Spotlight Obese Metabolism Obesity is one of the greatest worldwide health threats of our time. The sequelae of obesity, including diabetes and cardiovascular disease, are treated with drugs for which the balance between therapeutic and toxic effect is the result of the actions of multiple drug metabolizing enzymes and transporting proteins. Prior studies have indicated that expression of these key proteins is altered in obesity. Now, Cheng et al. [(2008) Mol. Pharm. 5, 77] explore in detail the mRNA and protein expression of a wide range of drug metabolizing and transport proteins in the ob/ob mouse model of obesity and diabetes. They show multiple and complex changes between obese and wild-type mice that differ between the sexes. For example, cytochrome
P450 2b10 expression decreases in females and increases in males while NAD(P)H:quinone oxidoreductase 1 increases in both sexes. mRNA expression of organic anionic transporting polypeptides 1A1 and 1A4 decreases in both sexes, but expression of the 1A4 protein increases. mRNA expression of multidrug resistance proteins (mrp) 1 and 6 decreases in females while mrp3, 4, and 5 increases in males. Protein expression increases for mrp4 in females and mrp1, 3, and 5 in males. The authors suggest that the changes indicate alterations in the function of the CAR, PPARR, and Nrf2 transcription factors in obesity. These results demonstrate a need to evaluate drug disposition and metabolism carefully in obese and diabetic patients. • Carol A. Rouzer
Transthyretin and the Brain As the understanding of toxic mechanisms of xenobiotics has grown, many have come to view toxicology as a “mature” field with few remaining new horizons. However, increasing appreciation of the toxicity of endogenously produced species provides novel challenges for toxicologists. An example is the neurotoxicity of aβ peptides, which accumulate in the plaques that are the hallmark of Alzheimer’s disease (AD). The exact means by which aβ aggregates lead to neurodegeneration is not fully understood. However, Buxbaum et al. [(2008) Proc. Natl. Acad. Sci. U.S.A. 105, 2681] present new information concerning the mechanism of aβ accumulation. Prior data suggested a relationship between AD and transthyretin (TTR), a thyroid hormone carrier protein that is produced primarily in the liver. TTR is also produced in the choroid plexus of the brain and is the only thyroid hormone carrier in that tissue. Human AD patients demonstrate decreased brain levels of TTR, and TTR forms aggregates with aβ peptides in vitro. These findings led to the hypothesis that binding of TTR prevents aβ aggregation and that decreased expression of TTR in AD patients promotes plaque formation. Buxbaum et al. tested this hypothesis using transgenic mice overexpressing human amyloid precursor protein (APP23), a model of AD. Overexpression of human TTR reduced the deficit in spatial strategy observed in APP23 transgenic mice at 15 months of age. Mice lacking the gene for mTTR demonstrated spatial strategy deficits as compared to wild-type mice as early as 5 months of age. Although overexpression of human APP23 did not exacerbate the deficits in mTTR-/- mice, histologic studies demonstrated the presence of cortical and hippocampal aβ deposits in APP23 transgenic mTTR-/mice but not in those expressing mTTR. 976
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Reproduced with permission from Buxbaum et al. [(2008) Proc. Natl. Acad. Sci. U.S.A. 105, 2681]. Copyright 2008 National Academy of Sciences U.S.A.
Surface plasmon resonance spectroscopy demonstrated interaction between TTR and aβ aggregates. TTR staining is detectable within the neurons of human and mouse brain, implying that TTR may be synthesized within the neurons themselves. The data support the hypothesis that TTR associates with newly forming aβ aggregates, preventing their progression to neurotoxic plaques. It should be noted, however, that in contrast to the APP23 transgenic mouse, TTR was not uniformly seen in the plaques of brains from human AD patients. This may mean that TTR plays no role in human aβ disposition, but it may also indicate that a deficiency in TTR in AD patients is at least, in part, responsible for aβ aggregation, plaque development, and toxicity. • Carol A. Rouzer
Fate of the Omega-3s Oxidative stress is a well-known mechanism of toxicity, resulting from the production of reactive oxidants in excess of a cell’s mechanisms to scavenge such species. When detoxification systems are overwhelmed, oxidative damage occurs to cellular constituents, including nucleic acids, proteins, and lipids. Particularly susceptible to such damage are the polyunsaturated fatty acids of membrane phospholipids. Free radical oxidation of arachidonic acid (AA), for example, leads to the formation of a wide range of oxygenated products, the isoprostanes. Isoprostane levels Published online 05/19/2008 •
DOI: 10.1021/tx800119b $40.75 © 2008 American Chemical Society
Spotlight are a reliable biomarker of oxidative stress in vivo, and considerable research has focused on the biological activities of individual isoprostane species. A growing body of evidence indicates that dietary supplementation with omega-3 fatty acids provides various health benefits, including amelioration of cardiovascular, neurodegenerative, and inflammatory diseases. The major component of most such dietary supplements is eicosapentaenoic acid (EPA). The higher degree of unsaturation of EPA vs AA (five vs four double bonds) renders EPA even more susceptible to oxidative damage. Now, Brooks et al. [(2008) J. Biol. Chem. Published online Feb. 10, DOI: 10.1074/ jbc.M800122200] explore the formation of a subclass of EPA-derived isoprostanes bearing an electrophilic cyclopentenone ring. The investigators first oxidized EPA in vitro and characterized the products by gas chromatography– mass spectrometry and by liquid chromatography-tandem mass spectrometry. Fragmentation analysis identified multiple positional isomers, and formation of glutathione conjugates confirmed the presence of the electrophilic cyclopentenone ring. Brooks et al. then demonstrated the presence of these species in phospholipids extracted from the livers of rats following dietary supplementation with EPAcontaining fish oil. The physiologic significance of these findings is not yet known. The formation of electrophilic isoprostanes from EPA and their increase following carbon tetrachloride treatment suggests a source of oxidative stress-related toxicity. On the other hand, cyclopentenone isoprostanes have been shown to have anti-inflammatory activities. If EPA-derived cyclopentenone isoprostanes have such beneficial effects, then the formation of these compounds would be protective, rather than toxic, and could partially explain EPA’s reported health benefits. • Carol A. Rouzer
Radical Thymines One-electron oxidation of DNA occurs through the action of ionizing radiation or through photochemical or chemical reactions. Removal of a single electron generates a radical cation, which can move great distances along the DNA strand before finally being trapped, usually by reaction with oxygen or water. The result is an oxidatively damaged site, most often a DNA base. The base most frequently oxidized under these conditions is guanine, a finding that has been attributed to guanine’s low oxidation potential. However, through their studies of the oxidation of DNA strands containing only adenine and thymine bases, Ghosh et al. [(2008) Org. Biomol. Chem. 6, 916] challenge that long-held view. They find that oxidation of these DNA strands occurs primarily at thymine, even though the oxidation potential of thymine is higher than that of adenine. They also report that oxidation requires a minimum of two adjacent thymine residues and increases with increasing numbers of adjacent
Published online 05/19/2008 • DOI: 10.1021/tx800119b © 2008 American Chemical Society
$40.75
thymines. Replacement of all thymines in a tandem grouping with uracil eliminates reaction at that position, and reaction can occur in a 3′-TU-5′ segment but not in a 3′UT-5′ segment. Chemical analysis of the products of oxidation as enzymatically released and modified 2′-deoxyribonucelosides reveals 5-formyl-2′-deoxyuridine (5-FormdUrd), cis and trans diastereomers of 5,6-dihydroxy-5,6-dihydrothymidine (ThGly), and 5-(hydroxymethyl)-2′-deoxyuridine (5HMdUrd) at 63, 13, 20, and 4%, respectively. The authors argue that the major factor determining the site of base modification is not the oxidation potential of the base but the activation energy for the reaction of the radical cation to form the final product. This argument is based on the Curtin-Hammett principle, which states that when reactions pass through multiple intermediates, each leading to a different product, the product distribution depends on the activation energy between each intermediate and its product rather than on the energy of each intermediate itself. They propose a mechanism for thymine oxidation in which the formation of a radical cation at the 5′-T of an adjacent pair first leads to reaction with water and oxygen across the 5,6-double bond, forming a 6-hydroxy-5,6-dihydrothymidine-5-peroxy radical. If there is an adjacent 3′-T, the peroxyl radical abstracts a hydrogen atom from the methyl group of that thymine residue, generating a radical, which will then go on to react with oxygen or water. The products will be ThGly at the 5′-T and either 5-FormdUrd or 5-HMdUrd at the 3′-T. Alternatively, if the radical cation is formed at the 3′-T of an adjacent pair and a 6-hydroxy-5,6-dihydrothymidine-5-peroxy radical is formed, it cannot abstract a hydrogen from the adjacent 5′-T, because it is too far away in the duplex structure. Thus, the only reaction possible is loss of a proton from the methyl group followed by reaction at that group, and the only products can be 5-FormdUrd or 5-HMdUrd at the 3′-T. In these experiments, oxidation was detected by base treatment leading to strand cleavage. The authors propose that such cleavage only occurs at ThGly bases, so, the only reactions detected were those in which the radical cation forms first at the 5′-T of an adjacent pair. This explains why no cleavage occurs when the 3′-T is replaced with uracil, since no methyl group is available for the hydrogen abstraction required for ThGly formation. However, replacement of the 5′-T with uracil does not block the reaction, because the methyl group is not required for reaction across the 5,6-double bond of the radical cation, and uracil glycol formation can still occur. These experiments highlight the complexity of DNA radical chemical reactions and demonstrate that widely held explanations for its chemical behavior must be subject to careful ongoing experimental evaluation. • Carol A. Rouzer TX800119B
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