Hepatic cytochrome P450 metabolism suppressed by mast cells in type 1 allergic mice.

Mast cells and Kupffer cells secrete interleukin (IL)-1ß, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α, which stimulate excess nitric oxide (NO) producing-inducible NO synthase (iNOS). Unlike Kupffer cells, immunoglobulin E-sensitized mast cells elicit sustained NO production. We investigated the participation of mast cell-released NO and cytokine-derived iNOS activation in type 1 allergy-suppressed hepatic cytochrome P450 (CYP) metabolism. Aminoguanidine, a selective iNOS inhibitor, completely suppressed serum nitrate plus nitrite (NOx) concentrations after primary and secondary sensitization of ICR mice and markedly attenuated allergy-suppressed hepatic CYP1A2, CYP2C, CYP2E1, and CYP3A activities. In the liver, primary and secondary sensitization enhanced iNOS-stimulating IFN-γ (5-15-fold) and TNF-α (3-5-fold) mRNA levels more than IL-1ß (2-fold) and F4/80-positive Kupffer cell (2-fold) mRNA levels. When mast cell-deficient (-/-) mice were sensitized, hepatic CYP activities were not suppressed. Serum NOx levels in the sensitized -/- mice were similar with those in saline-treated ICR and -/- mice. In the liver of -/- mice, secondary sensitization markedly enhanced mRNA expression of iNOS (20-fold), IFN-γ (15-fold), and TNF-α (3-fold). However, hepatic total NOS activities in -/- mice were not significantly different between saline treatment and sensitization. Similarly, primary and secondary ICR mice did not significantly enhance total NOS activities in the liver and hepatocytes. The total NOS activities observed did not relate to the high levels of iNOS, IFN-γ, and TNF-α mRNA in the liver. Hepatic c-kit-positive mast cells in sensitized ICR mice were maintained at control levels. Therefore, our data suggest that mast cell-released NO participates in type 1 allergy-suppressed CYP1A2, CYP2C, CYP2E1, and CYP3A metabolism.

Hepatic Flavin-Containing Monooxygenase 3 Enzyme Suppressed by Type 1 Allergy-Produced Nitric Oxide.

Flavin-containing monooxygenases (FMOs) are major mammalian non-cytochrome P450 oxidative enzymes. T helper 2 cell-activated allergic diseases produce excess levels of nitric oxide (NO) that modify the functions of proteins. However, it remains unclear whether allergy-induced NO affects the pharmacokinetics of drugs metabolized by FMOs. This study investigated alterations of hepatic microsomal FMO1 and FMO3 activities in type 1 allergic mice and further examined the interaction of FMO1 and FMO3 with allergy-induced NO. Imipramine (IMP; FMO1 substrate) N-oxidation activity was not altered in allergic mice with high serum NO and immunoglobulin E levels. At 7 days after primary sensitization (PS7) or secondary sensitization (SS7), benzydamine (BDZ; FMO1 and FMO3 substrate) N-oxygenation was significantly decreased to 70% of individual controls. The expression levels of FMO1 and FMO3 proteins were not significantly changed in the sensitized mice. Hepatic inducible NO synthase (iNOS) mRNA level increased 5-fold and 15-fold in PS7 and SS7 mice, respectively, and hepatic tumor necrosis factor-α levels were greatly enhanced. When a selective iNOS inhibitor was injected into allergic mice, serum NO levels and BDZ N-oxygenation activity returned to control levels. NO directly suppressed BDZ N-oxygenation, which was probably related to FMO3-dependent metabolism in comparison with IMP N-oxidation. In hepatic microsomes from PS7 and SS7 mice, the suppression of BDZ N-oxygenation was restored by ascorbate. Therefore, type 1 allergic mice had differentially suppressed FMO3-dependent BDZ N-oxygenation. The suppression of FMO3 metabolism related to reversible S-nitrosyl modifications of iNOS-derived NO. NO is expected to alter FMO3-metabolic capacity-limited drug pharmacokinetics in humans.

Whole-body insulin resistance is associated with elevated serum α-fetoprotein levels in patients with chronic hepatitis C.

OBJECTIVE: Little is known about the relationship between elevated serum α-fetoprotein (AFP) levels and insulin resistance, which adversely influence the clinical course of chronic hepatitis C (CHC). Therefore, we investigated the association between serum AFP and insulin resistance in patients with CHC. METHODS: We retrospectively investigated 300 patients with CHC without hepatoma who underwent liver biopsies and oral glucose tolerance tests. Patients taking antidiabetic drugs were excluded. We analyzed factors associated with elevated AFP levels (≥ 10.0 ng/mL) in 265 eligible patients. Twenty patients with a homeostasis model assessment for insulin resistance value of ≥ 2.0 and a whole-body insulin sensitivity index of <5.0 received prospective lifestyle intervention. RESULTS: A univariate analysis showed that the body mass index, platelet count, levels of albumin, aspartate aminotransferase, alanine aminotransferase and γ-glutamyl transpeptidase, glucose metabolism, hepatic inflammation, fibrosis and steatosis were associated with elevated AFP levels. In a multivariate analysis, a platelet count of < 15 × 10(4) /µL, aspartate aminotransferase level of ≥ 50 IU/L, γ-glutamyl transpeptidase level of ≥ 35 IU/L, whole-body insulin sensitivity index of <5.0 and stage 3-4 fibrosis were independently associated with an elevated AFP level. A Bayesian Network analysis showed that the aspartate aminotransferase level, whole-body insulin sensitivity index and hepatic fibrosis were directly associated with an elevated AFP level. The lifestyle intervention significantly improved the serum AFP level, homeostasis model assessment for insulin resistance and whole-body insulin sensitivity index. CONCLUSION: Whole-body insulin resistance is associated with an elevated serum AFP level in patients with CHC. Lifestyle interventions targeting insulin resistance can reduce the serum AFP level and may ameliorate the clinical course of CHC.

Subcutaneous panniculitis-like T-cell lymphoma (SPTCL) with hemophagocytosis (HPS): successful treatment using high-dose chemotherapy (BFM-NHL & ALL-90) and autologous peripheral blood stem cell transplantation.

Subcutaneous panniculitis-like T-cell lymphoma (SPTCL) is a rare form of non-Hodgkin lymphoma, in which lymphoma cells infiltrate preferentially into subcutaneous adipose tissue. Although various treatment trials for SPTCL have been attempted, no standardized therapy has been established. Here, we report a case of α/ß(+) T-cell-phenotype SPTCL (SPTCL-AB) with hemophagocytosis (HPS) in a 14-year-old girl, who presented with low-grade fever, general fatigue and chest swelling. Laboratory examinations revealed leukocytopenia, and bone marrow aspiration cytology showed HPS. The diagnosis of SPTCL-AB was made by biopsy on the basis of thickened subcutaneous tissue in the chest wall. Following high-dose chemotherapy (HDT) of BFM-NHL & ALL-90, autologous peripheral blood stem cell transplantation (auto-PBSCT) was performed. The patient responded to the treatment and has remained asymptomatic for 2 years. Our results suggest that a combination of HDT of BFM-NHL & ALL-90 and auto-SCT treatment is effective for SPTCL associated with HPS.

Downregulation of microRNA-211 is involved in expression of preferentially expressed antigen of melanoma in melanoma cells.

MicroRNAs (miRNAs) are small non-coding RNAs whose aberrations are involved in the initiation and progression of human cancers. To seek unique miRNAs contributing to melanoma tumorigenesis, we investigated the global miRNA expression profile of 7 melanoma cell lines and 3 primary cultures of neonatal human epidermal melanocytes (NHEMs) using the stem-loop real-time PCR method. We found 7 miRNAs that were commonly downregulated and 18 that were upregulated in all of the melanoma cell lines in comparison with the 3 primary cultures of NHEMs. We focused on one commonly downregulated miRNA (miR-211), and analyzed its relationship to the expression of preferentially expressed antigen of melanoma (PRAME) protein, which is a potential target of miR-211. We found that all melanoma cell lines exhibited marked down--regulation of miR-211 and upregulation of PRAME mRNA/protein expression in comparison with NHEMs (P<0.05). A significant inverse correlation between miR-211 and PRAME protein expression was found in melanoma cell lines and primary cultures of NHEMs (correlation coefficient of -0.733, P<0.05). We demonstrated that overexpression of miR-211 induced a reduction of PRAME protein levels, and confirmed the target specificity between miR-211 and PRAME by luciferase reporter assay. These results suggest that downregulation of miR-211 may be partly involved in aberrant expression of the PRAME protein in melanoma cells.

Angiotensin II and epidermal growth factor receptor cross-talk mediated by a disintegrin and metalloprotease accelerates tumor cell proliferation of hepatocellular carcinoma cell lines.

AIM: The cross-talk pathway between angiotensin II (AngII) and the epidermal growth factor receptor (EGFR) mediated by epidermal growth factor (EGF)-like ligands cleaved by a disintegrin and metalloprotease (ADAM) has been elucidated in several cell types. Even though the liver is a representative angiotensinogen-producing organ, such cross-talk has never been elucidated in hepatocellular carcinomas (HCCs). We investigated whether AngII exerted a mitogenic effect on HCC cell lines through the AngII-EGFR cross-talk pathway. METHODS: We determined the expression and/or phosphorylation status of AngII receptor type 1 (AGTR1), ADAM9, ADAM17, ERK1/2, STAT3, AKT and EGFR in five HCC cell lines using Western blotting. Proliferation and invasion activities were measured by ATP and Matrigel invasion assays, respectively. RESULTS: AGTR1 was expressed ubiquitously in HCC cell lines. EGFR expression in HepG2 was relatively weaker than that in the remaining HCC cell lines. The phosphorylation status of EGFR, ERK1/2, STAT3 and AKT was upregulated by AngII treatment in two EGFR-overexpressing cell lines (Huh7 and PLC/PRF/5), but not in HepG2 (showing weak EGFR expression). AngII stimulation significantly accelerated proliferation and invasion activities in Huh7 and PLC/PRF/5, and was inhibited by pretreatment with an ADAM inhibitor. A selective AGTR1 blocker significantly repressed proliferation activity in both cell lines, but did not significantly repress the invasion activity. Both chemical agents and neutralizing antibodies against ADAMs (ADAM9 and ADAM17) and EGF-like ligands suppressed EGFR transactivation and/or subsequent phosphorylation of ERK1/2, STAT3 and AKT. CONCLUSION: These results suggest that AngII-EGFR cross-talk signaling mediated by ADAMs is involved in the proliferation and invasion activities of several HCC cell lines.

Metabolism of nicotine in rat lung microvascular endothelial cells.

The aim of this study was to examine whether cultured rat lung microvascular endothelial cells (LMECs), which constitute the gas-blood barrier, have the ability to metabolize nicotine. Nicotine was biotransformed to cotinine and nicotine N'-oxide by cytochrome 450 (CYP) and flavin-containing monooxyganase (FMO), respectively, in rat LMECs. The intrinsic clearance (Vmax1/Km1) for the cotinine formation was about 20 times as high as that for the trans-nicotine N'-oxide formation in the low-Km phase, indicating that oxidation by CYP was much higher than that by FMO. On the other hand, as shown in Eadie-Hofstee plots, the formation of cis-nicotine N'-oxide was monophasic, whereas the plot for the trans-nicotine N'-oxide formation was clearly biphasic. These results suggest that nicotine N'-oxide was stereoselectively metabolized to cis and trans forms. However, in the high-Km phase there was no significant difference in N'-oxidation between the cis and trans forms. Moreover, we suggest that CYP2C11 and CYP3A2 are key players in the metabolism to cotinine of nicotine in rat LMECs using the respective enzyme inhibitors (tranylcypromine and troleandomycine). On the other hand, methimazole (5 microM) caused 73 and 45% decreases in the formation of N'-oxides of cis- and trans- enantiomers, respectively, demonstrating the presence of FMO in rat LMECs. These results suggest that rat LMEC enzymes can convert substrates of exogenous origin such as nicotine for detoxication, indicating LMECs are an important barrier for metabolic products, besides hepatic cells.