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.

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.

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.

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.

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.

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.

Relationship between blood plasma and synovial fluid metabolite concentrations in patients with osteoarthritis.

OBJECTIVE: To investigate the relationship between plasma and synovial fluid (SF) metabolite concentrations in patients with osteoarthritis (OA). METHODS: Blood plasma and SF samples were collected from patients with primary knee OA undergoing total knee arthroplasty. Metabolic profiling was performed by electrospray ionization tandem mass spectrometry using the AbsoluteIDQ kit. The profiling yielded 168 metabolite concentrations. Correlation analysis between SF and plasma metabolite concentrations was done on absolute concentrations as well as metabolite concentration ratios using Spearman's rank correlation (ρ) method. RESULTS: A total of 69 patients with knee OA were included, 30 men and 39 women, with an average age of 66 ± 8 years. For the absolute metabolite concentrations, the average ρ was 0.23 ± 0.13. Only 8 out of 168 metabolite concentrations had a ρ ≥ 0.45, with a p value ≤ 2.98 × 10(-4), statistically significant after correcting multiple testing with the Bonferroni method. For the metabolite ratios (n = 28,056), the average ρ was 0.29 ± 0.20. There were 4018 metabolite ratios with a ρ ≥ 0.52 and a p value ≤ 1.78 × 10(-6), significant after correcting multiple testing. Sex-separate analyses found no difference in ρ between men and women. Similarly, there was no difference in ρ between people younger and older than 65 years. CONCLUSION: Correlation between blood plasma and SF metabolite concentrations are modest. Metabolite ratios, which are considered proxies for enzymatic reaction rates and have higher correlations, should be considered when using blood plasma as a surrogate of SF in OA biomarker identification.

Partial Meal Replacement Plan and Quality of the Diet at 1 Year: Action for Health in Diabetes (Look AHEAD) Trial.

BACKGROUND: Little is known about diet quality with a reduced-energy, low-fat, partial meal replacement plan, especially in individuals with type 2 diabetes. The Action for Health in Diabetes (Look AHEAD) trial implemented a partial meal replacement plan in the Intensive Lifestyle Intervention. OBJECTIVE: To compare dietary intake and percent meeting fat-related and food group dietary recommendations in Intensive Lifestyle Intervention and Diabetes Support and Education groups at 12 months. DESIGN: A randomized controlled trial comparing Intensive Lifestyle Intervention with Diabetes Support and Education at 0 and 12 months. PARTICIPANTS/SETTING: From 16 US sites, the first 50% of participants (aged 45 to 76 years, overweight or obese, with type 2 diabetes) were invited to complete dietary assessments. Complete 0- and 12-month dietary assessments (collected between 2001 and 2004) were available for 2,397 participants (46.6% of total participants), with 1,186 randomized to Diabetes Support and Education group and 1,211 randomized to Intensive Lifestyle Intervention group. MAIN OUTCOME MEASURES: A food frequency questionnaire assessed intake: energy; percent energy from protein, fat, carbohydrate, polyunsaturated fatty acids, and saturated fats; trans-fatty acids; cholesterol; fiber; weekly meal replacements; and daily servings from food groups from the Food Guide Pyramid. STATISTICAL ANALYSES PERFORMED: Mixed-factor analyses of covariance, using Proc MIXED with a repeated statement, with age, sex, race/ethnicity, education, and income controlled. Unadjusted χ² tests compared percent meeting fat-related and food group recommendations at 12 months. RESULTS: At 12 months, Intensive Lifestyle Intervention participants had a significantly lower fat and cholesterol intake and greater fiber intake than Diabetes Support and Education participants. Intensive Lifestyle Intervention participants consumed more servings per day of fruits; vegetables; and milk, yogurt, and cheese; and fewer servings per day of fats, oils, and sweets than Diabetes Support and Education participants. A greater percentage of Intensive Lifestyle Intervention participants than Diabetes Support and Education participants met fat-related and most food group recommendations. Within Intensive Lifestyle Intervention, a greater percentage of participants consuming two or more meal replacements per day than participants consuming less than one meal replacement per day met most fat-related and food group recommendations. CONCLUSIONS: The partial meal replacement plan consumed by Intensive Lifestyle Intervention participants was related to superior diet quality.

Classification of osteoarthritis phenotypes by metabolomics analysis.

OBJECTIVES: To identify metabolic markers that can classify patients with osteoarthritis (OA) into subgroups. DESIGN: A case-only study design was utilised. PARTICIPANTS: Patients were recruited from those who underwent total knee or hip replacement surgery due to primary OA between November 2011 and December 2013 in St. Clare's Mercy Hospital and Health Science Centre General Hospital in St. John's, capital of Newfoundland and Labrador (NL), Canada. 38 men and 42 women were included in the study. The mean age was 65.2±8.7 years. OUTCOME MEASURES: Synovial fluid samples were collected at the time of their joint surgeries. Metabolic profiling was performed on the synovial fluid samples by the targeted metabolomics approach, and various analytic methods were utilised to identify metabolic markers for classifying subgroups of patients with OA. Potential confounders such as age, sex, body mass index (BMI) and comorbidities were considered in the analysis. RESULTS: Two distinct patient groups, A and B, were clearly identified in the 80 patients with OA. Patients in group A had a significantly higher concentration on 37 of 39 acylcarnitines, but the free carnitine was significantly lower in their synovial fluids than in those of patients in group B. The latter group was further subdivided into two subgroups, that is, B1 and B2. The corresponding metabolites that contributed to the grouping were 86 metabolites including 75 glycerophospholipids (6 lysophosphatidylcholines, 69 phosphatidylcholines), 9 sphingolipids, 1 biogenic amine and 1 acylcarnitine. The grouping was not associated with any known confounders including age, sex, BMI and comorbidities. The possible biological processes involved in these clusters are carnitine, lipid and collagen metabolism, respectively. CONCLUSIONS: The study demonstrated that OA consists of metabolically distinct subgroups. Identification of these distinct subgroups will help to unravel the pathogenesis and develop targeted therapies for OA.

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.

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.

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.

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.

Dioxin-dependent and dioxin-independent gene batteries: comparison of liver and kidney in AHR-null mice.

The aryl hydrocarbon receptor (AHR) is a widely expressed ligand-dependent transcription factor that mediates cellular responses to dioxins and other planar aromatic hydrocarbons. Ahr-null mice are refractory to the toxic effects of dioxin exposure. Although some mechanistic aspects of AHR activity are well understood, the tissue specificity of AHR effects remains unclear, both during development and following administration of exogenous ligands. To address the latter issue, we defined and compared transcriptional responses to dioxin exposure in the liver and kidney of wild-type and Ahr-null adult C57BL/6J mice treated with either 2,3,7,8-tetrachlorodibenzo-p-dioxin or corn-oil vehicle. In both tissues, essentially all effects of dioxin on hepatic mRNA levels were mediated by the AHR. Although 297 genes were altered by dioxin exposure in the liver, only 17 were changed in the kidney, including a number of well-established AHR target genes. Ahr genotype had a large effect in both tissues, profoundly remodeling both the renal and hepatic transcriptomes. Surprisingly, a large number of genes were affected by Ahr genotype in both tissues, suggesting the presence of a basal AHR gene battery. Alterations of the renal transcriptome in Ahr-null animals were associated with perturbation of specific functional pathways and enrichment of specific DNA motifs. Our results demonstrate the importance of intertissue comparisons, highlight the basal role of the AHR in liver and kidney, and support a role in development or normal physiology.

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.

CYP2D6 and DRD2 genes differentially impact pharmacodynamic sensitivity and time course of prolactin response to perphenazine.

OBJECTIVES: We observed that CYP2D6 contributes to pharmacodynamic tissue sensitivity to perphenazine as measured by the areas under the curve (AUCs) expressed as a ratio (prolactin-AUC0-6/perphenazine-AUC0-6) in Chinese Canadians [Pharmacogenetics and Genomics 2007; 17:339-347]. As genetic heterogeneity in drug targets can influence drug response, we sought to further evaluate the contribution of CYP2D6 to pharmacodynamic sensitivity in our previous study sample in tandem with DRD2, the primary molecular target for perphenazine. METHODS: Genotyping for DRD2 Taq1A, -141C ins/del and Ser311Cys functional polymorphisms was performed using PCR-restriction-fragment length polymorphism methods. RESULTS: After controlling for DRD2 polymorphisms, CYP2D6 was a significant predictor of pituitary pharmacodynamic tissue sensitivity to perphenazine (P=0.024; power=80.4%). Taq1A polymorphism significantly influenced the time course of prolactin response (P=0.039; power=70%). A1/A1 genotype displayed a higher prolactin elevation 2 h after perphenazine administration (P=0.02). Patients with -141C ins/ins genotype showed a strong trend toward a 38% larger prolactin AUC compared with the -141C ins/del genotypic group (P=0.07). CONCLUSIONS: CYP2D6 seems to be an independent contributor to pituitary pharmacodynamic tissue sensitivity to perphenazine after accounting for DRD2 functional polymorphisms. The A1 allele of the Taq1A polymorphism was previously shown to decrease D2 receptor density in vitro and in neuroimaging studies in vivo. At a given antipsychotic dose, individuals with A1 allele might thus achieve a higher DRD2 antipsychotic occupancy, which is consistent with an increased prolactin elevation in the A1/A1 genotype in this study. These findings provide a basis for further studies on the endogenous substrates of CYP2D6 and the rational selection of candidate genes for long-term consequences of antipsychotic-induced hyperprolactinemia (e.g. susceptibility to breast and prostate cancers).