In Vitro Effects of Paclitaxel and Cremophor EL on Human Riboflavin Transporter SLC52A2.

Paclitaxel, a mitotic inhibitor with anti-cancer effects, is dissolved in Cremophor EL (CrEL). However, peripheral neuropathy is a known side effect. As one of the mechanisms of the neuropathy, mitochondrial dysfunction has been proposed, while peroxidation products are involved in the cause of CrEL-induced neurotoxicity. Riboflavin is an essential nutrient required for ATP production in mitochondria and has an antioxidant role as a coenzyme for glutathione. Therefore, riboflavin transporters might play a key role to mitigate neuropathy. However, it is unclear whether paclitaxel and CrEL affect these transporters. In this study, human riboflavin transporter SLC52A2 was used to analyze the effects of paclitaxel and CrEL. CrEL, but not paclitaxel, inhibited uptake of riboflavin in human embryonic kidney 293 cells transfected with the SLC52A2 expression vector, suggesting that altered riboflavin disposition may be involved in the pathogenesis of paclitaxel/CrEL toxicity.

Riboflavin uptake transporter Slc52a2 (RFVT2) is upregulated in the mouse mammary gland during lactation.

While it is well recognized that riboflavin accumulates in breast milk as an essential vitamin for neonates, transport mechanisms for its milk excretion are not well characterized. The multidrug efflux transporter ABCG2 in the apical membrane of milk-producing mammary epithelial cells (MECs) is involved with riboflavin excretion. However, it is not clear whether MECs possess other riboflavin transport systems, which may facilitate its basolateral uptake into MECs. We report here that transcripts encoding the second (SLC52A2) and third (SLC52A3) member of the recently discovered family of SLC52A riboflavin uptake transporters are expressed in milk fat globules from human breast milk. Furthermore, Slc52a2 and Slc52a3 mRNA are upregulated in the mouse mammary gland during lactation. Importantly, the induction ofSlc52a2, which was the major Slc52a riboflavin transporter in the lactating mammary gland, was also observed at the protein level. Subcellular localization studies showed that green fluorescent protein-tagged mouse SLC52A2 mainly localized to the cell membrane, with no preferential distribution to the apical or basolateral membrane in polarized kidney MDCK cells. These results strongly implicate a potential role for SLC52A2 in riboflavin uptake by milk-producing MECs, a critical step in the transfer of riboflavin into breast milk.

Genome-wide DNA methylation study of hip and knee cartilage reveals embryonic organ and skeletal system morphogenesis as major pathways involved in osteoarthritis.

BACKGROUND: Evidence suggests that epigenetics plays a role in osteoarthrits (OA). The aim of the study was to describe the genome wide DNA methylation changes in hip and knee OA and identify novel genes and pathways involved in OA by comparing the DNA methylome of the hip and knee osteoarthritic cartilage tissues with those of OA-free individuals. METHODS: Cartilage samples were collected from hip or knee joint replacement patients either due to primary OA or hip fractures as controls. DNA was extracted from the collected cartilage and assayed by Illumina Infinium HumanMethylation450 BeadChip array, which allows for the analysis of >480,000 CpG sites. Student T-test was conducted for each CpG site and those sites with at least 10 % methylation difference and a p value <0.0005 were defined as differentially methylated regions (DMRs) for OA. A sub-analysis was also done for hip and knee OA separately. DAVID v6.7 was used for the functional annotation clustering of the DMR genes. Clustering analysis was done using multiple dimensional scaling and hierarchical clustering methods. RESULTS: The study included 5 patients with hip OA, 6 patients with knee OA and 7 hip cartilage samples from OA-free individuals. The comparisons of hip, knee and combined hip/knee OA patients with controls resulted in 26, 72, and 103 DMRs, respectively. The comparison between hip and knee OA revealed 67 DMRs. The overall number of the sites after considering the overlaps was 239, among which 151 sites were annotated to 145 genes. One-fifth of these genes were reported in previous studies. The functional annotation clustering of the identified genes revealed clusters significantly enriched in skeletal system morphogenesis and development. The analysis revealed significant difference among OA and OA-free cartilage, but less different between hip OA and knee OA. CONCLUSIONS: We found that a number of CpG sites and genes across the genome were differentially methylated in OA patients, a remarkable portion of which seem to be involved in potential etiologic mechanisms of OA. Genes involved in skeletal developmental pathways and embryonic organ morphogenesis may be a potential area for further OA studies.

Role of STAT5 and epigenetics in lactation-associated upregulation of multidrug transporter ABCG2 in the mammary gland.

The multidrug resistance efflux transporter ATP-binding cassette subfamily G member 2 (ABCG2) is not only overexpressed in certain drug-resistant cancers but is also highly expressed in the mammary gland during lactation, carrying xenobiotics and nutrients into milk. We sought to investigate the molecular mechanisms involved in the upregulation of ABCG2 during lactation. Expression profiling of different mouse Abcg2 mRNA isoforms (E1a, E1b, and E1c) revealed that E1b is predominantly expressed and induced in the lactating mouse mammary gland. Despite this induction, analyses of CpG methylation status and published ChIP-seq datasets reveal that E1b promoter sequences in the virgin gland are already hypomethylated and marked with the open chromatin histone mark H3K4me2. Using a forced-weaning model to shut down lactation, we found that within 24 h there was a significant reduction in Abcg2 mRNA expression and a loss of signal transducer and activator of transcription-5 (STAT5) occupancy at the mouse Abcg2 gene. Luciferase reporter assays further showed that some of these STAT5-binding regions that contained interferon-γ-activated sequence (GAS) motifs function as an enhancer after prolactin treatment. We conclude that Abcg2 is already poised for expression in the virgin mammary gland and that STAT5 plays an important role in Abcg2 expression during lactation.

Induction of multidrug resistance transporter ABCG2 by prolactin in human breast cancer cells.

The multidrug transporter, breast cancer resistance protein, ABCG2, is up-regulated in certain chemoresistant cancer cells and in the mammary gland during lactation. We investigated the role of the lactogenic hormone prolactin (PRL) in the regulation of ABCG2. PRL dose-dependently induced ABCG2 expression in T-47D human breast cancer cells. This induction was significantly reduced by short-interfering RNA-mediated knockdown of Janus kinase 2 (JAK2). Knockdown or pharmacologic inhibition of the down-stream signal transducer and activator of transcription-5 (STAT5) also blunted the induction of ABCG2 by PRL, suggesting a role for the JAK2/STAT5 pathway in PRL-induced ABCG2 expression. Corroborating these findings, we observed PRL-stimulated STAT5 recruitment to a region containing a putative γ-interferon activation sequence (GAS) element at -434 base pairs upstream of the ABCG2 transcription start site. Introduction of a single mutation to the -434 GAS element significantly attenuated PRL-stimulated activity of a luciferase reporter driven by the ABCG2 gene promoter and 5'-flanking region containing the -434 GAS motif. In addition, this GAS element showed strong copy number dependency in its response to PRL treatment. Interestingly, inhibitors against the mitogen-activated protein kinase and phosphoinositide-3-kinase signaling pathways significantly decreased the induction of ABCG2 by PRL without altering STAT5 recruitment to the GAS element. We conclude that the JAK2/STAT5 pathway is required but not sufficient for the induction of ABCG2 by PRL.

Identification of aryl hydrocarbon receptor binding targets in mouse hepatic tissue treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Genome-wide, promoter-focused ChIP-chip analysis of hepatic aryl hydrocarbon receptor (AHR) binding sites was conducted in 8-week old female C57BL/6 treated with 30 µg/kg/body weight 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) for 2 h and 24 h. These studies identified 1642 and 508 AHR-bound regions at 2h and 24h, respectively. A total of 430 AHR-bound regions were common between the two time points, corresponding to 403 unique genes. Comparison with previous AHR ChIP-chip studies in mouse hepatoma cells revealed that only 62 of the putative target genes overlapped with the 2 h AHR-bound regions in vivo. Transcription factor binding site analysis revealed an over-representation of aryl hydrocarbon response elements (AHREs) in AHR-bound regions with 53% (2 h) and 68% (24 h) of them containing at least one AHRE. In addition to AHREs, E2f-Myc activator motifs previously implicated in AHR function, as well as a number of other motifs, including Sp1, nuclear receptor subfamily 2 factor, and early growth response factor motifs were also identified. Expression microarray studies identified 133 unique genes differentially regulated after 4 h treatment with TCDD. Of which, 39 were identified as AHR-bound genes at 2 h. Ingenuity Pathway Analysis on the 39 AHR-bound TCDD responsive genes identified potential perturbation in biological processes such as lipid metabolism, drug metabolism, and endocrine system development as a result of TCDD-mediated AHR activation. Our findings identify direct AHR target genes in vivo, highlight in vitro and in vivo differences in AHR signaling and show that AHR recruitment does not necessarily result in changes in target gene expression.

Aryl hydrocarbon receptor is a transcriptional activator of the human breast cancer resistance protein (BCRP/ABCG2).

Breast cancer resistance protein (BCRP/ABCG2) is a membrane-bound efflux transporter important in cellular detoxification and multidrug resistance. Some aryl hydrocarbon receptor (AHR) agonists were reported to induce BCRP expression in human colon carcinoma cells. However, a direct involvement of AHR transcriptional regulation remains unexplored. In this study, we show that BCRP induction by AHR ligands occurs in human intestinal, liver, and mammary carcinoma cells and in primary colonocytes and hepatocytes. Increased BCRP transporter activity consistent with gene induction was also evident in the Caco2 subclone C2bbe1 cells. Using RNA interference and ectopic expression techniques to manipulate cellular AHR status, we confirmed AHR dependence of ABCG2 gene regulation. By gene promoter analysis, chromatin immunoprecipitation, and electrophoretic mobility shift assays, an active, proximal dioxin-response element at -194/-190 base pairs upstream of the transcription start site of the human ABCG2 gene was identified. Despite a common observation in human-derived cells, our in vitro and in vivo studies supported by phylogenetic footprinting analysis did not find that mouse Abcg2 is subject to AHR regulation. We conclude that AHR is a direct transcriptional regulator of human BCRP and provide an unprecedented role of AHR in cellular adaptive response and cytoprotection by up-regulating an important ATP-binding cassette efflux transporter.

Cigarette smoking, genetic variants in carcinogen-metabolizing enzymes, and colorectal cancer risk.

The risk of colorectal cancer associated with smoking is unclear and may be influenced by genetic variation in enzymes that metabolize cigarette carcinogens. The authors examined the colorectal cancer risk associated with smoking and 26 variants in carcinogen metabolism genes in 1,174 colorectal cancer cases and 1,293 population-based controls recruited in Canada by the Ontario Familial Colorectal Cancer Registry from 1997 to 2001. Adjusted odds ratios were calculated by multivariable logistic regression. Smoking for >27 years was associated with a statistically significant increased colorectal cancer risk (adjusted odds ratio (AOR) = 1.25, 95% confidence interval (CI): 1.02, 1.53) in all subjects. Colorectal cancer risk associated with smoking was higher in males for smoking status, duration, and intensity. The CYP1A1-3801-CC (AOR = 0.47, 95% CI: 0.23, 0.94) and CYP2C9-430-CT (AOR = 0.82, 95% CI: 0.68, 0.99) genotypes were associated with decreased risk, and the GSTM1-K173N-CG (AOR = 1.99, 95% CI: 1.21, 3.25) genotype was associated with an increased risk of colorectal cancer. Statistical interactions between smoking and genetic variants were assessed by comparing logistic regression models with and without a multiplicative interaction term. Significant interactions were observed between smoking status and SULT1A1-638 (P = 0.02), NAT2-857 (P = 0.01), and CYP1B1-4390 (P = 0.04) variants and between smoking duration and NAT1-1088 (P = 0.02), SULT1A1-638 (P = 0.04), and NAT1-acetylator (P = 0.03) status. These findings support the hypothesis that prolonged cigarette smoking is associated with increased risk of colorectal cancer and that this risk may be modified by variation in carcinogen metabolism genes.

Receptor- and reactive intermediate-mediated mechanisms of teratogenesis.

Drugs and environmental chemicals can adversely alter the development of the fetus at critical periods during pregnancy, resulting in death, or in structural and functional birth defects in the surviving offspring. This process of teratogenesis may not be evident until a decade or more after birth. Postnatal functional abnormalities include deficits in brain function, a variety of metabolic diseases, and cancer. Due to the high degree of fetal cellular division and differentiation, and to differences from the adult in many biochemical pathways, the fetus is highly susceptible to teratogens, typically at low exposure levels that do not harm the mother. Insights into the mechanisms of teratogenesis come primarily from animal models and in vitro systems, and involve either receptor-mediated or reactive intermediate-mediated processes. Receptor-mediated mechanisms involving the reversible binding of xenobiotic substrates to a specific receptor are exemplified herein by the interaction of the environmental chemical 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD or "dioxin") with the cytosolic aryl hydrocarbon receptor (AHR), which translocates to the nucleus and, in association with other proteins, binds to AH-responsive elements (AHREs) in numerous genes, initiating changes in gene transcription that can perturb development. Alternatively, many xenobiotics are bioactivated by fetal enzymes like the cytochromes P450 (CYPs) and prostaglandin H synthases (PHSs) to highly unstable electrophilic or free radical reactive intermediates. Electrophilic reactive intermediates can covalently (irreversibly) bind to and alter the function of essential cellular macromolecules (proteins, DNA), causing developmental anomalies. Free radical reactive intermediates can enhance the formation of reactive oxygen species (ROS), resulting in oxidative damage to cellular macromolecules and/or altered signal transduction. The teratogenicity of reactive intermediates is determined to a large extent by the balance among embryonic and fetal pathways of xenobiotic bioactivation, detoxification of the xenobiotic reactive intermediate, detoxification of ROS, and repair of oxidative macromolecular damage.

Adopting High-Resolution Allele Frequencies Substantially Expedites Variant Interpretation in Genetic Diagnostic Laboratories.

A cohort of 1242 individuals tested in a clinical diagnostic laboratory was used to test whether the filtering allele frequencies (FAFs)-based framework, recently recommended for MHY7-associated cardiomyopathy, is extendable to 45 cardiomyopathy genes. Statistical analysis revealed a threshold of 0.00164% for the extreme outlier allele frequencies (AFs), based on the Genome Aggregation Database (exome fraction) total AFs of 138 unique pathogenic and likely pathogenic variants; 135 of them (97.8%) had AFs of <0.004%, the recommended threshold to apply moderate pathogenicity evidence for MYH7-associated cardiomyopathy. Of the 460 cases reported with only variant(s) of unknown clinical significance (VUCSs), 97 (21%) solely had VUCSs with FAFs >0.03%, frequencies above which were estimated herein as strong evidence against pathogenicity. Interestingly, 74.5% (172/231) of the unique VUCSs with FAFs >0.03% had Genome Aggregation Database maximum allele frequencies across all populations AFs >0.1%, deemed herein as stand-alone evidence against pathogenicity. Accordingly, using an FAF threshold of >0.1%, compared with AF >1%, led us to issue considerably more (25.9% versus 41.3%) negative patient reports. Also, 82.7% (N = 629) of the unique classified benign or likely benign variants with AFs <1% had FAFs >0.1%, reinforcing the use of this filtering strategy. Together, these data demonstrate that implementing FAF thresholds may considerably decrease the amount of variant interpretations and significantly reduce the cost of genetic testing for clinical genetic laboratories, without compromising the accuracy of genetic diagnostic services.

Red meat intake, doneness, polymorphisms in genes that encode carcinogen-metabolizing enzymes, and colorectal cancer risk.

Colorectal cancer literature regarding the interaction between polymorphisms in carcinogen-metabolizing enzymes and red meat intake/doneness is inconsistent. A case-control study was conducted to evaluate the interaction between red meat consumption, doneness, and polymorphisms in carcinogen-metabolizing enzymes. Colorectal cancer cases diagnosed 1997 to 2000, ages 20 to 74 years, were identified through the population-based Ontario Cancer Registry and recruited by the Ontario Family Colorectal Cancer Registry. Controls were sex-matched and age group-matched random sample of Ontario population. Epidemiologic and food questionnaires were completed by 1,095 cases and 1,890 controls; blood was provided by 842 and 1,251, respectively. Multivariate logistic regression was used to obtain adjusted odds ratio (OR) estimates. Increased red meat intake was associated with increased colorectal cancer risk [OR (> 5 versus < or = 2 servings/wk), 1.67 (1.36-2.05)]. Colorectal cancer risk also increased significantly with well-done meat intake [OR (> 2 servings/wk well-done versus < or = 2 servings/wk rare-regular), 1.57 (1.27-1.93)]. We evaluated interactions between genetic variants in 15 enzymes involved in the metabolism of carcinogens in overcooked meat (cytochrome P450, glutathione S-transferase, UDP-glucuronosyltransferases, SULT, NAT, mEH, and AHR). CYP2C9 and NAT2 variants were associated with colorectal cancer risk. Red meat intake was associated with increased colorectal cancer risk regardless of genotypes; however, CYP1B1 combined variant and SULT1A1-638G>A variant significantly modified the association between red meat doneness intake and colorectal cancer risk. In conclusion, well-done red meat intake was associated with an increased risk of colorectal cancer regardless of carcinogen-metabolizing genotype, although our data suggest that persons with CYP1B1 and SULT1A1 variants had the highest colorectal cancer risk.

Aryl hydrocarbon receptor-dependent induction of flavin-containing monooxygenase mRNAs in mouse liver.

Flavin-containing monooxygenases (FMOs) are important in detoxication but generally are considered not to be inducible by xenobiotics. Our recent microarray studies revealed induction of FMO2 and FMO3 mRNAs by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in liver of mice with wild-type aryl hydrocarbon receptor (AHR) but not in Ahr-null mice. The aim of the present study was to delineate mechanisms of FMO regulation. In adult male mice, basal FMO3 mRNA is low but was induced 6-fold at 4 h and 6000-fold at 24 h. The ED50 was approximately 1 microg/kg for FMO2 and FMO3, similar to that for the classic AHR-regulated gene, Cyp1a1. In adult female mice basal FMO3 mRNA is high and was not induced at 4 h but was elevated 8-fold at 24 h. FMO5 mRNA was significantly down-regulated by TCDD in both male and female adult mice. Juvenile mice show no sex difference in response to TCDD; FMO3 was induced 4 to 6-fold by TCDD in both sexes. Chromatin immunoprecipitation demonstrated recruitment of AHR and aryl hydrocarbon nuclear translocator proteins to Fmo3 regulatory regions, suggesting that induction by TCDD is a primary AHR-mediated event. Although FMO2 and FMO3 mRNAs were highly induced by TCDD in adult males, overall FMO catalytic activity increased only modestly. In contrast to the striking up-regulation of FMO2 and FMO3 in mouse liver, TCDD has little effect on FMO mRNA in rat liver. However, FMO2 and FMO3 mRNAs were highly induced in transgenic mice that express wild-type rat AHR, indicating that lack of induction in rat is not due to an incompetent AHR in this species.

Effect of weight loss and nutritional intervention on arterial stiffness in type 2 diabetes.

OBJECTIVE: There is increased stiffness of the large central arteries in type 2 diabetic patients, and obesity is a risk factor. However, the effect of intentional weight loss on arterial stiffness is uncertain, and the purpose of the current study was to assess this effect. RESEARCH DESIGN AND METHODS: Arterial stiffness was assessed by measuring aortic pulse wave velocity (aPWV) at baseline and at completion of a 1-year weight loss intervention. Metabolic control of type 2 diabetes was also appraised. RESULTS: Mean weight loss at 1 year in 38 volunteers with type 2 diabetes was 7.8%. There were improvements in HbA1c, LDL cholesterol, homeostasis model assessment of insulin resistance, and inflammatory markers (plasminogen activator inhibitor-1, tumor necrosis factor-alpha, interleukin-6, and C-reactive protein). There was also a significant improvement in aPWV at completion of weight loss intervention, from 740 to 690 cm/s (P < 0.05). CONCLUSIONS: Moderate weight loss improves arterial stiffness in type 2 diabetes.

Regulating the regulator: factors that control levels and activity of the aryl hydrocarbon receptor.

The aryl hydrocarbon receptor (AHR) participates in a wide range of critical cellular events in response to endogenous signals or xenobiotic chemicals. Hence, it is important that AHR levels and activity themselves be well controlled in target tissues. The AHR is essentially ubiquitous in its distribution in mammalian tissues. However, levels of the receptor vary widely across different tissues and among different cell types. AHR levels and activity are modulated by exposure to the receptor's own ligands and are influenced by other xenobiotic chemicals. Many different factors impinge on AHR levels and AHR activity. These factors may alter responsiveness of downstream pathways, thereby affecting normal physiologic functions as well as responses to toxic environmental chemicals such as dioxins. Our commentary appraises the current literature on factors that regulate AHR levels/activity and attempts to identify fruitful strategies towards discovery of key pathways by which AHR levels are modulated in response to endogenous signals and in response to xenobiotic chemicals. An extraordinarily large number of agents alter the level or activity of the AHR. We have not yet entered an age of enlightenment sufficient to achieve true understanding of the interplay of mechanisms that regulate AHR expression in space and in time.

Gas-phase ambient air contaminants exhibit significant dioxin-like and estrogen-like activity in vitro.

Several adverse health effects, such as respiratory and cardiovascular morbidity, have been linked to exposure to particulate matter in ambient air; however, the biologic activity of gas-phase ambient organic air contaminants has not been examined as thoroughly. Using aryl hydrocarbon receptor (AHR)-based and estrogen receptor (ER)-based cell bioassay systems, we assessed the dioxin-like and estrogenic activities of gas-phase organic ambient air contaminants compared with those of particulate-phase contaminants using samples collected between seasons over 2 years from an urban and a rural location in the Greater Toronto Area, Canada. The concentration of the sum (Sigma) of polycyclic aromatic hydrocarbons, which was highest in the gas phase, was 10-100 times more abundant than that of Sigmapolychlorinated biphenyls, Sigmanitro-polycyclic aromatic hydrocarbons, and Sigmaorganochlorine pesticides, and 10(3) to 10(4) times more abundant than Sigmapolychlorinated dibenzo-p-dioxins/dibenzofurans. Gas-phase samples induced significant AHR- and ER-dependent gene expression. The activity of the gas-phase samples was greater than that of the particulate-phase samples in the estrogen assay and, in one case, in the AHR assay. We found no strong associations between either summer or winter seasons or urban or rural locations in the relative efficacy of the extracts in either the ER or AHR assay despite differences in chemical composition, concentrations, and abundance. Our results suggest that mechanistic studies of the health effects of ambient air must consider gas and particulate phases because chemicals present in both phases can affect AHR and ER signaling pathways.

In utero exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin induces amphiregulin gene expression in the developing mouse ureter.

Exposure to the environmental contaminant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), produces hydronephrosis in developing mice, the etiology of which involves hyperplasia within the ureteric luminal epithelium. Dysregulation of epidermal growth factor receptor (EGFR), EGF, and transforming growth factor-alpha expression has been implicated as playing a role in TCDD-induced hydronephrosis. In this study, changes in the expression of genes encoding the EGFR and its cognate ligands in response to TCDD were evaluated within the developing ureter. C57BL/6 dams were injected ip with 30 mug/kg TCDD on gestational day (GD) 13 or 16 and fetal tissues removed on GD 17. Aryl hydrocarbon receptor (AHR) and AHR nuclear translocator messenger RNA (mRNA) were expressed in control and treated fetal tissues at GD 14 and 17. Prototypical AHR target genes, Cyp1a1, Cyp1a2, and Cyp1b1 were upregulated in TCDD-exposed fetal tissues, demonstrating AHR transcriptional activity at these developmental stages. Amphiregulin (AREG) and epiregulin, ligands for the EGFR, were induced at the transcriptional level in ureters of fetuses exposed to TCDD for 24 h. AREG mRNA was also induced by TCDD dose- and time-dependently in the mouse hepatoma cell line Hepa-1c1c7 (Hepa-1), mimicking the induction patterns of CYP1A1 mRNA. Other AHR ligands also induced AREG mRNA in Hepa-1 cells. Furthermore, variant Hepa-1 cells (TAOBP(r)c1 cells) virtually deficient in the AHR failed to display an increase in AREG mRNA in response to TCDD. Taken together, these data suggest that the AHR cross talks with the EGFR signaling pathway by directly inducing the expression of growth factors that are important for EGFR signaling in the developing mouse ureter.

SMAD3 Is Upregulated in Human Osteoarthritic Cartilage Independent of the Promoter DNA Methylation.

OBJECTIVE: To compare SMAD3 gene expression between human osteoarthritic and healthy cartilage and to examine whether expression is regulated by the promoter DNA methylation of the gene. METHODS: Human cartilage samples were collected from patients undergoing total hip/knee joint replacement surgery due to primary osteoarthritis (OA), and from patients with hip fractures as controls. DNA/RNA was extracted from the cartilage tissues. Real-time quantitative PCR was performed to measure gene expression, and Sequenom EpiTyper was used to assay DNA methylation. Mann-Whitney test was used to compare the methylation and expression levels between OA cases and controls. Spearman rank correlation coefficient was calculated to examine the association between the methylation and gene expression. RESULTS: A total of 58 patients with OA (36 women, 22 men; mean age 64 ± 9 yrs) and 55 controls (43 women, 12 men; mean age 79 ± 10 yrs) were studied. SMAD3 expression was on average 83% higher in OA cartilage than in controls (p = 0.0005). No difference was observed for DNA methylation levels in the SMAD3 promoter region between OA cases and controls. No correlation was found between SMAD3 expression and promoter DNA methylation. CONCLUSION: Our study demonstrates that SMAD3 is significantly overexpressed in OA. This overexpression cannot be explained by DNA methylation in the promoter region. The results suggest that the transforming growth factor-ß/SMAD3 pathway may be overactivated in OA cartilage and has potential in developing targeted therapies for OA.

Induction of cytochrome P450 1A by cow milk-based formula: a comparative study between human milk and formula.

During the treatment of neonatal apnea, formula-fed infants, compared to breastfed infants, show nearly three-fold increase in clearance of caffeine, a substrate of cytochrome P450 1A (CYP1A) and in part CYP3A4. However, human milk is known to contain higher concentrations of environmental pollutants than infant formula, which are potent CYP1A inducers. To gain insight into the mechanism underlying this apparent contradiction, we characterized CYP1A and CYP3A4 induction by human milk and cow milk-based infant formula. The mRNA and protein expression of CYP1A1/1A2 were significantly induced by cow milk-based formula, but not by human milk, in HepG2 cells. Luciferase reporter assay demonstrated that cow milk-based formula but not human milk activated aryl hydrocarbon receptor (AhR) significantly. The cotreatment of 3,4-dimethoxyflavone, an AhR antagonist, abolished the formula-induced CYP1A expression. In addition, AhR activation by dibenzo[a,h]anthracene, a potent AhR agonist, was significantly suppressed by infant formula and even more by human milk. In contrast, CYP3A4 mRNA expression was only mildly induced by formula and human milk. Consistently, neither formula nor human milk substantially activated pregnane X receptor (PXR). Effects of whey and soy protein-based formulas on the AhR-CYP1A and the PXR-CYP3A4 pathways were similar to those of cow milk-based formula. In conclusion, infant formula, but not human milk, enhances in vitro CYP1A expression via an AhR-mediated pathway, providing a potential mechanistic basis for the increased caffeine elimination in formula-fed infants.

Effects of moderate weight loss and orlistat on insulin resistance, regional adiposity, and fatty acids in type 2 diabetes.

OBJECTIVE: Moderate weight loss is recommended for overweight and obese patients with type 2 diabetes, and conjunctive use of weight loss medication has been advocated. The current study examined weight loss-dependent and -independent effects of the intestinal lipase inhibitor orlistat at 6 months of treatment, using behavioral intervention (Int) combined with randomized, double-blinded, placebo (P)-controlled treatment with orlistat (O). RESEARCH DESIGN AND METHODS: Metabolic control, insulin sensitivity (IS), regional fat distribution, and fat content in liver and muscle were measured in 39 volunteers with type 2 diabetes in whom all antidiabetic medication was withdrawn 1 month preceding randomization. Weight loss was equivalent in the Int+O and Int+P groups, respectively (-10.3 +/- 1.3 vs. -8.9 +/- 1.1%), and there were identical decreases in visceral adipose tissue (VAT), fat mass (FM), thigh adiposity, and hepatic steatosis. RESULTS: Weight loss resulted in substantial improvement (P < 0.001) in HbA(1c) (-1.6 +/- 0.3 vs. -1.0 +/- 0.4%; NS between groups). IS improved significantly more with orlistat (Delta2.2 +/- 0.4 vs. Delta1.2 +/- 0.4 mg. min(-1). kg(-1) fat-free mass [FFM]; P < 0.05), and plasma free fatty acid (FFA) levels were strongly correlated with IS (r = 0.56; P < 0.001). Orlistat caused greater reductions in fasting plasma FFA (Delta-154 +/- 22 vs. Delta-51 +/- 33 micro mol/l; P < 0.05), insulin-suppressed FFA (Delta-119 +/- 23 vs. Delta-87 +/- 34 micro mol/l; P < 0.05), and fasting plasma glucose (FPG; -62 +/- 9 vs. -32 +/- 8 mg/dl; P = 0.02). Changes in HbA(1c) were correlated with DeltaIS (r = -0.41; P < 0.01) but not with weight loss per se. CONCLUSIONS: At equivalent weight loss, conjunctive use of orlistat resulted in greater improvement in FFA levels and IS.

Overexpression of MMP13 in human osteoarthritic cartilage is associated with the SMAD-independent TGF-ß signalling pathway.

INTRODUCTION: In vitro and animal model of osteoarthritis (OA) studies suggest that TGF-ß signalling is involved in OA, but human data is limited. We undertook this study to elucidate the role of TGF-ß signalling pathway in OA by comparing the expression levels of TGFB1 and BMP2 as ligands, SMAD3 as an intracellular mediator, and MMP13 as a targeted gene between human osteoarthritic and healthy cartilage. METHODS: Human cartilage samples were collected from patients undergoing total hip/knee joint replacement surgery due to primary OA or hip fractures as controls. RNA was extracted from the cartilage tissues. Real-time quantitative PCR was performed to measure gene expression. Mann-Whitney test was utilized to compare the expression levels of TGFB1, BMP2, SMAD3 and MMP13 in human cartilage between OA cases and controls. Spearman's rank correlation coefficient (rho) was calculated to examine the correlation between the expression levels of the four genes studied and non-parametric regression was used to adjust for covariates. RESULTS: A total of 32 OA cases (25 hip OA and 7 knee OA) and 21 healthy controls were included. The expression of TGFB1, SMAD3, and MMP13 were on average 70%, 46%, and 355% higher, respectively, whereas the expression of BMP2 was 88% lower, in OA-affected cartilage than that of controls (all p < 0.03), but no difference was observed between hip and knee OA (all p > 0.4). The expression of TGFB1 was correlated with the expression of SMAD3 (rho = 0.50, p = 0.003) and MMP13 (rho = 0.46, p = 0.007) in OA-affected cartilage and the significance became stronger after adjustment for age, sex, and BMI. The expression of BMP2 was negatively correlated with both TGFB1 (rho = -0.50, p = 0.02) and MMP13 (rho = -0.48, p = 0.02) in healthy cartilage, but the significance was altered after adjustment for the covariates. There was no correlation between the expression of SMAD3 and MMP13. CONCLUSIONS: Our results demonstrate that MMP13 expression is associated with an increased expression of TGFB1 in OA-affected cartilage, possibly through SMAD-independent TGF-ß pathway. Furthermore, TGF-ß/SMAD3 is overactivated in OA cartilage; yet, the consequence of this overactivation remains to be established.