Directional Drug Transport through Membrane-Supported Monolayers of Human Liver-Derived Cell Lines.

The aim of this work is to develop a new assay system for screening biliary excretion drugs. When monolayers of human liver-derived cell lines HepG2 and Huh-7 were grown on an insert membrane, the efflux ratio (ER: ratio of the apparent permeability coefficient in the basal-to-apical direction (Papp,B-to-A) to that in the apical to basal direction (Papp,A-to-B)) of sulfobromophthalein (BSP), a model substrate of multidrug resistance-associated protein 2 (MRP2), was greater than 1.0, indicating transport of BSP in the efflux direction. The efflux transport was significantly suppressed by MK-571, an inhibitor of MRPs, in both cell lines. Expression of MRP2 mRNA in HepG2 and Huh-7 was 3.5- and 1.4-fold higher, respectively, than in primary human hepatocytes, while expression of P-glycoprotein and breast cancer resistance protein mRNAs was markedly lower, supporting the idea that MRP2 is the main mediator of directional BSP transport in this assay system. The advantage of our system is the potential to quantitatively evaluate biliary excretion of MRP2 substrates in vitro.

Evaluation system for cell-permeable CYP3A4 inhibitory activity using 1α,25-dihydroxy-vitamin D3-induced intestinal cell lines.

We developed an assay system to evaluate the cytochrome P450 (CYP) 3A4-inhibitory activity of compounds, taking account of their cellular permeability, using intestine-derived cell lines pre-treated with the CYP3A4 inducer 1α,25-dihydroxy-vitamin D3 (250 nM).Ketoconazole (KTZ), saquinavir (SQV), naringin, naringenin (NGE), bergamottin (BG), 6',7'-dihydroxybergamottin (DHBG), epigallocatechin gallate (EGCG), and resveratrol (RES) were evaluated as known CYP3A4 inhibitors. The apparent IC50 (IC50,app) values of known inhibitors were determined in Caco-2 cells with 10 µM midazolam as a CYP3A4 substrate, and compared with the IC50 values in a human liver microsome assay.SQV and BG with high lipophilicity and good membrane permeability show similar concentrations inside and outside the cells, and consequently IC50,app and IC50 are similar.KTZ, EGCG, DHBG, NGE, and RES showed a difference between IC50 and IC50,app. This is considered to result from a difference between the intracellular and extracellular concentrations of the compound, which is likely due to the involvement of efflux and/or influx transporters.This method to evaluate CYP inhibition taking account of membrane permeation should be helpful to assess the potential clinical relevance of drug-drug or drug-food interactions in the gastrointestinal tract.

Triphenyltin disrupts intracellular Zn2+ homeostasis in rat thymic lymphocytes.

Triphenyltin (TPT), previously used as an agricultural fungicide and industrial antifoulant, is now considered an environmental pollutant. The effect of TPT on human health is concerning due to its presence as a contaminant in seafood. In this study, the changes in intracellular Zn2+ concentration ([Zn2+]i) and cellular content of nonprotein thiols ([NPT]i) induced by triphenyltin chloride (TPTCH), were measured in rat thymic lymphocytes. This was studied by flow-cytometry using the fluorescent probes FluoZin-3-AM and 5-chloromethylfluorescein diacetate (5-CMF-DA). Incubation with TPTCH, at 0.1 µM or more (up to 3 µM), increased [Zn2+]i in a concentration-dependent manner. The TPTCH-induced elevation in [Zn2+]i was due to the increase in membrane Zn2+ permeability and intracellular Zn2+ release. Incubation with TPTCH at 0.3 µM significantly increased [NPT]i levels, whereas the addition of an intracellular Zn2+ chelator had no effect on the same. TPT at higher concentrations (1 or 3 µM) reduced [NPT]i. TPT may disturb intracellular Zn2+ signaling in lymphocytes that disturbs cellular functions.

Non-invasive X-ray micro-computed tomographic evaluation of indomethacin on urethane-induced lung carcinogenesis in mice.

BACKGROUND: Lung cancer is the leading cause of cancer-related death worldwide. We previously reported that respiration-gated X-ray micro-computed tomography (micro-CT) is a useful tool for analyzing lung tumor development in animal models. MATERIALS AND METHODS: Lung tumors were induced by a single intraperitoneal injection (250 mg/kg) of urethane in male A/J mice, followed by indomethacin treatment at 5 ppm in the diet. The mice were scanned by micro-CT every 4 weeks from 10 to 26 weeks after urethane administration. RESULTS: Total incidence and multiplicity of lung tumors were not significantly reduced by indomethacin treatment, as compared with untreated mice. However, the incidence of adenocarcinoma tended to be reduced by indomethacin treatment. Moreover, the size of lung tumors, especially adenomas, was suppressed by indomethacin treatment. Micro-CT analysis revealed that indomethacin effectively suppressed tumor development after urethane treatment for 10 weeks. CONCLUSION: These findings indicate that indomethacin suppresses lung carcinogenesis in mice and micro-CT is a useful non-invasive imaging approach for evaluating the characteristics and suppression of lung tumors in mice treated with cancer chemopreventive agents.

Suppressive effect of pioglitazone, a PPAR gamma ligand, on azoxymethane-induced colon aberrant crypt foci in KK-Ay mice.

Obesity is an established risk factor for colorectal cancer. Pioglitazone is a peroxisome proliferator- activated receptor γ(PPARγ) agonist that induces differentiation in adipocytes and induces growth arrest and/or apoptosis in vitro in several cancer cell lines. In the present study, we investigated the effect of pioglitazone on the development of azoxymethane-induced colon aberrant crypt foci (ACF) in KK-Ay obesity and diabetes model mice, and tried to clarify mechanisms by which the PPARγ ligand inhibits ACF development. Administration of 800 ppm pioglitazone reduced the number of colon ACF / mouse to 30% of those in untreated mice and improved hypertrophic changes of adipocytes in KK-Ay mice with significant reduction of serum triglyceride and insulin levels. Moreover, mRNA levels of adipocytokines, such as leptin, monocyte chemoattractant protein-1 and plasminogen activator inhibitor-1, in the visceral fat were decreased. PCNA immunohistochemistry revealed that pioglitazone treatment suppressed cell proliferation in the colorectal epithelium with elevation of p27 and p53 gene expression. These results suggest that pioglitazone prevented obesity-associated colon carcinogenesis through improvement of dysregulated adipocytokine levels and high serum levels of triglyceride and insulin, and increase of p27 and p53 mRNA levels in the colorectal mucosa. These data indicate that pioglitazone warrants attention as a potential chemopreventive agent against obesity-associated colorectal cancer.

Inhibition of intestinal polyp formation by pitavastatin, a HMG-CoA reductase inhibitor.

It has been suggested that hyperlipidemia is positively associated with colon carcinogenesis. Statins, 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase inhibitors, reduce serum lipid levels. In this study, we clarified the effects of a novel chemically synthesized statin, pitavastatin, on intestinal polyp formation in Min mice, and further examined serum lipid and adipocytokine levels, and proinflammatory and adipocytokine gene levels in intestinal mucosa of Min mice. Treatment with pitavastatin at doses of 20 and 40 ppm decreased the total number of polyps dose-dependently to 85.2% and 65.8% (P < 0.05) of the untreated value, respectively. Serum levels of total cholesterol and triglyceride were slightly reduced and those of IL-6, leptin, and MCP-1 were decreased by 40-ppm pitavastatin treatment. mRNA expression levels of cyclooxygenase-2, IL-6, inducible nitric oxide (iNOS), MCP-1, and Pai-1 were significantly reduced in intestinal nonpolyp parts by pitavastatin treatment. Among them, iNOS mRNA levels were also reduced in the intestinal polyps. Moreover, oxidative stress represented by 8-nitroguanosine in the small intestinal epithelial cells was reduced by pitavastatin treatment. Related to these proinflammatory genes, PPARγ activity was activated in the intestinal nonpolyp parts and in the liver of Min mice with pitavastatin treatment. These results indicated that pitavastatin has potential benefit for the suppression of intestinal polyp development.

High susceptibility to azoxymethane-induced colorectal carcinogenesis in obese KK-Ay mice.

Obesity is associated with colon carcinogenesis. However, not much information is available regarding the mechanisms of obesity-associated colorectal cancer, and there are only few useful animal models for investigating the underlying mechanism between obesity and colorectal cancer. KK-A(y) mice exhibit severe obesity. Amount of visceral fat assessed by micro-computed tomography was almost 15 times higher than that of same aged C57BL/6J mice. Treatment with azoxymethane (AOM; 200 µg/mouse injected once a week for 3 times) resulted in markedly increased colon aberrant crypt foci (ACF) development (≈70 ACF/mouse) in KK-A(y) mice compared with lean C57BL/6J mice (≈9 ACF/mouse). Moreover, administration of AOM at a dose of 200 µg/mouse once a week for 6 times developed colorectal adenocarcinomas within only 7 weeks after the last AOM injection. The incidence of adenocarcinoma was 88% in KK-A(y) mice and was markedly higher than the 4% observed in C57BL/6J mice. The number of tumors/mouse was 7.80 in KK-A(y) mice and also markedly higher than the 0.12 in the C57BL/6J case. Interestingly, adenocarcinomas were observed in most of the AOM-treated KK-A(y) mice along with remarkable tumor angiogenesis, and some showed submucosal invasion. These results indicate that the KK-A(y) mouse, featuring intact leptin and leptin receptor Ob-Rbl, could be a useful animal model to investigate obesity-associated cancer.

Overexpression of low-density lipoprotein receptor and lipid accumulation in intestinal polyps in Min mice.

Apc-deficient Min mice feature low expression of lipoprotein lipase (LPL), high concentration of serum triglyceride (TG), fatty change of the liver and large numbers of intestinal polyps. We have reported that induction of LPL expression reduces serum lipid, especially TG, improves fatty change of the liver and inhibits intestinal polyp formation in the mice. In this study, fatty change/lipid accumulation in intestinal mucosa and polyps in Min mice were analyzed by Oil-red O staining and electron microscopy. A number of large lipid droplets were found in the epithelia of the upper part of polyps. On the other hand, small lipid droplets were only slightly observed at the tip of the villi in non-tumoros parts of the small intestine of Min mice and in the villi of wild-type mice. Moreover, low-density lipoprotein receptor (LDLR) was overexpressed in the area where lipid droplets were observed. The expression levels of LDLR mRNA in the intestinal polyps of Min mice were approximately 3 times higher compared to those in the non-tumoros parts. Remarkable expression of cyclooxygenase-2 was mainly distributed in stromal cells and some in epithelial cells. It is speculated that lipid accumulation in the intestinal polyps may play an important role in intestinal polyp formation in Apc-deficient mice.