A Revised Classification of Primary Iron Overload Syndromes.

Background and Aims: The clinical introduction of hepcidin25 (Hep25) has led to a more detailed understanding of its relationship with ferroportin (FP) and divalent metal transporter1 in primary iron overload syndromes (PIOSs). In 2012, we proposed a classification of PIOSs based on the Hep25/FP system, which consists of prehepatic aceruloplasminemia, hepatic hemochromatosis (HC), and posthepatic FP disease (FP-D). However, in consideration of accumulated evidence on PIOSs, we aimed to renew the classification. Methods: We reviewed the 2012 classification and retrospectively renewed it according to new information on PIOSs. Results: Iron-loading anemia was included in PIOSs as a prehepatic form because of the newly discovered erythroferrone-induced suppression of Hep25, and the state of traditional FP-D was remodeled as the BIOIRON proposal. The key molecules responsible for prehepatic PIOSs are low transferrin saturation in aceruloplasminemia and increased erythroferrone production by erythroblasts in iron-loading anemia. Hepatic PIOSs comprise four genotypes of HC, in each of which the synthesis of Hep25 is inappropriately reduced in the liver. Hepatic Hep25 synthesis is adequate in posthepatic PIOSs; however, two mutant FP molecules may resist Hep25 differently, resulting in SLC40A1-HC and FP-D, respectively. PIOS phenotypes are diagnosed using laboratory tests, including circulating Hep25, followed by suitable treatments. Direct sequencing of the candidate genes may be outsourced to gene centers when needed. Laboratory kits for the prevalent mutations, such as C282Y, may be the first choice for a genetic analysis of HC in Caucasians. Conclusions: The revised classification may be useful worldwide.

Hyperglycaemia Aggravates Oxidised Low-Density Lipoprotein-Induced Schwann Cell Death via Hyperactivation of Toll-like Receptor 4.

Increased low-density lipoprotein levels are risk factors for diabetic neuropathy. Diabetes mellitus is associated with elevated metabolic stress, leading to oxidised low-density lipoprotein formation. Therefore, it is important to investigate the mechanisms underlying the pathogenesis of diabetic neuropathy in diabetes complicated by dyslipidaemia with increased levels of oxidised low-density lipoprotein. Here, we examined the effects of hyperglycaemia and oxidised low-density lipoprotein treatment on Schwann cell death and its underlying mechanisms. Immortalised mouse Schwann cells were treated with oxidised low-density lipoprotein under normo- or hyperglycaemic conditions. We observed that oxidised low-density lipoprotein-induced cell death increased under hyperglycaemic conditions compared with normoglycaemic conditions. Moreover, hyperglycaemia and oxidised low-density lipoprotein treatment synergistically upregulated the gene and protein expression of toll-like receptor 4. Pre-treatment with TAK-242, a selective toll-like receptor 4 signalling inhibitor, attenuated hyperglycaemia- and oxidised low-density lipoprotein-induced cell death and apoptotic caspase-3 pathway. Our findings suggest that the hyperactivation of toll-like receptor 4 signalling by hyperglycaemia and elevated oxidised low-density lipoprotein levels synergistically exacerbated diabetic neuropathy; thus, it can be a potential therapeutic target for diabetic neuropathy.

A 70-year-old Woman with Asymptomatic Ferroportin Disease.

A 59-year-old Japanese woman presented with hyperferritinemia. We decided against iron removal treatment because there were no symptoms or signs of iron-induced organ damage. A follow-up study revealed a gradual increase in transferrin saturation. The patient underwent a second examination at 66 years old. A liver biopsy showed substantial iron deposits in hepatocytes and Kupffer cells but no inflammation or fibrosis. Serum hepcidin-25 levels were highly parallel with hyperferritinemia. A genetic analysis revealed a G80S mutation in SLC40A1. These features are compatible with those of ferroportin disease. The patient remained asymptomatic at 70 years old, suggesting that the iron-loading condition may have been benign.

One-step embedding method for maintaining orientation of pathological tissue specimens using agar thin films.

Pathological histology examination involves handling a variety of specimens that are cut according to regulations and placed in cassettes. Tissue fragments in the cassettes are then diagnosed after processing, embedding, thin sectioning, staining and other procedures using a processing machine. Maintaining tissue fragment order and orientation during these processes is important for accurate diagnosis. In this study, we present a method of maintaining tissue fragment order and orientation using a thin film of ultra-high-strength agar and evaluate its usefulness during tissue sectioning.Cassettes were prepared, each containing three pieces of porcine liver, and compared embedding time with and without agar thin films (ATFs). Embedding was performed by three medical laboratory scientists with different levels of experience.To enable one-step tissue sample embedding, ATFs were integrated with samples in the cassettes. This resulted in an average reduction of 6.22 s of embedding time per cassette compared with traditional embedding methods.Through the use of ATFs, tissue fragment order and orientation is maintained, and embedding process time shortened. Additionally, ATFs are easily prepared and stored in 10% neutral buffered formalin over extended periods, allowing for immediate use during sectioning. This method is ideal to implement in busy pathology laboratories.

Contrast-enhanced ultrasound imaging for monitoring the efficacy of near-infrared photoimmunotherapy.

BACKGROUND: Near-infrared photoimmunotherapy (NIR-PIT) is a promising cancer therapy combining NIR-light irradiation with an antibody and IR700DX, a light-sensitive substance, to destroy tumours. However, homogeneous irradiation is difficult because the light varies depending on the distance and tissue environment. Therefore, markers that indicate sufficient irradiation are necessary. Nanoparticles sized 10∼200 nm show enhanced permeation and retention within tumours, which is further enhanced via NIR-PIT (super enhanced permeability and retention, SUPR). We aimed to monitor the effectiveness of NIR-PIT by measuring SUPR. METHODS: A xenograft mouse tumour model was established by inoculating human cancer cells in both buttocks of Balb/C-nu/nu mice, and NIR-PIT was performed on only one side. To evaluate SUPR, fluorescent signal examination was performed using QD800-fluorescent nanoparticles and NIR-fluorescent poly (d,l-lactide-co-glycolic acid) (NIR-PLGA) microparticles. Harmonic signals were evaluated using micro-bubbles of the contrast agent Sonazoid and contrast-enhanced ultrasound (CEUS) imaging. The correlation between SUPR immediately after treatment and NIR-PIT effectiveness on the day after treatment was evaluated. FINDINGS: QD800 fluorescent signals persisted only in the treated tumours, and the intensity of remaining signals showed high positive correlation with the therapeutic effect. NIR-PLGA fluorescent signals and Sonazoid-derived harmonic signals remained for a longer time in the treated tumours than in the controls, and the kE value of the two-compartment model correlated with NIR-PIT effectiveness. INTERPRETATION: SUPR measurement using Sonazoid and CEUS imaging could be easily adapted for clinical use as a therapeutic image-based biomarker for monitoring and confirming of NIR-PIT efficacy. FUNDING: This research was supported by ARIM JAPAN of MEXT, the Program for Developing Next-generation Researchers (Japan Science and Technology Agency), KAKEN (18K15923, 21K07217) (JSPS), CREST (JPMJCR19H2, JST), and FOREST-Souhatsu (JST). Mochida Memorial Foundation for Medical and Pharmaceutical Research; Takeda Science Foundation; The Japan Health Foundation; and Princess Takamatsu Cancer Research Fund. Funders only provided financial support and had no role in the study design, data collection, data analysis, interpretation, and writing of the report.

Association between dietary intake of n-3 polyunsaturated fatty acids and risk of colorectal cancer in the Japanese population: The Japan Collaborative Cohort Study.

BACKGROUND: Epidemiological studies of the dietary intake of specific n-3 polyunsaturated fatty acids (PUFA) and anatomical subsite-specific colorectal cancer (CRC) are limited. We examined the prospective associations of total n-3 PUFA, marine-derived n-3 PUFA [combined eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), and docosahexaenoic acid (DHA)], and alpha-linolenic acid (ALA) as plant-derived n-3 PUFA with the risk of CRC by subsite in the Japan Collaborative Cohort Study. METHODS: The participants completed a self-administered food frequency questionnaire and had no prior history of CRC. Cox proportional hazards model was used to determine the associations between n-3 PUFAs intake and CRC risk overall and by anatomical subsite. RESULTS: During the median 13.8-year follow-up period, 699 of the 42,536 participants aged 40-79 years developed incident CRC. An inverse association was found between dietary ALA intake and the risk of distal colon cancer; the multivariable hazard ratios and 95% confidence intervals for the highest quartiles (Q4) were 0.41 (0.21-0.81; p trend = 0.01) compared with the lowest quartiles (Q1). Marine n-3 PUFA intake was not associated with CRC risk in the overall or anatomical subsite-specific analyses. CONCLUSION: Our findings suggest that higher ALA intake may be beneficial for lowering the risk of distal colon cancer.

Docosahexaenoic Acid Suppresses Oxidative Stress-Induced Autophagy and Cell Death via the AMPK-Dependent Signaling Pathway in Immortalized Fischer Rat Schwann Cells 1.

Autophagy is the process by which intracellular components are degraded by lysosomes. It is also activated by oxidative stress; hence, autophagy is thought to be closely related to oxidative stress, one of the major causes of diabetic neuropathy. We previously reported that docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) induced antioxidant enzymes and protected Schwann cells from oxidative stress. However, the relationship between autophagy and oxidative stress-induced cell death in diabetic neuropathy has not been elucidated. Treatment with tert-butyl hydroperoxide (tBHP) decreased the cell survival rate, as measured by an MTT assay in immortalized Fischer rat Schwann cells 1 (IFRS1). A DHA pretreatment significantly prevented tBHP-induced cytotoxicity. tBHP increased autophagy, which was revealed by the ratio of the initiation markers, AMP-activated protein kinase, and UNC51-like kinase phosphorylation. Conversely, the DHA pretreatment suppressed excessive tBHP-induced autophagy signaling. Autophagosomes induced by tBHP in IFRS1 cells were decreased to control levels by the DHA pretreatment whereas autolysosomes were only partially decreased. These results suggest that DHA attenuated excessive autophagy induced by oxidative stress in Schwann cells and may be useful to prevent or reduce cell death in vitro. However, its potentiality to treat diabetic neuropathy must be validated in in vivo studies.

Role of pyruvate in maintaining cell viability and energy production under high-glucose conditions.

Pyruvate functions as a key molecule in energy production and as an antioxidant. The efficacy of pyruvate supplementation in diabetic retinopathy and nephropathy has been shown in animal models; however, its significance in the functional maintenance of neurons and Schwann cells under diabetic conditions remains unknown. We observed rapid and extensive cell death under high-glucose (> 10 mM) and pyruvate-starved conditions. Exposure of Schwann cells to these conditions led to a significant decrease in glycolytic flux, mitochondrial respiration and ATP production, accompanied by enhanced collateral glycolysis pathways (e.g., polyol pathway). Cell death could be prevented by supplementation with 2-oxoglutarate (a TCA cycle intermediate), benfotiamine (the vitamin B1 derivative that suppresses the collateral pathways), or the poly (ADP-ribose) polymerase (PARP) inhibitor, rucaparib. Our findings suggest that exogenous pyruvate plays a pivotal role in maintaining glycolysis-TCA cycle flux and ATP production under high-glucose conditions by suppressing PARP activity.

CDKN1C-mediated growth inhibition by an EZH1/2 dual inhibitor overcomes resistance of mantle cell lymphoma to ibrutinib.

Mantle cell lymphoma (MCL) is a rare subtype of non-Hodgkin's lymphoma, which is characterized by overexpression of cyclin D1. Although novel drugs, such as ibrutinib, show promising clinical outcomes, relapsed MCL often acquires drug resistance. Therefore, alternative approaches for refractory and relapsed MCL are needed. Here, we examined whether a novel inhibitor of enhancer of zeste homologs 1 and 2 (EZH1/2), OR-S1 (a close analog of the clinical-stage compound valemetostat), had an antitumor effect on MCL cells. In an ibrutinib-resistant MCL patient-derived xenograft (PDX) mouse model, OR-S1 treatment by oral administration significantly inhibited MCL tumor growth, whereas ibrutinib did not. In vitro growth assays showed that compared with an established EZH2-specific inhibitor GSK126, OR-S1 had a marked antitumor effect on MCL cell lines. Furthermore, comprehensive gene expression analysis was performed using OR-S1-sensitive or insensitive MCL cell lines and showed that OR-S1 treatment modulated B-cell activation, differentiation, and cell cycle. In addition, we identified Cyclin Dependent Kinase Inhibitor 1C (CDKN1C, also known as p57, KIP2), which contributes to cell cycle arrest, as a direct target of EZH1/2 and showed that its expression influenced MCL cell proliferation. These results suggest that EZH1/2 may be a potential novel target for the treatment of aggressive ibrutinib-resistant MCL via CDKN1C-mediated cell cycle arrest.

Long-term phlebotomy successfully alleviated hepatic iron accumulation in a ferroportin disease patient with a mutation in SLC40A1: a case report.

BACKGROUND: Hereditary hemochromatosis is a heterogenous group of inherited iron-overload conditions that is characterized by increased intestinal absorption and deposition in vital organs. Hepcidin is a soluble regulator that acts to attenuate both intestinal iron absorption and iron release from reticuloendothelial macrophages through internalization of ferroportin-1, an iron exporter. Ferroportin disease is hereditary hemochromatosis which is affected by SLC40A1, a gene coding ferroportin-1, and phenotypically classified into two forms (classical and nonclassical). In nonclassical form, ferroportin mutations are responsible for a gain of function with full iron export capability but insensitivity to downregulation by hepcidin. Here, we report a case of nonclassical ferroportin disease. CASE PRESENTATION: A 46-year-old Japanese man showed elevated serum iron (284 µg/dl), ferritin (1722 ng/ml), transferrin saturation ratio (91.3%), and hepcidin-25 level (139.6 ng/ml). Magnetic resonance imaging (MRI) demonstrated a marked reduction in the signal intensity of the liver in T1- and T2-weighted images. The liver histology exhibited a large amount of iron that had accumulated predominantly in hepatocytes. We identified a heterozygous 1520A > G (p.H507R) mutation in the SLC40A1 gene. Phlebotomy (400 ml at a time) was monthly performed for 3 years in this patient. Importantly, the serum hepcidin level (1.0 ng/ml) was normal when the serum ferritin level was normal and hepatic iron accumulation was remarkably reduced after 3 years of phlebotomy. CONCLUSIONS: The present case demonstrated for the first time that there was a correlation between hepatic iron levels as measured by MRI and serum hepcidin levels through long-term phlebotomy in a patient with ferroportin disease with the p.H507R mutation of in SLC40A1.

Diagnostic performance of 68Ga-DOTATOC PET/CT in tumor-induced osteomalacia.

OBJECTIVE: Tumor-induced osteomalacia (TIO) is caused by typically small tumors that secrete fibroblast growth factor 23 (FGF23). As tumor resection is the only effective treatment for TIO, it is important to detect the culprit tumor. We aimed to assess the utility of 68Gallium-DOTA-D-Phe(1)-Tyr(3)-octreotide (68Ga-DOTATOC) PET/CT in TIO and the correlation between biochemical parameters and the PET/CT results. METHODS: Thirty-five patients with clinically suspected TIO who had undergone 68Ga-DOTATOC PET/CT were retrospectively analyzed. 68Ga-DOTATOC PET/CT results were compared with biochemical parameters and the final diagnosis, including histopathology. RESULTS: 68Ga-DOTATOC PET/CT detected focal uptake consistent with TIO in 21/35 patients, one of which was considered false positive. In 16 patients, the cause of osteomalacia was confirmed histologically as phosphaturic mesenchymal tumor (n = 15) or fibrous dysplasia (n = 1). The other four patients were judged clinically as true positive by subsequent MRI and the clinical course. Overall, the detection rate of 68Ga-DOTATOC PET/CT was 57% (20/35). Median tumor maximum standardized uptake value (SUVmax) was 6.9 (range 1.5-37.7). There was no significant difference in serum intact FGF23 level between DOTATOC-positive and DOTATOC-negative cases, and no significant correlation was observed between intact FGF23 level and tumor SUVmax. CONCLUSIONS: 68Ga-DOTATOC PET/CT was clinically useful in detecting culprit tumors and subsequent patient management in TIO.

A Japanese female with chronic mild anemia and primary iron overloading disease.

A 65-year-old woman died of congestive heart failure and diabetes mellitus. She had a history of mild anemia since adolescence, but received neither iron supplementation nor transfusion. The cirrhotic liver obtained at autopsy contained a large amount of iron. The heart and pancreas also had excess iron. Her iron overload may be due to excess iron absorption in the gut because of the absence of an iatrogenic background such as transfusion or iron supplementation.

Correction: The Smart Life Stay (SLS) program: effects of a lifestyle intervention program in combination with health tourism and health guidance for type 2 diabetes.

Following publication, the authors asked to add the following institution to the affiliations of author Madoka Matsushita: Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine Both the PDF and HTML versions of the Article have been updated accordingly.

The Smart Life Stay (SLS) program: effects of a lifestyle intervention program in combination with health tourism and health guidance for type 2 diabetes.

BACKGROUND: The aim of this study was to determine the effectiveness of the Smart Life Stay (SLS) program, which is an experience-oriented stayover program, in combination with health tourism and mandatory health guidance on glucose metabolism after 2 years. METHODS: The participants of the SLS program (n = 792) were recruited from a database of 23 medical insurers. They underwent a mandatory health examination termed Specific Health Checkups in 2014. The participants were included if they had diabetes or were at a high risk of diabetes and if they satisfied the following inclusion criteria: (1) body mass index (BMI; kg/m2) > 25, or (2) waist circumference (WC; cm) > 85 for men and > 90 for women, or (3) hemoglobin A1c (HbA1c; %) > 5.6, or (4) fasting plasma glucose (FPG; mg/dl) > 100. Individuals who corresponded to one or more items were included as study participants. The control subjects (n = 3645) were nonparticipants of the program who were selected from the database and met the inclusion criteria. The lifestyle changes and changes in mean BMI, WC, FPG, and HbA1c in both groups from baseline to 2-year follow-up were compared by inverse probability weighting of a propensity score. RESULTS: The percentage of people who exercised regularly increased significantly in the SLS group compared with the control group. In the SLS group, BW, BMI, and WC significantly decreased by 1.75 kg, 0.60 kg/m2, and 1.45 cm, respectively, whereas in the control group, WC, FPG, and HbA1c increased significantly by 0.38 cm, 3.37 mg/dl, and 0.12%, respectively. The comparison between groups revealed that the BW, BMI, WC, FPG, and HbA1c improved significantly in the SLS group. CONCLUSIONS: The SLS program is suggested to help improve glucose metabolism. This program could be a feasible option as a lifestyle intervention program for diabetes.

Follicular dendritic cell-secreted protein gene expression is upregulated and spread in nifedipine-induced gingival overgrowth.

Follicular dendritic cell-secreted protein (FDC-SP) is secreted protein expressed in follicular dendritic cells, periodontal ligament and junctional epithelium (JE). Its expression could be controlled during inflammatory process of gingiva; however, responsible mechanism for gingival overgrowth and involvement of FDC-SP in clinical condition is still unclear. We hypothesized that JE-specific genes are associated with the initiation of drug-induced gingival enlargement (DIGE) called gingival overgrowth, and investigated the changes of JE-specific gene's expression and their localization in overgrown gingiva from the patients. Immunohistochemical analysis revealed that the FDC-SP localization was spread in overgrown gingival tissues. FDC-SP mRNA levels in GE1 and Ca9-22 cells were increased by time-dependent nifedipine treatments, similar to other JE-specific genes, such as Amelotin (Amtn) and Lamininß3 subunit (Lamß3), whereas type 4 collagen (Col4) mRNA levels were decreased. Immunocytochemical analysis showed that FDC-SP, AMTN, and Lamß3 protein levels were increased in GE1 and Ca9-22 cells. Transient transfection analyses were performed using luciferase constructs including various lengths of human FDC-SP gene promoter, nifedipine increased luciferase activities of -345 and -948FDC-SP constructs. These results raise the possibility that the nifedipine-induced FDC-SP may be related to the mechanism responsible for gingival overgrowth does not occur at edentulous jaw ridges.

Prognostic Value of Quantitative Parameters of 18F-FDG PET/CT for Patients With Angiosarcoma.

OBJECTIVE. The purpose of this study was to evaluate quantitative parameters in 18F-FDG PET/CT in terms of correlation with histologic grade and overall survival in patients with angiosarcoma. MATERIALS AND METHODS. The cases of 16 patients with histologically confirmed angiosarcoma who had undergone pretreatment FDG PET/CT were retrospectively analyzed. Maximum standardized uptake value for the primary tumor (pSUVmax), metabolic tumor volume (MTV) and total lesion glycolysis (TLG) for the whole body, tumor-to-blood ratio (TBR) for the primary tumor, and summed ratios of tumor-to-blood glycolytic activity for all lesions (whole-body TLG ratio) were calculated. Tumors were divided into high grade and low grade, according to the pathologic results. Correlations between these metabolic parameters and tumor grade were investigated. The prognostic value of these parameters and various clinicopathologic factors with respect to overall survival was assessed with the Cox proportional hazards regression model. RESULTS. Histopathologic examination revealed 10 high-grade and six low-grade tumors. Among the quantitative parameters, pSUVmax (p < 0.0001) and primary TBR (p = 0.0003) were significantly higher for high-grade tumors than for low-grade tumors. Ten patients died during follow-up (median survival time, 19.6 months). Higher pSUVmax (p = 0.040), MTV (p = 0.016), whole-body TLG (p = 0.010), primary TBR (p = 0.019), and whole-body TLG ratio (p = 0.007) correlated significantly with poorer overall survival. Single lesion at initial diagnosis (p = 0.0008) and performance of curative surgery (p = 0.0008) were strong favorable prognostic factors for overall survival, but histologic grade was not identified as a significant predictor. CONCLUSION. In angiosarcoma, high-grade tumors had significantly higher pSUVmax and primary TBR at FDG PET/CT. All quantitative parameters evaluated in this study were found to be significant prognostic factors for overall survival.

EBV LMP1 in Gingival Epithelium Potentially Contributes to Human Chronic Periodontitis via Inducible IL8 Production.

BACKGROUND/AIM: Human chronic periodontitis is a major health problem. Although some oral bacteria have been reported to be putative pathogens, Epstein-Barr virus (EBV) is reported to be associated with the progression of periodontitis. However, the role of EBV in the aetiology of periodontitis is unknown. Therefore, we investigated periodontal pathogenesis of EBV to confirm whether EBV-encoded latent membrane protein 1 (LMP1) induces Interleukin-8 (IL8) production in human gingival cells. MATERIALS AND METHODS: Real-time polymerase chain reaction, luciferase assay, enzyme-linked immunosorbent assay (ELISA), and western blotting were performed for determining IL8 mRNA expression, nuclear factor kappa B (NF-ĸB) transcription, IL8 production, and the phosphorylation of NF-ĸB p65 and Inhibitor of kappa B alpha (IĸBα), respectively, in Ca9-22 human gingival epithelial cells. Two LMP1 mutants lacking C-terminal activating region (CATR) domains responsible for activating NF-ĸB were used. RESULTS: Extremely high IL8 production was induced by LMP1 in time- and dose-dependent manner, where simultaneous phosphorylation of NF-κB p65 and IĸBα and transcription of NF-ĸB were observed. On the contrary, IL8 production and NF-ĸB transcription were drastically inhibited by dominant negative mutant of IĸBα. Moreover, the LMP1 mutants failed to induce IL8 production. CONCLUSION: Our findings suggest that due to CATR domains, LMP1 contributes to the progression of periodontitis via IL8 production attributable to NF-ĸB activation.

Smoking results in accumulation of ectopic fat in the liver.

OBJECTIVE: An association between smoking and nonalcoholic fatty liver disease has been reported. However, objective quantification of intrahepatic fat via magnetic resonance spectroscopy (MRS) in relation to smoking has rarely been performed in previous studies. Moreover, the possible pathways via which smoking could induce ectopic fat accumulation have not yet been addressed. The current study aimed to examine the association between smoking status and intrahepatic fat quantity and explore the possible mediating effects of triglycerides (TG) and adiponectin. SUBJECTS AND METHODS: Magnetic resonance imager (MRI) spectra were analyzed to quantify intrahepatic fat in 45 men who were on average 62.3 years of age. Smoking status and alcohol intake were self-reported. Accelerometers were used to record daily total physical activity. Fasting blood TG and adiponectin levels were measured enzymatically. Differences in mean intrahepatic fat values according to smoking status were assessed using analysis of covariance. RESULTS: A stepwise increase in mean intrahepatic fat was observed between never, former, and current smokers, respectively, independent of age, physical activity, alcohol intake, and body mass index (BMI) (P=0.005). Adjustment for TG and adiponectin significantly attenuated this association (P=0.074). CONCLUSION: Current smoking was significantly associated with increased intrahepatic fat, which may be a result of adipocyte dysfunction, manifested as high circulating TG concentrations and low adiponectin levels.

Effect of inter-individual variability in human liver cytochrome P450 isozymes on cyclophosphamide-induced micronucleus formation.

We investigated the relationship between metabolic activities of cytochrome P450 (CYP) isozymes present in microsomal fractions derived from the livers of 78 donors and micronucleus induction by cyclophosphamide (CPA). Consequently, a wide inter-individual variation in CYP activities was observed among the 78 donors. The CYP activities were partially correlated with the metabolic phenotypes predicted for the donors based on their single nucleotide polymorphisms. In addition, CPA induced micronucleus formation was seen for 47 out of 52 donors whose samples were tested with CPA doses ranging from 18.8 to 100 µg/mL. The CPA dose at which micronucleated cells were observed varied among the donors. Furthermore, a close correlation was identified between the catalytic activities of the CYP2B6, CYP2C9, CYP2C19, and CYP3A4 isozymes and micronucleus induction by CPA. To elucidate the mechanism underlying CPA-induced micronucleus formation in vitro tests were conducted on expression systems of CYP2B6, CYP2C9, CYP2C19, CYP2D6, and CYP3A4. Additionally, the metabolites of CPA generated by the expression systems were quantified by a liquid chromatography tandem mass spectrometer. Interestingly, several metabolites including the 4-hydroxyl form of CPA (4-OH-CPA) and phosphamide mustard were detected in the CYP2B6, CYP2C19, and CYP3A4 expression systems, but not in the CYP2C9 and CYP2D6 system. The presence of these metabolites was correlated with micronucleus induction by CPA. The absence of CPA metabolites in the CYP2C9 expression system might be associated with the lower 4-hydroxylase activity of this system. The present results suggest that inter-individual variability in the metabolic capacity of each donor was associated with potential micronucleus induction due to CPA. Additionally, CPA metabolites like 4-OH-CPA and phosphamide mustard produced by human CYP2B6, CYP2C9, CYP2C19, and CYP3A4 are suggested to be major determinants of micronucleus induction by CPA.