Systemic mitochondrial dysfunction and the etiology of Alzheimer's disease and down syndrome dementia.

Increasing evidence is implicating mitochondrial dysfunction as a central factor in the etiology of Alzheimer's disease (AD). The most significant risk factor in AD is advanced age and an important neuropathological correlate of AD is the deposition of amyloid-beta peptide (Abeta40 and Abeta42) in the brain. An AD-like dementia is also common in older individuals with Down syndrome (DS), though with a much earlier onset. We have shown that somatic mitochondrial DNA (mtDNA) control region (CR) mutations accumulate with age in post-mitotic tissues including the brain and that the level of mtDNA mutations is markedly elevated in the brains of AD patients. The elevated mtDNA CR mutations in AD brains are associated with a reduction in the mtDNA copy number and in the mtDNA L-strand transcript levels. We now show that mtDNA CR mutations increase with age in control brains; that they are markedly elevated in the brains of AD and DS and dementia (DSAD) patients; and that the increased mtDNA CR mutation rate in DSAD brains is associated with reduced mtDNA copy number and L-strand transcripts. The increased mtDNA CR mutation rate is also seen in peripheral blood DNA and in lymphoblastoid cell DNAs of AD and DSAD patients, and distinctive somatic mtDNA mutations, often at high heteroplasmy levels, are seen in AD and DSAD brain and blood cells DNA. In aging, DS, and DSAD, the mtDNA mutation level is positively correlated with beta-secretase activity and mtDNA copy number is inversely correlated with insoluble Abeta40 and Abeta42 levels. Therefore, mtDNA alterations may be responsible for both age-related dementia and the associated neuropathological changes observed in AD and DSAD.

Growth and angiogenesis are inhibited in vivo in developing tissues by pyrazine and its derivatives.

Sidestream cigarette smoke solution was previously screened to identify the groups of chemicals in smoke that inhibit growth and angiogenesis in the chick chorioallantoic membrane (CAM). Pyrazine and several pyrazine derivatives were identified as a major chemical group in this screen. In the current study, purified pyrazine and six pyrazine derivatives identified in the screen were tested in dose response experiments to measure their effects on CAM growth, embryo growth, and angiogenesis. Chemicals or control medium were placed on CAMs in ovo on day 5 of development, and results were evaluated on day 6. Of the chemicals tested, pyrazine was the most potent and inhibited both CAM and embryo growth at picomolar doses. 2-Ethylpyrazine and 2,3-dimethylpyrazine were inhibitory at nanomolar doses. Inhibition of growth by pyrazine was correlated with inhibition of DNA synthesis. The pattern of blood vessel development in CAMs was disturbed by micromolar doses of pyrazine and 2,3-dimethylpyrazine. Migration of mesodermal blood vessels to the ectoderm of CAMs and their subsequent differentiation into the capillary plexus was impaired by nanomolar doses of pyrazine. In summary, these data show that pyrazine and some of its derivatives inhibit growth and certain processes important in angiogenesis at very low doses. Since pyrazine and some of its derivatives are considered safe food additives, further toxicological testing of pyrazine, in particular on developing tissues, should be done to fully evaluate its safety as a consumer product additive.

Identification of pyridine compounds in cigarette smoke solution that inhibit growth of the chick chorioallantoic membrane.

Based on prior work, we hypothesized that cigarette smoke contains chemicals that can inhibit growth of the chick chorioallantoic membrane (CAM). In this study, gas chromatography and mass spectrometry were used to identify 12 pyridine derivatives in the inhibitory fractions of smoke eluted from solid phase extraction cartridges. These pyridine derivatives were further studied individually in dose response experiments to determine their effects on CAM growth. A correlation was observed between the functional group substitutions on pyridine and the relative toxicity of each pyridine derivative. In the CAM growth assay, pyridine derivatives with single methyl or single ethyl substitutions had lowest observed adverse effect levels (LOAELs) of 5 x 10(-9) and 5 x 10(-12) M, respectively. Other pyridine derivatives and pyridine itself had LOAELs in the micromolar range. One of the most inhibitory derivatives, 3-ethylpyridine, was studied further and inhibited cell proliferation, as measured by BrdU incorporation. Since 3-ethylpyridine inhibited growth at picomolar doses and is added to consumer products including cosmetics, food, drinks, and tobacco, it will be important to perform further toxicological testing to determine its effect on human health.

Reactive carbonyls from tobacco smoke increase arterial endothelial layer injury.

We hypothesized that reactive carbonyls generated from smoke exposure cause increased arterial low-density lipoprotein (LDL) accumulation and endothelial layer permeability. In addition, we hypothesized that estrogen supplementation was protective against chronic environmental tobacco smoke (ETS) exposure to the artery wall. Quantitative fluorescence microscopy was used to determine artery injury after exposure. For our chronic studies, ovariectomized rats treated with subcutaneous placebo or 17beta-estradiol pellets were exposed to ETS or filtered air for 6 wk. ETS exposure increased carotid artery LDL accumulation more than fourfold compared with filtered air exposure, an effect largely mediated by increased permeability. No protective effect of estradiol was observed. Acute ETS exposure of a buffer solution containing LDL resulted in a more than sixfold increase in the highly reactive carbonyl glyoxal. Perfusion of this solution through carotid arteries resulted in a 105% increase in permeability. Moreover, perfusion of glyoxal alone caused a 50% increase in carotid artery permeability. This endothelial damage and changes in lipid accumulation may serve as an initiating event in atheroma formation in individuals exposed to ETS.

Mainstream and sidestream cigarette smoke inhibit growth and angiogenesis in the day 5 chick chorioallantoic membrane.

The purpose of this study was to test the hypothesis that components in mainstream (MS) and sidestream (SS) cigarette smoke inhibit growth and angiogenesis using the chick chorioallantoic membrane (CAM). Varying doses of whole or gas-phase MS and SS smoke solutions were placed on day 5 CAMs, and their effects on angiogenesis were evaluated on day 6. All parameters evaluated (CAM area, major blood vessel area, major blood vessel diameter, blood vessel pattern formation, and capillary plexus formation) were inhibited to different degrees in a dose-dependent manner by both MS and SS smoke treatment. Inhibition of growth and vessel development was correlated with inhibition of cell proliferation. Inhibition of capillary plexus formation was caused by failure of mesodermal blood vessels to migrate to the ectoderm. SS smoke solution was more inhibitory than MS smoke solution in all assays, except for capillary plexus formation. In all assays, the toxicants in SS smoke partitioned mainly with the gas phase, whereas those in MS smoke were deduced to be mainly in the particulate phase in the proliferation-dependent assays (CAM area, blood vessel area, blood vessel diameter) and in both the gas and particulate phase in the pattern formation and plexus formation assays. Some of the inhibitory doses of MS and SS smoke solutions had nicotine concentrations within the range found in human smokers. Taken together, these data demonstrate that exposure to complex mixtures of chemicals in MS and SS cigarette smoke adversely affect growth, vessel development, vessel migration, and cell proliferation.

Capillary plexus development in the day five to day six chick chorioallantoic membrane is inhibited by cytochalasin D and suramin.

The chick chorioallantoic membrane (CAM) is a valuable model for evaluating angiogenesis and vasculogenesis. Our purpose was to characterize the formation of the CAM vasculature, in particular the capillary plexus, between days five and six after fertilization and to examine the mode of action of cytochalasin D and suramin on vascular development during this interval. The CAM increased 20-fold in size between days five and six, during which time the capillary plexus forms by both migration of mesodermal blood vessels toward the ectoderm and by the formation of new vessels from angioblasts near the ectoderm. Between days five and six, the CAM becomes thinner, and the density of the mesodermal cells decreases. To determine the mode of action of anti-angiogenic drugs on the day five to day six CAM, various concentrations of cytochalasin D or suramin were added directly to day five CAMs, and their effects were evaluated on day six. Both drugs significantly inhibited CAM growth, altered branching patterns of the major vessels, decreased area of the major vessels, and inhibited the formation of the capillary plexus by inhibiting both vasculogenesis and the migration of mesodermal blood vessels to the ectoderm. Cytochalasin D also inhibited compartmentalization of the plexus. Cytochalasin D and suramin were inhibitory at similar doses. This study provides new information on early CAM development, establishes the mode of action and dose dependency of cytochalasin D and suramin on day five to day six CAMs, and demonstrates that the day five to day six CAM provides a useful assay to examine the effect of anti-angiogenic drugs on blood vessel development, including capillary plexus formation.