European Journal of Pharmacology: Environmental Toxicology and Pharmacology最新文献

In anesthetized rats, stimulation of the arterial chemoreceptors excites reticulospinal vasomotor neurons of the nucleus rostroventrolateral reticularis of the medulla oblongata, via activating the neuronal NMDA receptors. Excitation of these neurons is responsible for reflex increases in sympathetic neuronal activity and arterial pressure. Additional doses (0.12–0.24 g/kg, i.v.) of urethane dose-dependently reduced the elevations in the discharge rate of reticulospinal vasomotor and sympathetic neurons and of arterial pressure, elicited by stimulating the carotid chemoreceptors with intra-carotid injections of sodium cyanide (100 nmol/10 μl), without affecting discharges of the carotid chemoafferents. Microiontophoresis of urethane onto the reticulospinal vasomotor neurons reversibly inhibited the excitation elicited by stimulation of the chemoreceptors and by iontophoretically applied L-glutamate. The results indicate that the suppression of chemoreflexes by excessive amount of urethane is central and one of its actions is blocking excitatory amino acid transmission onto these vasomotor neurons.

Experiments were carried out to clarify the influence of K⁺-induced chronic membrane depolarization on cytotoxicity and changes in extracellular glutamate, as induced by hypoxia in serum-free cortical cultures. Excitotoxic cell death was examined by measuring lactic dehydrogenase (LDH) activity released into the culture medium. In cultures grown in the presence of 25 mM K⁺, morphological injury occured during a 4 h exposure to hypoxia, together with a substantial efflux of LDH. In hypoxic cultures, extracellular glutamate concentrations were elevated and these responses were absent in cultures grown in physiological medium (K⁺=5.4 mM), even with 16 h of hypoxia. In cultures at 25 mM K⁺, the cytotoxicity induced by hypoxia was attenuated by NMDA receptor antagonists, in a concentration dependent manner. We also examined the effects of excitatory amino acids, agonists of the main glutamate receptor classes (glutamate, NMDA, kainate, and AMPA). In both 5.4 mM and 25 mM K⁺ cultures, a dose dependent release of LDH was induced by a long exposure to glutamate receptor agonists, although the release of LDH in the 5.4 mM K⁺ was less than that in the 25 mM K⁺ cultures. Despite of the expression of the glutamate receptor in the 5.4 mM K⁺ cultures, hypoxic neuronal damage did not occur. These results suggest that when cultures grown in a chronically depolarizing environment are exposed to hypoxia, they are damaged by an excitotoxic mechanism in which the main cause seems to be the glutamate released into the medium at high extracellular levels.

Both ozone and allergen inhalation increase the capacity to produce oxygen radicals by bronchoalveolar lavage cells in dogs. The purpose of these studies was to determine whether inhaled corticosteroids inhibits these increases in oxygen radical production from bronchoalveolar lavage cells. Six random source dogs were studied after dry air or ozone inhalation (3 ppm, 30 min). Seven random source dogs were studied after diluent or allergen inhalation. The dogs inhaled budesonide (2.74 mg/day) or lactose powder, twice daily for 7 days before ozone and allergen. 90 min after ozone or dry air, and 24 h after Ascaris suum or diluent a bronchoalveolar lavage was carried out. Spontaneous luminol-enhanced chemiluminescence was measured from bronchoalveolar lavage cells (4 × 10⁶ cells) for 10 min, followed by a measurement of phorbol myristate acetate (PMA 2.4 μmol/I) stimulated chemiluminescence for 10 min. Both ozone and allergen inhalation caused an increase in PMA stimulated chemiluminescence (P < 0.05). Budesonide pretreatment inhibited ozone-induced (P < 0.008), but not allergen-induced PMA stimulated chemiluminescence (P > 0.90). Both ozone and allergen inhalation caused an increase in the bronchoalveolar lavage neutrophils. Budesonide pretreatment significantly inhibited the ozone-induced (P = 0.007), but not the ascaris-induced neutrophil influx (P = 0.93). These results demonstrate that ozone, but not allergen, stimulated oxygen radical release and neutrophil influx are attenuated by inhaled corticosteroids. This suggests that luminol-enhanced chemiluminescence from bronchoalveolar lavage cells measures oxygen radicals derived from neutrophils, and that ozone-and allergen-induced bronchoalveolar lavage neutrophilia are caused by different mechanisms.

Perturbations in cellular Ca²⁺ homeostasis can lead to oxidative stress whereas nitric oxide has been shown to inactive oxygen radicals. Therefore the effects of inhibition of nitric oxide (NO) synthase activity on Ca²⁺-mediated disruption to rabbit dispersed gastric mucosal cells have been examined. Addition of the Ca²⁺ ionophore A23187 (3–25 μM) to the incubation medium induced a concentration-dependent increase in cell damage is assessed by trypan blue dye uptake and decreased cellular metabolic activity as estimated by alamar blue absorbance. These responses were exacerbated by inhibition of NO synthase activity with $\text{N}{}^{\mathrm{<mtext>G</mtext>}}\text{-}\text{monomethyl-}\text{L}\text{-arginine}$ (300 μM). The deleterious effects of ionophore A23187 and $\text{N}{}^{\mathrm{<mtext>G</mtext>}}\text{-}\text{monomethyl-}\text{L}\text{-arginine}$ were ameliorated by addition of the NO donor S-nitroso-acetyl-penicillamine to the cell suspension. An increase in cellular Ca²⁺ in response to ionophore A23187 (12.5 μM) resulted in enhaced 2′7′-dichlorofluorescein fluorescence suggesting an elevation in oxidative stress. Ca²⁺-mediated cell injury was abolished by the oxygen radical scavengers, catalase and 2′,2′-dipyridyl. However, the cytotoxic effect of combined treatment with A23187 and $\text{N}{}^{\mathrm{<mtext>G</mtext>}}\text{-}\text{monomethyl-}\text{L}\text{-arginine}$ was not reduced by administration of oxygen radical scavengers. $\text{N}{}^{\mathrm{<mtext>G</mtext>}}\text{-}\text{monomethyl-}\text{L}\text{-arginine}$ treatment exacerbated the increase in cytosolic Ca²⁺ in response to ionophore A23187 as assessed by indo-1 fluorescence. Furthermore this increase in cytosolic Ca²⁺ was reduced by addition of S-nitroso-acetyl-penicillamine to the incubation medium. These data suggest that NO synthase inhibition in gastric mucosal cells exacerbates the damaging actions of the Ca²⁺ ionophore A23187. The increase in cell damage in response to the NO synthase inhibitor $\text{N}{}^{\mathrm{<mtext>G</mtext>}}\text{-}\text{monomethyl-}\text{L}\text{-arginine}$ does not appear to be mediated by an increase in oxidative stress and may be associated in part with changes in cellular Ca²⁺ flux.

The effect of environmental temperature on lead accumulation in tissues of mice repeatedly treated with lead acetate (2 mg/kg per day and 5 mg/kg per day) for 3 or 6 weeks was studied. In blood, kidney and liver, the amount of lead accumulated after 3 weeks of treatment was markedly higher in animals exposed to 22°C than those maintained at 35°C. Conversely, when the treatment was extended to 6 weeks, lead concentrations in the liver and kidney were equal or higher respectively, in the mice exposed to 35°C. In the brain, lead concentration was lower than that found in kidney and liver and it was independent of dose and ambient temperature after 3 weeks of treatment but increased when the treatment period was extended up to 6 weeks, the accumulation of lead being higher at 35°C than at 22°C. These results demonstrate that environmental temperature influences the amount of lead accumulated in some rodent tissues, and that the duration of the treatment modifies the effect produced by temperature, suggesting that the changes elicited during the period of acclimation to the hot environment could be responsible for these findings.

The activation of glutamate receptors by endogenous glutamate has been implicated in the processes that underlie cell loss associated with ischemia and trauma and in the development of some neurodegenerative diseases. The antagonism of NMDA-sensitive glutamate receptors may therefore have therapeutic applications. The present study compared the side effects and neuroprotective potency of 1-aminoadamantane hydrochloride (amantadine), 1-amino-3,5-dimethyladamantane hydrochloride (memantine), and (+)-5-methyl-10,11-dihydro-5H-dibenzocyclohepten-5,10-imine maleate ((+)-MK-801) against NMDA injected directly into the nucleus basalis manocellularis of rats. Each drug significantly attenuated the loss of nucleus basalis magnocellularis cholinergic cells. The ED₅₀s were respectively 0.077, 2.81 and 43.5 mg/kg for (+)-MK-801, memantine and amantadine, giving a relative potency ratio of 1:36:565. The ration of the ED₅₀ for the side effects observed, including ataxia, myorelaxation and stereotypy, and the ED₅₀ for neuroprotective ability, was highest for memantine and the lowest for (+)-MK-801. The results suggest that a potential neuroprotective action of NMDA receptor antagonists, memantine and amantadine in particular, can be seen at low doses lacking side effects.

We studied the effects of ozone exposure and dexamethasone on inducible nitric oxide synthase (iNOS) gene expression in Brown Norway rats in vivo. Using a murine iNOS cDNA probe, we detected a 4.4 kb iNOS mRNA by Northern analysis in rat lung. The iNOS signal was weak in control lungs, but increased in lungs exposed to ozone (3 ppm, 6 h). Ozone-induced iNOS mRNA expression was time-dependent, with maximal expression at 2 h, declining by 8 and increasing again at 24 h postexposure. Dexamethasone significantly reduced the iNOS mRNA expression in the lungs of both controls and ozone-exposed rats. These results demonstrate that ozone inhalation induces iNOS expression in vivo, thus providing evidence at the molecular level for the possible involvement of nitric oxide generation in ozone-induced pulmonary inflammation or lung damage.

The therapeutic application of zinc sulphate as an antidote to acetaminophen overdose was examined in ICR mice. Hepatotoxicity was induced by a single oral dose of acetaminophen (750 mg/kg). Various treatments (normal saline, 15 or 30 mg/kg zinc sulphate, 150 mg/kg N-acetylcysteine, 15 mg/kg zinc sulphate + 150 mg/kg N-acetylcysteine) were given i.p. 1 h after acetaminophen overdose. Serum alanine aminotransferase, hepatic glutathione and malondialdehyde levels were measured before experiments and at various intervals after the administration of acetaminophen. Serum acetaminophen levels were also measured at different tt different time intervals. Zinc sulphate showed protection by dose-dependently reducing alanine aminotransferase and malondialdehyde levels. The drug also partially prevented the depletion of hepatic glutathione. These effects were not as good as those of N-acetylcysteine. However, the combination of zinc sulphate with N-acetylcysteine produced even better protective affects. Furthermore, drug treatments did not affect serum acetaminophen levels. It is concluded that both drugs attenuate acetaminophen-induced hepatic toxicity, and the action is likely to be mediated through replenishment of hepatic glutathione levels. The use of zinc sulphate alone or in combination with N-acetylcysteine could be another alternative for the treatment of acetaminophen overdose in view of possible side effects produced by N-acetylcysteine.

We reported previously that 18 compounds varying in general anesthetic potency by up to 66000-fold inhibited, at anesthetic concentrations, the metabolism of arachidonic acid and aminopyrine by cytochrome P450 monooxygenases in rat liver microsomes. Now, we report that P450-mediated para-hydroxylation of aniline is more sensitive to the anesthetics. The K_i values for enzyme inhibition for seven compounds were close to and for seven compounds 5–40 times less than their respective anesthetic potencies. Endogenous substrates with an aniline-like binding mode to P450 include histamine and related imidazoles. Acetone and each of the halogenated compounds, halothane, enflurane, and chloroform, stimulated aniline hydroxylase activity at concentrations well below and above their EC₅₀ values. These potent actions on the universal P450 isozymes may contribute to pharmacological effects of the anesthetics associated with levels of drug well below concentrations that effect general anesthesia.

Expression of human cytochrome P450 (CYP) in heterologous cells is a means of specifically studying the role of these enzymes in drug metabolism. The complete cDNA encoding CYP3A4 (PCN1) was inserted into an expression vector containing the strong myeloproliferative sarcoma virus promoter in combination with the enhancer of the cytomegalovirus and stably expressed in V79 Chinese hamster cells. The presence of genomically integrated CYP3A4 cDNA cell clones was confirmed by polymerase chain reaction analysis. Transcription was detected by reverse transcribed polymerase chain reaction analysis. Functional expression could be demonstrated by conversion of testosterone to the specific 6β-hydroxylated product. In recombinant V79 cells expressing CYP3A4 about 6% of the substrate was converted to 6β-hydroxytestosterone. The metabolism of two dopaminergic ergot derivatives was investigated in live recombinant V79 cells. Both lisuride and terguride were monodeethylated.