Новые публикации по токсикологии и смежным дисциплинам
(по страницам журнала
Toxicology and Applied Pharmacology, v 156, N 1, 1999)

Effects of U-83836E on Glutamate-Induced Neurotoxicity in Dissociated Rat Cerebellar Granule Cells

Francesc X. Sureda, Cecilia Gabriel, David Pubill, Merce Pallas, Elena Escubedo, Jorge Camarasa, Antonio Camins

The effects of the lazaroid compound U-83836E on the glutamate-induced production of reactive oxygen species (ROS) were studied in dissociated rat cerebellar granule cells by flow cytometry. U-83836E completely inhibited ROS production with an estimated IC₅₀ value of 21.7 ± 9.1 nM. However, U-83836E did not inhibit the glutamate-evoked decrease in mitochondrial membrane potential (MMP). Nevertheless, U-83836E (10 nM to 10 µM) prevented cell death induced by 10 mM of glutamate. At concentrations above 10 µM, U-83836E by itself showed slight cytotoxicity, which was significant at a 100 µM concentration. U-83836E (25 to 200 µM) also increased the cytosolic calcium levels in a concentration-dependent manner. Our results indicate that the cytotoxic effects found at micromolar concentrations of U-83836E could be explained by an increase in [Ca²⁺]i. Finally, since U-83836E did not prevent the MMP decrease evoked by glutamate, it is suggested that antioxidant pharmacotherapy would not be sufficient to block the neurotoxic effects of glutamate. Copyright 1999 Academic Press.

Toxicology and Applied Pharmacology, v 156, n 1, April 1, 1999, p1-5 (ID taap.1998.8613)

Differential Susceptibility of Brain Areas to Cyanide Involves Different Modes of Cell Death

Edward M. Mills, Palur G. Gunasekar, L. Li, Joseph L. Borowitz, Gary E. Isom

We have demonstrated that cyanide (KCN) induces selective degeneration of dopaminergic neurons in mice and apoptotic cell death in cultured neurons. In the present study the mode of cyanide-induced cell death was determined in the susceptible brain areas. Mice were treated with KCN (6 mg/kg ip) or vehicle (saline) twice daily for 1 to 12 days. After 3 days of KCN treatment, two separate lesions were observed in coronal brain sections. Widespread DNA fragmentation in parietal and suprarhinal regions of the motor cortex was observed by the in situ terminal deoxynucleotide transferase nick-end labeling (TUNEL) technique. Pyknosis and chromatin condensation, morphological hallmarks of apoptotic cells, were observed in TUNEL-positive regions. On the other hand, in the substantia nigra (SN), KCN produced a progressive, bilateral necrotic lesion that was evident by 3 days of treatment. The SN lesion was circumscribed by a prominent ring of glial infiltration, as determined by glial-acidic fibrillary protein (GFAP) immunostaining. The extent of the SN lesion steadily increased with treatment duration, and DNA fragmentation was not observed over the 1- to 12-day period. On the other hand, cortical apoptosis was not associated with necrotic cell loss or astrogliosis. Pretreatment of animals with the antioxidant a-phenyl-tert-butyl nitrone (PBN) for 7 days prior to and during 3 days of KCN administration markedly reduced cortical DNA fragmentation whereas the PBN treatment did not influence the SN necrosis or astrocytic gliosis. Except for moderate GFAP immunostaining in corpus callosum, other brain areas were not affected by cyanide. It is concluded that KCN-induced neuronal loss involves selective activation of necrosis or apoptosis in different neuronal populations, and involves divergent mechanisms and sensitivity to antioxidants. Copyright 1999 Academic Press.

Toxicology and Applied Pharmacology, v 156, n 1, April 1, 1999, p6-16 (ID taap.1999.8630)

Activation of Poly [ADP-Ribose] Polymerase in Endothelial Cells and Keratinocytes: Role in an in Vitro Model of Sulfur Mustard-Mediated Vesication

Daniel B. Hinshaw, Irfan J. Lodhi, Lauren L. Hurley, Kevin B. Atkins, Milena I. Dabrowska

Although endothelial cells and keratinocytes appear to be the primary cellular targets of sulfur mustard (SM), the role of the nuclear enzyme poly (ADP-ribose) polymerase (PARP) in SM-induced vesication has not been clearly defined. PARP is thought to play a crucial role in DNA repair mechanisms following exposure to alkylating agents like SM. Using a combination of fluorescence microscopy and biochemical assays, we tested the hypothesis that SM causes activation of PARP in endothelial cells and keratinocytes with subsequent loss of nicotinamide adenine dinucleotide (NAD) and depletion of adenosine triphosphate (ATP) levels. To determine if PARP activation accounts for SM-induced vesication, keratinocyte adherence and permeability of endothelial monolayers were measured as in vitro correlates of vesication. As early as 2 to 3 h after exposure to SM concentrations as low as 250 µM, dramatic changes were induced in keratinocyte morphology and microfilament architecture. Exposure to 500 µM SM induced a fourfold increase in PARP activity in endothelial cells, and a two- to threefold increase in keratinocytes. SM induced a dose-related loss of NAD⁺ in both endothelial cells and keratinocytes. ATP levels fell to 50% of control levels in response to SM concentrations 500 µM. SM concentrations 250 µM significantly reduced keratinocyte adherence as early as 3 h after exposure. Endothelial monolayer permeability increased substantially with concentrations of SM > 250 µM. These observations support the hypothesis that the pathogenic events necessary for SM-induced vesication (i.e., capillary leak and loss of keratinocyte adherence) at higher vesicating doses of SM ( 500 µM) may depend on NAD loss with PARP activation and subsequent ATP-dependent effects on microfilament architecture. Vesication developing as a result of exposure to lower concentrations of SM presumably occurs by mechanisms that do not depend on loss of cellular ATP (e.g., apoptosis and direct SM-mediated damage to integrins and the basement membrane). Copyright 1999 Academic Press.

Toxicology and Applied Pharmacology, v 156, n 1, April 1, 1999, p17-29 (ID taap.1999.8634)

Induction of Altered Hepatic Foci by a Mixture of Dioxin-like Compounds with and without 2,2',4,4',5,5'-Hexachlorobiphenyl in Female Sprague-Dawley Rats

Simone A. van der Plas, Marie Haag-Gronlund, Gunilla Scheu, Lars Warngard, Martin van den Berg, Piet Wester, Jan H. Koeman, Abraham Brouwer

The hepatic tumor-promoting activity of a mixture of polyhalogenated aromatic hydrocarbons (PHAHs) was studied in a medium term two-stage initiation/promotion bioassay in female Sprague-Dawley rats. The PHAH mixture contained 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 1,2,3,7,8-pentachlorodibenzo-p-dioxin (PeCDD), 2,3,4,7,8-pentachlorodibenzofuran (PeCDF), 3,3',4,4',5-pentachlorobiphenyl (PCB 126), 2,3',4,4',5-pentachlorobiphenyl (PCB 118), 2,3,3',4,4',5-hexachlorobiphenyl (PCB 156), 2,2',4,4',5,5'-hexachlorobiphenyl (PCB 153) and covered > 90% of the total toxic equivalents (TEQ) present in Baltic herring. To determine possible interactive effects of di-ortho-substituted PCBs, the PHAH mixture was tested with (PHAH⁺) and without (PHAH^-) PCB 153. Rats were initiated by a diethylnitrosamine injection (30 mg/kg body wt i.p.) 24 h after a partial 23 hepatectomy. Six weeks after initiation, the PHAH mixtures were administered once a week by subcutaneous injections for 20 weeks. Treatment with the PHAH mixtures caused liver enlargement and an increased activity of the hepatic cytochrome P-4501A1/2 and P-4502B1/2. All PHAH exposure groups exhibited an increased occurrence of hepatic foci positive for the placental form of glutathione-S-transferase. In the PHAH-group dosed 1 µg TEQ/kg body wt/week, the volume fraction of the liver occupied by foci was significantly lower compared to the TEQ equivalent dosed TCDD group (3.8 vs 8.7%). The volume fraction was significantly increased in the groups treated with 0.5, 1, or 2 µg TEQ/kg body wt/week of the PHAH+ mixture (4.5, 5.2, and 6.6%, respectively) compared to the corn oil group (2.0%), but to a lower extent than expected on basis of the TEQ doses. Overall, the TEQ-based administered dose overestimated the observed tumor-promoting effects of this PHAH mixture. The applicability of the toxic equivalency factor concept, the role of differences in toxicokinetic properties and interactive effects of PCB 153 on hepatic deposition of the dioxin-like congeners are discussed. Copyright 1999 Academic Press.

Toxicology and Applied Pharmacology, v 156, n 1, April 1, 1999, p30-39 (ID taap.1999.8629)

Analysis of Differential Effects of Pb2+ on Protein Kinase C Isozymes

Xiaoyan Sun, Xintian Tian, Jose L. Tomsig, Janusz B. Suszkiw

Protein kinase C has been implicated as a cellular target for Pb²⁺ toxicity. We have previously proposed that Pb²⁺ modulates PKC activity by interacting with multiple sites within the enzyme. In order to further characterize the Pb-PKC interactions we compared the effects of Pb²⁺ on the CA-dependent and -independent protein kinase C isozymes using recombinant human PKC-alpha, PKC-epsilon, and PKC-zeta as well as the catalytic fragment of bovine brain protein kinase C, the PKC-M. The results demonstrate that, whereas at pM concentrations Pb²⁺ activates PKC-alpha half maximally (KAct ~ 2 pM), it has no effect on PKC-epsilon, PKC-zeta, or PKC-M activities. The activation of PKC-alpha by Pb²⁺ is additive with Ca²⁺ in a manner indicating interaction with half of the calcium activation sites. In the micromolar range of concentrations, Pb²⁺ inhibits all PKCs with estimated K0.5 of 1.0, 2.3, 28, and 93 µM for PKC-M, PKC-alpha, PKC-epsilon, and PKC-zeta, respectively. Examination of Pb²⁺ effects on PKC-M kinetics indicates a mixed type inhibition with respect to ATP and noncompetitive inhibition with respect to histone. Taken together with the results of our previous study (Tomsig and Suszkiw, J. Neurochem. 64, 2667-2673, 1995) and the evidence for the existence of two Ca²⁺ coordination sites Ca1 and Ca2 within the C2 domain (Shao et al., Science [Washington, D.C.] 273, 248-251, 1996), the results of the current study provide further support for a multisite Pb-PKC interaction scheme wherein lead (1) partially activates the enzyme through pM-affinity interactions with the Ca1 site and inhibits the divalent cation-dependent activity through nM-affinity interactions with Ca2 site in the C2 domain and (2) inhibits the constitutive kinase activity through µM-affinity interactions with the catalytic domain. The concentration dependence of the differential effects of Pb²⁺ on the calcium-dependent and -independent PKCs underscores the importance of the C2 motif as a high affinity molecular target for Pb²⁺. Copyright 1999 Academic Press

Toxicology and Applied Pharmacology, v 156, n 1, April 1, 1999, p40-45 (ID taap.1999.8622)

Induction of Apoptosis and Changes in Nuclear G-actin Are Mediated by Different Pathways: The Effect of Inhibitors of Protein and RNA Synthesis in Isolated Rat Hepatocytes

Irma Meijerman, W. Marty Blom, Hans J. G. M. de Bont, Gerard J. Mulder, J. Fred Nagelkerke

Stressor-induced changes in the cytoskeleton, of which actin is a major component, may lead to apoptosis. The role of drug-induced changes in nuclear G-actin and apoptosis was studied in freshly isolated hepatocytes. Several protein synthesis inhibitors, cycloheximide, puromycin, and emetine, induced 10 to 15% apoptosis in hepatocytes after 4 h, as was determined by changes in nuclear morphology and flow cytometric analysis of Annexin V-positive cells. Apoptosis induced by protein synthesis inhibition could be prevented by the caspase inhibitors Z-Val-Ala-DL-Asp fluormethylketone (zVAD-fmk) and Ac-Asp-Glu-Val-Asp-aldehyde (DEVD-cho).Several (chemical) stressors cause a rapid increase in nuclear G-actin staining in hepatocytes or cell lines (Meijerman et al., Biochem. Biophys. Res. Commun. 240, 697-700, 1997). The protein synthesis inhibitors also increased G-actin staining in nuclei after 2 h; this could not be inhibited by zVAD-fmk or DEVD-cho. Changes in the cytosolic F-actin pattern did not occur until nuclear G-actin staining had already increased. The mRNA synthesis inhibitor actinomycin D, also increased nuclear G-actin staining, but did not induce apoptosis within the studied time frame. The results suggest that the induction of apoptosis and the increased nuclear staining of G-actin by protein synthesis inhibition are differently controlled. Copyright 1999 Academic Press

Toxicology and Applied Pharmacology, v 156, n 1, April 1, 1999, p46-55 (ID taap.1998.8616)

Antagonism of Paraoxon Intoxication by Recombinant Phosphotriesterase Encapsulated within Sterically Stabilized Liposomes

I. Petrikovics, K. Hong, G. Omburo, Q. Z. Hu, L. Pei, W. D. McGuinn, D. Sylvester, C. Tamulinas, D. Papahadjopoulos, J. C. Jaszberenyi, J. L. Way

This investigation effort is focused on increasing organophosphate (OP) degradation by phosphotriesterase to antagonize OP intoxication. For these studies, sterically stabilized liposomes encapsulating recombinant phosphotriesterase were employed. This enzyme was obtained from Flavobacterium sp. and was expressed in Escherichia coli. It has a broad substrate specificity, which includes parathion, paraoxon, soman, sarin, diisopropylfluorophosphate, and other organophosphorous compounds. Paraoxon is rapidly hydrolyzed by phosphotriesterase to the less toxic 4-nitrophenol and diethylphosphate. This enzyme was isolated and purified over 1600-fold and subsequently encapsulated within sterically stabilized liposomes (SL). The properties of this encapsulated phosphotriesterase were investigated. When these liposomes containing phosphotriesterase were incubated with paraoxon, it readily degraded the paraoxon. Hydrolysis of paraoxon did not occur when these sterically stabilized liposomes contained no phosphotriesterase. These sterically stabilized liposomes (SL) containing phosphotriesterases (SL)* were employed as a carrier model to antagonize the toxic effects of paraoxon by hydrolyzing it to the less toxic 4-nitrophenol and diethylphosphate. This enzyme-SL complex (SL)* was administered intravenously to mice either alone or in combination with pralidoxime (2-PAM) and/or atropine intraperitoneally. These results indicate that this carrier model system provides a striking enhanced protective effects against the lethal effects of paraoxon. Moreover when these carrier liposomes were administered with 2-PAM and/or atropine, a dramatic enhanced protection was observed. Copyright 1999 Academic Press.

Toxicology and Applied Pharmacology, v 156, n 1, April 1, 1999, p56-63 (ID taap.1998.8620)

Sarin-like and Soman-like Organophosphorous Agents Activate PLC gamma in Rat Brains

Hitoshi Niijima, Masataka Nagao, Makoto Nakajima, Takehiko Takatori, Yukimasa Matsuda, Hirotaro Iwase, Masahiko Kobayashi

We report that there is a time-related change in the phospholipase C (PLC) activities of rat brain cytosol and membrane fractions after iv injection of a soman-like or a sarin-like organophosphorous agent (bis(isopropyl methyl)phosphonate [BIMP] and bis(pinacolyl methyl)phosphonate [BPMP]). PLC gamma was activated in the brain cytosol fraction from BPMP-injected rats. The phosphorylating activity of rat brain membrane fractions were enhanced by BPMP treatment. The brain membrane fractions from BPMP-treated rats phosphorylated several proteins, including supposedly PLC gamma in the brain cytosol fraction from control rats in vitro. These results suggest that soman and sarin may stimulate a membrane tyrosine kinase, including growth factor receptors, directly or indirectly. Copyright 1999 Academic Press.

Toxicology and Applied Pharmacology, v 156, n 1, April 1, 1999, p64-69 (ID taap.1998.8628)