Acute toxicity of several organophosphorous insecticides and protection by cholinergic antagonists and 2-PAM on Artemia salina larvae

Western blot analysis and indirect immunofluorescence microscopy were used to evaluate the fate of the aryl-hydrocarbon receptor (AhR) and aryl-hydrocarbon receptor nuclear translocator (Arnt) protein in culture cell models exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In wild-type (WT) murine Hepa-1c1c7 cells, AhR protein was depleted by 85% after 4 hr of TCDD treatment as measured in total cell lysates. In contrast, the concentration of Arnt protein was unaffected by TCDD treatment in WT cells. Analysis of the AhR with immunofluorescence microscopy revealed that nuclear translocation of the liganded AhR preceded its depletion from cells. AhR protein was depleted from Hepa-1 type I variants (that contain a concentration of AhR that is 10% of WT) with a similar time course and to the same maximal level observed in WT cells (85%). The EC50 for AhR depletion in Hepa-1 cells was 39 pm TCDD and correspond to the EC50 for induction of P4501A1 protein. Murine embryonic fibroblasts (NIH-3T3), rat aortic smooth muscle cells (A7), and murine skeletal muscle cells (C2C12) all exhibited >90% depletion of the AhR after 2-4 hr of TCDD treatment. Arnt concentration was not affected by TCDD in these cell lines. These results indicate that the liganded AhR is rapidly depleted within the nuclear compartment of hepatic and nonhepatic cells in a manner independent of the Arnt protein.     

Metabolic activation and carcinogen-DNA adduct detection in human larynx

Putative carcinogen-DNA adducts in human larynx tissues (n = 25) from smoker and non/ex-smoker patients were examined by 32P-postlabeling and compared with the metabolic activation capacity of larynx microsomes and cytosols from the same tissues. Hydrophobic DNA adducts were evident only in smokers, and chromatographic profiles of the adducts were similar using either the butanol extraction or nuclease P1 enhancement method, which suggested that the adducts may be derived from polycyclic aromatic hydrocarbons but not aromatic amines. Immunoblots of larynx microsomes using anti-cytochrome P450 1A1/1A2, 2C, 3A4, 2E1, and 2A6 antibodies showed intensities ranging from 1-10% of that typically observed with human liver microsomes. Enzymatic assays of larynx microsomes showed appreciable activity for benzo(a)pyrene hydroxylation (P450 1A1 and 2C) but not for 4-aminobiphenyl N-oxidation (P450 1A2), which indicated that the observed immunoreactivity was for P450 1A1; this represents the highest level of this P450 yet detected in human extrahepatic tissues. Accordingly, total DNA adduct levels in the larynx correlated strongly with levels of P450 2C, 1A1, and 3A4 but not with P450 2E1 or 2A6. Larynx cytosols also showed appreciable aromatic amine N-acetyl-transferase activity for p-aminobenzoic acid (NAT-1) but not for sulfamethazine (NAT-2); however, NAT-1 activity was not correlated with total DNA adducts, which is again consistent with the lack of aromatic amine-DNA adducts detected by 32P-postlabeling. Thus, these results suggest that the DNA adducts detected in human larynx are largely derived from metabolic activation of polycyclic aromatic hydrocarbons in cigarette smoke by P450 2C, 3A4, and/or 1A1.     

Homology between the seed cytolysin enterolobin and bacterial aerolysins

Enterolobin, a 55-kDa cytolytic, inflammatory, and insecticidal protein isolated from seeds of the Brazilian tree Enterolobium contortisiliquum (Leguminosae-Mimosoideae) has been further purified and partially sequenced by using both manual and automated methods. A computational search of enterolobin partial amino acid sequence against the PIR database revealed possible sequence similarities with aerolysins, cytolytic proteins from Aeromonas species. An alignment of enterolobin partial sequence to the amino acid sequences of A. hydrophila and A. sobria aerolysins showed several similar regions with many residue identities. The seed protein enterolobin and the bacterial aerolysins may be homologous proteins despite the distant phylogenetic relationship.     

Use of [6,6-2H2]glucose and of low-enrichment [U-13C6]-glucose for sequential or simultaneous measurements of glucose turnover by gas chromatography-mass spectrometry

We developed gas chromatography-mass spectrometric methods for assaying the enrichment of 99 at.% [6,6-2H2]glucose and 30 at.% [U-13C6]glucose, although both tracers are mostly M + 2. 13C enrichment is determined either by the C-1 to C-5 fragment of glucose aldonitrile pentaacetate or by oxidation of glucose to glucarate. 2H enrichment is assayed as the difference between the 13C enrichment of glucarate and the 2H + 13C enrichment of glucose. The techniques, which were validated in in vivo experiments, are applicable to the determination of simultaneous or sequential measurements of the rate of glucose appearance before and after an intervention. They could also be applied to the simultaneous determination of (i) gluconeogenesis by incorporation of a 13C-labeled precursor into glucose and (ii) the rate of glucose appearance by [6,6-2H2]glucose infusion.     

Selective loss of [3H]kainic acid and [3H]AMPA binding in layer VI of frontal cortex in Huntington's disease

Excitatory amino acid neurotoxicity has been proposed to cause the neostriatal neuronal degeneration of Huntington's disease (HD); N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), and kainate receptors have been hypothesized to play important roles in this process. We have recently reported a loss of neurons in layer VI of the cerebral cortex in HD. Using quantitative autoradiographic methods, we have now measured NMDA, AMPA, and kainate receptor binding in the frontal cerebral cortex of the brains of controls and individuals with HD. We find no change in NMDA receptor binding but a selective decrease in kainate and AMPA receptor binding in layer VI. These data suggest that cerebral cortical neurons possessing kainate or AMPA receptors may be selectively vulnerable in individuals with HD.     

Provisional bilateral symmetry in Xenopus eggs is established during maturation

Dorsal-ventral patterning in the Xenopus egg becomes established midway through the first cell cycle during a 30 degree rotation of the subcortical yolk mass relative to the egg cortex. This 'rotation of symmetrisation' is microtubule dependent, and its direction is thought to be cued by the usually eccentric sperm centrosome. The fact that parthenogenetically activated eggs also undergo a directed rotation, despite the absence of a sperm centrosome, suggests that an endogenous asymmetry in the unfertilised egg supports the directed polymerisation of microtubules in the vegetal cortex, in the way that an eccentric sperm centrosome would in fertilised eggs. Consistent with this idea, we noticed that the maturation spot is usually located an average of more than 15 degrees from the geometric centre of the pigmented animal hemisphere. In parthenogenetically activated eggs, this eccentric maturation spot can be used to predict the direction of rotation. Although in most fertilised eggs the yolk mass rotates toward the sperm entry point (SEP) meridian, occasionally this relationship is perturbed significantly; in such eggs, the maturation spot is never on the same side of the egg as the SEP. In oocytes tilted 90 degrees from upright during maturation in vitro, the maturation spot developed 15 degrees or more from the centre of the pigmented hemisphere, always displaced towards the point on the equator that was up during maturation. This experimentally demonstrated lability is consistent with an off-axis oocyte orientation during oogenesis determining its eccentric maturation spot position, and, in turn, its endogenous rotational bias.