Toxicological responses to acetaminophen (APAP) overdose were evaluated in human hepatocytes transplanted chimeric mice using 2-dimensional gel electrophoresis (2DE)-based proteomics and 1H-nuclear magnetic resonance (NMR)-based metabonomics. Huge variations, which were supported by histopathological findings, were observed in proteins expression in chimeric mice liver. The proteomic analysis of the livers showed that the proteins involved in the pathways of lipid/fatty acid metabolism, glycolysis and energy metabolism/production were affected. In addition, oxidative stress-related proteins showed altered expression. The metabonomic analysis of urine and plasma revealed alterations of endogenous metabolites, which were the intermediates involved in the tricarboxylic acid (TCA) cycle. Those findings were already confirmed in normal mice. We hypothesized that the mechanism of APAP-induced effects on chimeric mice liver was in accordance with the mechanism observed in normal mice. Therefore, these toxicopanomic approaches successfully revealed that the mechanisms in humans were identical with "known" APAP-induced hepatotoxicity detected in chimeric mice. Further investigations are needed to detect idiosyncratic hepatotoxicity in humans using chimeric mice.
Mitemcinal (GM-611) is a novel erythromycin-derived prokinetic agent that acts as an agonist at the motilin receptor. Erythromycin has shown QT prolongation and torsades de pointes (TdP) in humans and cisapride, a second class of prokinetic agents typified by the 5-HT4 receptor agonist, has been terminated due to TdP. In this study an extended series of safety pharmacology protocols and evaluations have been undertaken to assess the potential risk of mitemcinal on QT prolongation or proarrhythmic effects. Mitemcinal and its metabolites, GM-577 and GM-625, inhibited the human ether-a-go-go-related gene (HERG) tail current in a concentration-dependent manner with IC50 values of 20.2, 41.7, and 55.0 μM, respectively. Administration of 10 mg/kg mitemcinal in anesthetized guinea pigs resulted in a slight prolongation of the monophasic action potential (MAP) duration during atrial pacing at the plasma concentration of mitemcinal 1.1 μM, with low maximum increases in MAPD70 (6.6%) and MAPD90 (4.6%) relative to vehicle. A 10-min infusion of 20 mg/kg of mitemcinal in a proarrhythmic rabbit model did not evoke TdP even when QT and corrected QT (QTc) intervals were significantly prolonged. In this study, the Cmax plasma-free concentration of mitemcinal indicates that the prolongation was more than 400-fold that of the therapeutic dose. Our findings of a wide safety margin and the absence of TdP within this margin suggest that mitemcinal may provide sufficient safety in clinical use.
Mitemcinal (GM-611) is a novel erythromycin-derived prokinetic agent that acts as an agonist at the motilin receptor. We investigated the QT-prolonging effects of mitemcinal using a halothane-anesthetized canine model. Intravenous administration of mitemcinal at doses of more than 8.3 mg/kg per 10 min significantly prolonged the QT interval corrected by Fridericia's corrections. Mitemcinal exhibited a bradycardiac effect and produced significantly greater prolongation in monophasic action potential duration (MAP90) at sinus rhythm compared with MAP90 at pacing and showed reverse use-dependent prolongation of repolarization, suggesting that the negative chronotropic effect of mitemcinal potentiates the prolongation of the repolarization period. A technique using MAP/pacing electrodes allowed measurements of both MAP90 and effective refractory period (ERP) simultaneously at the same ventricular site. Although mitemcinal slightly prolonged the MAP90(CL400) and ERP in comparison with the control group at the dose of 25 mg/kg per 10 min, the terminal repolarization period, the difference between MAP90(CL400) and ERP, did not increase suggesting the absence of a proarrhythmic effect even with a 7000-fold for the therapeutic blood concentration as free level. The electrophysiological results from mitemcinal in this study indicate that the risk of serious arrhythmia such as torsades de pointes, a major clinical concern related to QT interval prolongation, might be low.
The aim of this work was to typify the mechanisms involved in gonyautoxins intestinal permeability. For this purpose, permeability of gonyautoxins through intestinal epithelium, and their effect on transepithelial resistance was investigated in excised human jejunal segments. The isolated mucosa segments were mounted in a Ussing chamber and experiments performed under voltage-controlled conditions. Organic gonyautoxin cations were applied in the apical side and samples collected in the basolateral side. Results show that gonyautoxin 2/3 epimers (GTX 2/3) permeate the intestine through a paracellular pathway and, to reach the resolution of the technique we used, no evidence was found of any other transport mechanism involved in the process. A model was developed, according to which tight junctions undergo a toxin concentration and time-dependent change, while transepithelial resistance shows a modest decrease.
To evaluate the effects of aging on DNA damage, spontaneous and chemical-induced DNA damage and its repair were examined using comet assays at pH 9, 12.1 and 13, and an 8-OH-dG assay in the liver and kidney of young (9-week-old) and aged (20-month-old) rats. Additionally, blood chemistry was examined to investigate any correlation between vital functions and age-dependent DNA damage. DNA migration at pH 13 and 8-OH-dG levels increased in the liver and/or kidney of aged rats, but DNA migration did not increase at pH 9 or 12.1; that is, alkali-labile sites and 8-OH-dG were concomitantly accumulated in aged rats. These results suggest that 8-OH-dG production caused by reactive oxygen species exceeded glycosylation and that the glycosylation activity is far more than the AP endonucleation in aged rats. Methyl methanesulfonate (MMS, 80 mg/kg, i.p.) increased DNA migration at pH 12.1 and 13 in the liver and kidney at 3 and 24 hr after treatment in young and aged rats. The DNA damage in aged rats was less and decreased more slowly compared with young rats. The pictures of MMS-induced DNA migrations at pH 12.1 and 13 were very similar to each other. These results suggest that the adduct glycosylation and repair of the single-strand breaks (SSBs) of aged rats are less than those of young rats, although AP endonucleation is sufficient to remove the AP sites. N-nitrosodiethylamine (160 mg/kg, i.p.) increased DNA migration at pH 12.1 and 13 in the liver and kidney at 3 and 24 hr in young rats and at pH 12.1 and 13 in the kidney at 24 hr in aged rats. These results showed that SSBs were predominantly detected as chemical-induced DNA damage and DNA repairs such as N-glycosylase, DNA polymerase and DNA ligase, and that the metabolic activation declined in aged rats. Aspartate aminotransferase, alanine aminotransferase, total bilirubin, total cholesterol, total protein, globulin, creatinine and chloride age-dependently increased and alkaline phosphates, albumin/globulin ratio, inorganic phosphorus and potassium age-dependently decreased, and these changes were correlated with the DNA migration at pH 13 and/or 8-OH-dG. These results suggest that the activity of DNA repair and metabolic activation enzymes declines in aged rats and that the accumulation of spontaneous DNA damage may affect vital functions.
Alpha-hexachlorocyclohexane (alpha-HCH) is a stereoisomer of gamma-HCH, the active ingredient of lindane (> 99% gamma-HCH). In the present study, cDNA microarray technology was employed to identify changes in gene expression associated with toxicity in livers of male Fischer 344 rats after treatment with alpha-HCH (2, 20 mg/kg/day) and lindane (1, 10 mg/kg/day) by daily oral gavage for up to 28 days. Liver samples were obtained after 1, 3, 7, 14 and 28 days and compared for gene expression profiles. The dose of the alpha-HCH was higher than that of lindane and toxicity was greater, but the numbers of probe sets with differences in expression were fewer for the alpha-HCH-treated group except on Day 3. Only very few probe sets with differences in expression overlapped between alpha-HCH and lindane at each time point and the gene expression profiles were very dissimilar. Important liver-based differences in expression between alpha-HCH and lindane might possibly account for hepato-carcinogenicity of alpha-HCH.
The purpose of this study was to establish the simultaneous measurement of nucleated cell counts and cellular differentials in rat bone marrow examination. The bone marrow cells were stained with an anthraquinone fluorescent DNA stain (DRAQ5) and fluorescence-labeled antibodies, and were analyzed quantitatively using a flow cytometer in the presence of internal standard beads. DRAQ5 distinguished populations of nucleated cells. The absolute counts of nucleated cells were determined using an internal standard, and were equivalent to that measured by the electrical resistance method. The population of nucleated cells was classified into myeloids and erythroids by labeling with CD11b/c and CD71 antibodies, respectively. In a separate examination, T- and B-lymphocytes were also classified by labeling with CD3 and CD45RA antibodies, respectively. The classification of each cell lineage was identical with that of the alternative flow-cytometric method in which cells were differentiated according to cellular size and the fluorescence of a peroxidase indicator, 2',7'-dichlorofluorescin. The ratios of cell lineage, together with myeloid/erythroid ratio (ME), were the same as those obtained by a manual microscopic method. The present flow cytometric method enables the simultaneous measurement of the total nucleated cell counts and cellular differentials of rat bone marrow cells, allowing for rapid and highly quantitative bone marrow examination in rats.
An embryonic stem (ES) cell differentiation model would facilitate analysis of developmental processes at the cellular level and the effects of embryotoxic and teratogenic factors in vitro. We explored the use of differentiation of embryoid bodies (EBs) from cynomolgus monkey ES cells for embryotoxicity testing. We determined the mRNA expression of various genes using real-time RT-PCR. Oct-3/4 expression was almost completely suppressed on day 14, suggesting that ES cells reached differentiated status in around 14 days. mRNA expression of E-cadherin, connexin 43, caveolin-1, and argininosuccinate synthetase was reproducibly suppressed during EB differentiation in 7−32% of ES cells in three separate experiments. Although these may not be "general stemness marker genes" such as Oct-3/4, they could play a role in readying stem cells for differentiation in response to deletion of signals from feeder cells. Next, we examined the effects of bisphenol A (BPA) on the mRNA expression of several differentiation marker genes for ES cells. That of PAX-6, an ectoderm marker, with 0, 0.1, and 10 μM BPA in 21-day EBs was 3,500%, 6,668%, and 8,394%, respectively, compared with ES cells. The difference between doses of 0 and 10 μM BPA in 21-day EBs was statistically significant (p=0.049). Pax-6 activation in the presence of BPA may interfere with the development of eyes, sensory organs, and certain neural and epidermal tissues usually derived from ectodermal tissues. Differentiation of EBs from cynomolgus monkey ES cells could be a useful model for detecting gene expression changes in response to chemical exposure.
Cisplatin (CDDP)-induced acute toxicity was investigated in an experimental model of liver fibrosis produced through repeated intraperitoneal injections of swine serum in rats. A significant increase in level of hepatic markers, such as plasma ASAT, LDH, glucose, total cholesterol and bile acid levels, and a significant decrease in the plasma triacylglycerol level were observed. Slight histological changes, such as necrosis, vacuolar degeneration, and the proliferation of bile ducts were observed as compared with the control fibrotic rats. On the other hand, a significant increase in levels of renal markers, such as plasma BUN and creatinine levels as well as more remarkable tubular degeneration were observed. From these results, CDDP's hepatotoxicity was slight while its nephrotoxicity was more extensive in fibrotic rats.
Metallothionein (MT) is known to play an important role in the resistance of tumor cells to cis-diamminedichloroplatinum (II) (cisplatin). To identify non-MT factors of cisplatin resistance, we characterized cisplatin-resistant cell lines derived from MT-null cells. All of the cisplatin-resistant MT- null cell lines, namely MKCr-1, -3, -4, -12, and -13, showed strong cisplatin resistance and decreased platinum accumulation. Some multidrug resistance proteins (MRPs) have been reported to contribute to cisplatin resistance. However, no significant difference of the MRPs was observed in any of these cell lines. The MKCrs showed cross-resistance to other metals such as arsenite, arsenate, cadmium and antimony. The arsenate and arsenite sensitivities were highly correlated with sensitivity to cisplatin. In addition, the degree of arsenite accumulation was correlated with the degree of cisplatin accumulation. These results suggest that the cisplatin resistance was strongly correlated with the arsenite transport mechanism in these cells.