Quantitative investigation on the metabolism of 1,3-butadiene and of its oxidized metabolites in once-through perfused livers of mice and rats.

The industrial chemical 1,3-butadiene (BD) is a potent carcinogen in mice and a weak one in rats. This difference is generally related to species-specific burdens by the metabolites 1,2-epoxy-3-butene (EB), 1,2:3,4-diepoxybutane (DEB), and 3,4-epoxy-1,2-butanediol (EBD), which are all formed in the liver. Only limited data exist on BD metabolism in the rodent liver. Therefore, metabolism of BD, its epoxides, and the intermediate 3-butene-1,2-diol (B-diol) was studied in once-through perfused livers of male B6C3F1 mice and Sprague-Dawley rats. In BD perfusions, predominantly EB and B-diol were found (both species). DEB and EBD were additionally detected in mouse livers. Metabolism of BD showed saturation kinetics (both species). In EB perfusions, B-diol, EBD, and DEB were formed with B-diol being the major metabolite. Net formation of DEB was larger in mouse than in rat livers. In both species, hepatic clearance (Cl(H)) of EB was slightly smaller than the perfusion flow. In DEB perfusions, EBD was formed as a major metabolite. Cl(H) of DEB was 61% (mouse) and 73% (rat) of the perfusion flow. In the B-diol-perfused rat liver, EBD was formed as a minor metabolite. Cl(H) of B-diol was 53% (mouse) and 34% (rat) of the perfusion flow. In EBD-perfused rat livers, Cl(H) of EBD represented only 22% of the perfusion flow. There is evidence for qualitative species differences with regard to the enzymes involved in BD metabolism. The first quantitative findings in whole livers showing intrahepatic first-pass metabolism of BD and EB metabolites will improve the risk estimation of BD.

Intramyocardial transplantation of autologous CD34+ stem cells for intractable angina: a phase I/IIa double-blind, randomized controlled trial.

BACKGROUND: A growing population of patients with coronary artery disease experiences angina that is not amenable to revascularization and is refractory to medical therapy. Preclinical studies have indicated that human CD34+ stem cells induce neovascularization in ischemic myocardium, which enhances perfusion and function. METHODS AND RESULTS: Twenty-four patients (19 men and 5 women aged 48 to 84 years) with Canadian Cardiovascular Society class 3 or 4 angina who were undergoing optimal medical treatment and who were not candidates for mechanical revascularization were enrolled in a double-blind, randomized (3:1), placebo-controlled dose-escalating study. Patients received granulocyte colony-stimulating factor 5 microg x kg(-1) x d(-1) for 5 days with leukapheresis on the fifth day. Selection of CD34+ cells was performed with a Food and Drug Administration-approved device. Electromechanical mapping was performed to identify ischemic but viable regions of myocardium for injection of cells (versus saline). The total dose of cells was distributed in 10 intramyocardial, transendocardial injections. Patients were required to have an implantable cardioverter-defibrillator or to temporarily wear a LifeVest wearable defibrillator. No incidence was observed of myocardial infarction induced by mobilization or intramyocardial injection. The intramyocardial injection of cells or saline did not result in cardiac enzyme elevation, perforation, or pericardial effusion. No incidence of ventricular tachycardia or ventricular fibrillation occurred during the administration of granulocyte colony-stimulating factor or intramyocardial injections. One patient with a history of sudden cardiac death/ventricular tachycardia/ventricular fibrillation had catheter-induced ventricular tachycardia during mapping that required cardioversion. Serious adverse events were evenly distributed. Efficacy parameters including angina frequency, nitroglycerine usage, exercise time, and Canadian Cardiovascular Society class showed trends that favored CD34+ cell-treated patients versus control subjects given placebo. CONCLUSIONS: A randomized trial of intramyocardial injection of autologous CD34+ cells in patients with intractable angina was completed that provides evidence for feasibility, safety, and bioactivity. A larger phase IIb study is currently under way to further evaluate this therapy.

CHIP and HSPs interact with beta-APP in a proteasome-dependent manner and influence Abeta metabolism.

The C-terminus Hsp70 interacting protein (CHIP) has dual function as both co-chaperone and ubiquitin ligase. CHIP is increasingly implicated in the biology of polyglutamine expansion disorders, Parkinson's disease and tau protein in Alzheimer's disease. We investigated the involvement of CHIP in the metabolism of the beta-amyloid precursor protein and its derivative beta-amyloid (Abeta). Using immunoprecipitation, fluorescence localization and crosslinking methods, endogenous CHIP and betaAPP interact in brain and cultured skeletal myotubes as well as when they are expressed in stable HEK cell lines. Their interaction is confined to Golgi and ER compartments. In the presence of the proteasome inhibitor with MG132, endogenous and expressed betaAPP levels are significantly increased and accordingly, the interaction with CHIP enhanced. Concurrently, levels of Hsp70 were most consistently induced by proteasome inhibition among the various heat shock proteins (HSPs) tested. Thus, complexes of CHIP, Hsp70 and holo-betaAPP (as well as C-terminal fragments) were stabilized by the action of MG132. Moreover, CHIP itself is shown to both increase cellular holo-betaAPP levels and protect it from oxidative stress and degradation. Interestingly, CHIP also promotes the association of ubiquitin with betaAPP, implying that a smaller pool of betaAPP is destined for proteasomal processing. In neuronal cultures, CHIP and Hsp70/90 expression reduce steady-state cellular Abeta levels and hasten its degradation in pulse-chase experiments. The functional significance of CHIP and HSP interactions, especially with Hsp70, was tested using siRNA and in neuronal cells where protection from Abeta-induced toxicity is shown. We conclude that CHIP, as a bimolecular switch, interacts with HSP to stabilize normal holo-betaAPP on the one hand while also assisting in the ubiquitination of a subpopulation of betaAPP molecules that are destined for proteasome degradation. CHIP also hastens the clearance of Abeta in a manner consistent with its known neuroprotective properties.

Metabolism of 1,3-butadiene to toxicologically relevant metabolites in single-exposed mice and rats.

1,3-Butadiene (BD) was carcinogenic in rodents. This effect is related to reactive metabolites such as 1,2-epoxy-3-butene (EB) and especially 1,2:3,4-diepoxybutane (DEB). A third mutagenic epoxide, 3,4-epoxy-1,2-butanediol (EBD), can be formed from DEB and from 3-butene-1,2-diol (B-diol), the hydrolysis product of EB. In BD exposed rodents, only blood concentrations of EB and DEB have been published. Direct determinations of EBD and B-diol in blood are missing. In order to investigate the BD-dependent blood burden by all of these metabolites, we exposed male B6C3F1 mice and male Sprague-Dawley rats in closed chambers over 6-8h to constant atmospheric BD concentrations. BD and exhaled EB were measured in chamber atmospheres during the BD exposures. EB blood concentrations were obtained as the product of the atmospheric EB concentration at steady state with the EB blood-to-air partition coefficient. B-diol, EBD, and DEB were determined in blood collected immediately at the end of BD exposures up to 1200 ppm (B-diol, EBD) and 1280 ppm (DEB). Analysis of BD was done by GC/FID, of EB, DEB, and B-diol by GC/MS, and of EBD by LC/MS/MS. EB blood concentrations increased with BD concentrations amounting to 2.6 micromol/l (rat) and 23.5 micromol/l (mouse) at 2000 ppm BD and to 4.6 micromol/l in rats exposed to 10000 ppm BD. DEB (detection limit 0.01 micromol/l) was found only in blood of mice rising to 3.2 micromol/l at 1280 ppm BD. B-diol and EBD were quantitatively predominant in both species. B-diol increased in both species with the BD exposure concentration reaching 60 micromol/l at 1200 ppm BD. EBD reached maximum concentrations of 9.5 micromol/l at 150 ppm BD (rat) and of 42 micromol/l at 300 ppm BD (mouse). At higher BD concentrations EBD blood concentrations decreased again. This picture probably results from a competitive inhibition of the EBD producing CYP450 by BD, which occurs in both species.

Differential effects of mitochondrial heat shock protein 60 and related molecular chaperones to prevent intracellular beta-amyloid-induced inhibition of complex IV and limit apoptosis.

Defects in mitochondrial oxidative metabolism, in particular decreased activity of cytochrome c oxidase, have been reported in Alzheimer disease tissue and in cultured cells that overexpress amyloid precursor protein. Mitochondrial dysfunction contributes to neurodegeneration in Alzheimer disease partly through formation of reactive oxygen species and the release of sequestered molecules that initiate programmed cell death pathways. The heat shock proteins (HSP) are cytoprotective against a number of stressors, including accumulations of misfolded proteins and reactive oxygen species. We reported on the property of Hsp70 to protect cultured neurons from cell death caused by intraneuronal beta-amyloid. Here we demonstrate that Hsp60, Hsp70, and Hsp90 both alone and in combination provide differential protection against intracellular beta-amyloid stress through the maintenance of mitochondrial oxidative phosphorylation and functionality of tricarboxylic acid cycle enzymes. Notably, beta-amyloid was found to selectively inhibit complex IV activity, an effect selectively neutralized by Hsp60. The combined effect of HSPs was to reduce the free radical burden, preserve ATP generation, decrease cytochrome c release, and prevent caspase-9 activation, all important mediators of beta-amyloid-induced neuronal dysfunction and death.

Parkin protects against mitochondrial toxins and beta-amyloid accumulation in skeletal muscle cells.

Mutations in the ubiquitin ligase-encoding Parkin gene have been implicated in the pathogenesis of autosomal recessive Parkinson disease. Outside of the central nervous system, Parkin is prominently expressed in skeletal muscle. We have found accumulations of Parkin protein in skeletal muscle biopsies taken from patients with inclusion body myositis, a degenerative disorder in which intramyofiber accumulations of the beta-amyloid peptide are pathognomonic. In comparing primary cultures of skeletal muscle derived from parkin knock-out and wild-type mice, we have found the absence of parkin to result in greater sensitivity to mitochondrial stressors rotenone and carbonyl cyanide 3-chlorophenylhydrazone, without any alteration in sensitivity to calcium ionophore or hydrogen peroxide. Utilizing viral expression constructs coding for the Alzheimer disease and inclusion body myositis-linked beta-amyloid precursor protein and for its metabolic byproducts A beta42 and C100, we found that parkin knock-out muscle cells are also more sensitive to the toxic effects of intracellular A beta. We also constructed a lentiviral system to overexpress wild-type Parkin and have shown that boosting the levels of parkin expression in normal skeletal muscle cultures provides substantial protection against both mitochondrial toxins and overexpressed beta-amyloid. Correspondingly, exogenous Parkin significantly lowered A beta levels. These data support the hypothesis that in myocytes parkin has dual properties in the maintenance of skeletal muscle mitochondrial homeostasis and in the regulation of A beta levels.

Dissociation of ERK and Akt signaling in endothelial cell angiogenic responses to beta-amyloid.

Cerebrovascular deposits of beta-amyloid (Abeta) peptides are found in Alzheimer's disease and cerebral amyloid angiopathy with stroke or dementia. Dysregulations of angiogenesis, the blood-brain barrier and other critical endothelial cell (EC) functions have been implicated in aggravating chronic hypoperfusion in AD brain. We have used cultured ECs to model the effects of beta-amyloid on the activated phosphorylation states of multifunctional serine/threonine kinases since these are differentially involved in the survival, proliferation and migration aspects of angiogenesis. Serum-starved EC cultures containing amyloid-beta peptides underwent a 2- to 3-fold increase in nuclear pyknosis. Under growth conditions with sublethal doses of beta-amyloid, loss of cell membrane integrity and inhibition of cell proliferation were observed. By contrast, cell migration was the most sensitive to Abeta since inhibition was significant already at 1 muM (P = 0.01, migration vs. proliferation). In previous work, intracellular Abeta accumulation was shown toxic to ECs and Akt function. Here, extracellular Abeta peptides do not alter Akt activation, resulting instead in proportionate decreases in the phosphorylations of the MAPKs: ERK1/2 and p38 (starting at 1 microM). This inhibitory action occurs proximal to MEK1/2 activation, possibly through interference with growth factor receptor coupling. Levels of phospho-JNK remained unchanged. Addition of PD98059, but not LY294002, resulted in a similar decrease in activated ERK1/2 levels and inhibition of EC migration. Transfection of ERK1/2 into Abeta-poisoned ECs functionally rescued migration. The marked effect of extracellular Abeta on the migration component of angiogenesis is associated with inhibition of MAPK signaling, while Akt-dependent cell survival appears more affected by cellular Abeta.