were administered selenite subcutaneously 4 hours after the first hesperetin injection. On Days 2, 4, and 6, cataract grades were evaluated using slit-lamp biomicroscopy. The amount of a-crystallin and chaperone activity in water-soluble fraction were measured after animals sacrificed. Results. G3 on day 4 had developed significant cataract, as an average cataract grading of 4.6 ± 0.2. In contrast, G4 had less severe central opacities and lower stage cataracts than G3, as an average cataract grading of 2.4 ± 0.4. The a-crystallin levels in G3 lenses were lower than in G1, but the same as G4. Additionally, chaperone activity was weaker in G3 lenses than G1, but the same as in G4. Conclusions. Our results suggest that hesperetin can prevent the decreasing lens chaperone activity and a-crystallin water solubility by administered of selenite.

Emerging evidence indicates that stress hormone glucocorticoids (GC) are an important modulator of brain development and function. To investigate whether GCs modulate neurosteroid biosynthesis in neural cells, we studied the effects of GCs on steroidogenic gene expression in human glioma GI-1 cells. The GC dexamethasone (Dex) reduced steroidogenic acute regulatory protein (StAR), CYP11A1 and 3β-hydroxysteroid dehydrogenase gene expression in a dose- and GC receptor-dependent manner. In addition to its effects on steroidogenic gene expression, Dex also reduced de novo synthesis of progesterone (PROG). Furthermore, Dex inhibited all-trans retinoic acid (ATRA) and vitamin D 3-induced steroidogenic gene expression and PROG production. This suggests that GC regulates steroidogenic gene expression in neural cells via cross-talk with the two fat-soluble vitamins, A and D. The relationship between the effects of GCs on neurosteroid biosynthesis and on cognitive behaviors and hippocampal neural activity is also discussed herein. © 2014 The Pharmaceutical Society of Japan.

We examined the effect of herbal teas (including black and green teas) on conjugation reactions within a human colon carcinoma cell line, Caco-2. After adding herbal tea to the culture medium of Caco-2 cells, accumulation of 1-naphthyl sulfate and glucuronide was determined within the medium by analytical HPLC prior to 24 hr. A reduction in sulfoconjugation (< 50% of the control value) was observed in cells treated with 5% solutions of green tea, jasmine tea or black tea. Mild induction of sulfoconjugation (20-30%) was detected in cells exposed to 5% solutions of hibiscus or lemongrass tea. In addition, a slight reduction (20-30%) in glucuronic acid conjugation (glucuronidation) was observed in cells treated with rose red, peppermint, lavender or St. John's wort. At low concentrations, ranging from 0.1-1.0%, several herbal teas resulted in weak but significant induction (10-20%) of both types of conjugation reactions. These results suggest that herbal teas modify conjugation reactions within intestinal epithelial cells, thereby potentially affecting the bioavailability and toxicity of therapeutic drugs and environmental chemicals.

© 2015 Elsevier Inc. All rights reserved. Recent epidemiological studies have shown that moderate coffee consumption is associated with a lower risk of certain types of cancers, particularly colon cancer in postmenopausal women. To elucidate the molecular basis of the preventive action of coffee toward colon cancer, we focused on the effects of coffee on the metabolism of estrogens by sulfation, a reaction that inactivates estrogens. Estrogens are sulfonated by estrogen sulfotransferase (SULT1E1), and the sulfonated estrogens are reversely hydrolyzed by steroid sulfatase (STS). Sulfonated estrogens are pumped out of the cells by an ABC transporter, breast cancer resistance protein (BCRP). Intracellular concentrations of active estrogens may be determined by the balance of these activities. In human colon cancer Caco-2 cells, coffee decreases the expression of SULT1E1 and STS, whereas it increases BCRP expression. Activities of these three proteins change according to the expression level of each gene. Active constituents of coffee extract can be generated during the roasting process of coffee beans at different times. Modulation of the expression of these genes is likely to be mediated via activation of transcription factors such as NFκB (. BCRP) and stimulating protein 1 (. SULT1E1). These results suggest that coffee consumption may modulate the inactivation of estrogens by sulfation in human colon cells.

Estradiol benzoate (EB) and testosterone propionate (TP) were administered to male and female Wistar rats, and their effects on the zonal expression of hepatic phenol sulfotransferase (P-ST) activities were determined at pH 5.5 and 7.4. Cytosolic fractions from periportal (PP) and perivenous (PV) hepatocytes were prepared by the dual-digitonin-pulse perfusion technique. In control rats, P-ST activities assayed at pH 5.5 and 7.4 were higher in males than in females, and were higher in the PV fraction than in the PP fraction in both sexes. P-ST activities were increased by the administration of TP in the PP and PV fractions of females, whereas the same treatment diminished the enzyme activities in both fractions of the males. EB administration gave reduced P-ST activity at pH 5.5 of both fractions, irrespective of sex, but not a marked difference at pH 7.4. Chromatofocusing of PP and PV fractions revealed the presence of P-ST isoforms eluted at approx. pH 8.0 (peak 1), 7.5 (peak 2), 7.0 (peak 3) and 6.0 (peak 4). In male rats, peak 3, which showed high enzyme activity at pH 5.5 in the PP and PV fractions, was markedly decreased by EB treatment, whereas in females, peak 3 was present only in the PV fraction and was not affected by EB administration. TP treatment did not show remarkable changes in P-ST peaks in the males, while peaks 2 and 3 were increased in the females. Immunoblot analysis revealed the presence of multiple P-ST isoforms which showed different immunoreactivity and electrochemical properties.