Microcystins (MCs) produced by cyanobacteria are strong hepatotoxins and classified as possible carcinogens. MCs pose a considerable threat to consumers of tainted drinking and surface waters, but the photochemical fate of dissolved MCs in the environment has received limited attention. MCs are released into the environment upon cell lysis along with photoactive pigments including phycocyanin and chlorophyll a. The concentrations of MCs and pigments are expected to be greatest during a bloom event. These blooms occur in sunlit surface water and thus MCs can undergo a variety of solar initiated or photosensitized transformations. We report herein the role of oxygen, sensitizer, and light on the photochemical fate of MCs. The phycocyanin photosensitized transformation of MCs is elucidated, and photosensitized isomerization plays an important role in the process. The UV-A portion of sunlight was simulated using 350 nm light and the phototransformations of three MC variants (-LR, -RR, -LF) were investigated. Singlet oxygen leads to photooxidation of phycocyanin, the predominant pigment of cyanobacteria, hence, reducing the phototransformation rate of MCs. The phototransformation rate of MC-LR increases as pH decreases. The pH effect may be the result of MCs association with phycocyanin. Our results indicate photosensitized processes may play a key role in the photochemical transformation of MCs in the natural water.
Background: Human CYP4F2 metabolizes vitamin E, but its role in vivo is unknown. Results: Disruption of Cyp4f14, a murine ortholog of CYP4F2, severely alters vitamin E metabolism and status. Conclusion: CYP4F14 is an important regulator of vitamin E status, and its absence reveals counterbalancing mechanisms. Significance: An understanding of the fate of vitamin E is needed to predict and interpret its biological activities.
Vitamin E, a family of plant-derived lipophilic compounds that consists of tocopherols (TOH) and tocotrienols (T3), is considered to be the most important group of lipophilic antioxidants. These structurally related compounds differ only in the number and placement of methyl groups on the polar head of the molecule and in the presence of double bonds in the side chain. Interestingly, although the typical American diet contains 2-4 times as much ␥ -TOH as ␣ -TOH ( 1, 2 ), ␣ -TOH is present in the serum and tissues at levels 5-6 times that of ␥ -TOH ( 3 ). This preferential accumulation of ␣ -TOH in tissues, termed the ␣ -TOH phenotype, is widely conserved in the animal kingdom and occurs despite the fact that all forms of vitamin E exhibit roughly similar radical scavenging activities ( 4, 5 ). Although in vitro and animal studies have suggested both benefi cial ( 6-9 ) and detrimental ( 10 ) biological actions of non-␣ -TOH forms of vitamin E, the biological advantage of the ␣ -TOH phenotype remains elusive.The microsomal cytochrome P450 (CYP) enzymes catalyze a vast number of catalytic reactions, including the metabolism of lipids, steroids, and xenobiotics. These endoplasmic reticulum-bound enzymes require NADPH and the coenzyme cytochrome P450 reductase (CPR) for the source and transfer of electrons to the CYP enzyme. We previously identifi ed cytochrome P450 4F2 (CYP4F2) as a human vitamin E--hydroxylase ( 11 ), catalyzing the hydroxylation of one of the terminal methyl groups of the hydrophobic side chain. This -hydroxylation can be followed by oxidation to the corresponding carboxyl form and a series of side-chain shortening steps, ultimately leading to the formation of the 3 ′ and 5 ′ carboxychromanol metabolites that can be excreted in the urine ( 12-14 ). CYP4F2 displays substrate preference, such that Abstract The widely conserved preferential accumulation of ␣ -tocopherol ( ␣ -TOH) in tissues occurs, in part, from selective postabsorptive catabolism of non-␣ -TOH forms via the vitamin E--oxidation pathway. We previously showed that global disruption of CYP4F14, the major but not the only mouse TOH--hydroxylase, resulted in hyper-accumulation of ␥ -TOH in mice fed a soybean oil diet. In the current study, supplementation of Cyp4f14 ؊ / ؊ mice with high levels of ␦ -and ␥ -TOH exacerbated tissue enrichment of these forms of vitamin E. However, at high dietary levels of TOH, mechanisms other than -hydroxylation dominate in resisting diet-induced accumulation of non-␣ -TOH. These include TOH metabolism via -1/ -2 oxidation and fecal elimination of unmetabolized TOH. The -1 and -2 fecal metabolites of ␥ -and ␣ -TOH were observed in human fecal material. Mice lacking all liver microsomal CYP activity due to disruption of cytochrome P450 reductase revealed the presence of extra-hepatic -, -1, and -2 TOH hydroxylase activities. TOH--hydroxylase activity was exhibited by microsomes from mouse and human small intestine; murine activity was entirely due to CYP4F14. These fi ndings shed new light on the role of T...
Human cytochrome P450 4F2 (CYP4F2) catalyzes the ω-hydroxylation of the side chain of tocopherols (TOH) and tocotrienols (T3), the first step in their catabolism to polar metabolites excreted in urine. CYP4F2, in conjunction with α-TOH transfer protein, results in the conserved phenotype of selective retention of α-TOH. The purpose of this work was to determine the functional consequences of 2 common genetic variants in the human CYP4F2 gene on vitamin E-ω-hydroxylase specific activity using the 6 major dietary TOH and T3 as substrate. CYP4F2-mediated ω-hydroxylase specific activity was measured in microsomal preparations from insect cells that express wild-type or polymorphic variants of the human CYP4F2 protein. The W12G variant exhibited a greater enzyme specific activity (pmol product · min(-1) · pmol CYP4F2(-1)) compared with wild-type enzyme for both TOH and T3, 230-275% of wild-type toward α, γ, and δ-TOH and 350% of wild-type toward α, γ, and δ-T3. In contrast, the V433M variant had lower enzyme specific activity toward TOH (42-66% of wild type) but was without a significant effect on the metabolism of T3. Because CYP4F2 is the only enzyme currently shown to metabolize vitamin E in humans, the observed substrate-dependent alterations in enzyme activity associated with these genetic variants may result in alterations in vitamin E status in individuals carrying these mutations and constitute a source of variability in vitamin E status.
Due to the heterogeneous nature of breast cancer and the widespread use of single-gene studies, there is limited knowledge of multi-gene, locus-specific DNA methylation patterns in relation to molecular subtype and clinical features. We, therefore, quantified DNA methylation of 70 candidate gene loci in 140 breast tumors and matched normal tissues and determined associations with gene expression and tumor subtype. Using Sequenom’s EpiTYPER platform, approximately 1,200 CpGs were interrogated and revealed six DNA methylation patterns in breast tumors relative to matched normal tissue. Differential methylation of several gene loci was observed within all molecular subtypes, while other patterns were subtype-dependent. Methylation of numerous gene loci was inversely correlated with gene expression, and in some cases, this correlation was only observed within specific breast tumor subtypes. Our findings were validated on a larger set of tumors and matched adjacent normal tissue from The Cancer Genome Atlas dataset, which utilized methylation data derived from both Illumina Infinium 27 and 450 k arrays. These findings highlight the need to control for subtype when interpreting DNA methylation results, and the importance of interrogating multiple CpGs across varied gene regions.Electronic supplementary materialThe online version of this article (doi:10.1007/s10549-013-2738-0) contains supplementary material, which is available to authorized users.
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