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

In vitro effects of cadmium ion on vasoconstriction, particularly on vasoconstriction independent of extracellular Ca²⁺, were investigated using isolated rat aorta. Aorta incubation with CdCl₂ (0.01, 0.1 mM) significantly attenuated contractile responses to KCl and phenylephrine in the medium containing normal Ca²⁺ (2.5 mM). The contractile response to phenylephrine in the presence of calcium channel antagonists, nifedipine (1 μM) or verapamil (1 μM), was markedly inhibited by CdCl₂ (0.1 mM). In the medium without Ca²⁺, phenylephrine (10 μM) induced a phasic contraction, which was markedly inhibited by CdCl₂ (0.1 mM)/ In the medium without Ca²⁺, phorbol 12-myristate 13-acetate (1 μM) and okadaic acid (10 μM) caused tonic contractile responses, which were strongly attenuated by CdCl₂ (0.1 mM) pretreatment. Contractile response to sodium fluoride (5 ∼ 15 mM) in the absence of extracellular Ca²⁺ was strongly attenuated by CdCl₂ (0.1 mM) pretreatment. These results suggest that cadmium ion depresses an extracellular Ca²⁺-independent component of agonist-induced vasoconstriction by hindering an intracellular contractile mechanism(s).

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 3,3′,4,4′,5-pentachlorobiphenyl (PCB 126), 2,3,3′,4,4′,5-hexachlorobiphenyl (PCB 156) on thyroid hormone metabolism were studied in 13-week feeding studies in female Sprague-Dawley rats. The diets were supplemented with the compounds tested at concentrations ranging from 0.2 to 20 μg/kg diet for TCDD, 7 to 180 μg/kg diet for PCB 126, or 12 mg/kg diet for PCB 156, respectively. Significant correlations were found for all three compounds between reductions in plasma total thyroxine (TT₄) levels and inductions of the microsomal phase II enzyme UDP-glucuronosyltransferase by using T₄ as a substrate (T₄UGT). Furthermore, the coinduction of certain phase I and II isozymes, i.c., cytochrome P450 1A1 (CYP1A1) and UGT1A1, by these compounds, clearly suggests the involvement of an Ah receptor-mediated mechanism in the disturbance of thyroid hormone metabolism by these polyhalogenated aromatic compounds. These results provide a mechanistic base for the use of certain effects on thyroid hormone metabolism by polyhalogenated aromatic compounds in risk assessment. By using these effects, potencies of PCB 126 and PCB 156 relative to TCDD ranged from 0.008 to 0.1 for PCB 126, and from 0.00007 to 0.004 for PCB 156, respectively. These values correspond very well with relative potencies of PCB 126 and PCB 156 by using some other well-known Ah receptor-mediated toxic and biochemical parameters.

A monoclonal antibody against tetrodotoxin was produced. Tetrodotoxin coupled with keyhole limpet hemocyanin was used as an immunogen to BALB/c mice. These mice had no clinical signs for the toxicity of tetrodotoxin during the immunization. The reason may be that the guanidyl group of tetrodotoxin which is an important group for the toxicity was hidden by coupling with keyhole limpet hemocyanin. The monoclonal antibody was highly specific for tetrodotoxin and had no cross-reaction to tetrodotoxin derivatives, paralytic shellfish toxins, keyhole limpet hemocyanin and crude proteins from various organs of puffer fish. Also, tetrodotoxin was neutralized in vitro by this antibody. From the fact that the structural difference between tetrodotoxin and anhydro-tetrodotoxin is recognized by this antibody, it was suggested that this antibody reacted with the OH-groups on C-4 and/or C-9 of tetrodotoxin. In addition, the results from immunization and neutralization tests demonstrated that tetrodotoxin became non-toxic even when one of the active groups of tetrodoxin was coupled by a molecule.

Evidence from our previous work suggests that neurogenic mediators contribute to the inflammation following ultraviolet (UV) irradiation of the skin. We have investigated whether calcitonin gene-related peptide (CGRP), substance P and nitric oxide (NO) participate in the cutaneous inflammatory reaction of the rat hind paw and ear to UV irradiation. Skin blood flow was measured by laser Doppler technique. Oedema was quantified using a spring loaded micrometer to measure ear thickness. UV irradiation of the rat skin lead to a long lasting increase in skin blood flow. This increase was dose dependently attenuated by the CGRP receptor antagonist CGRP-(8–37) (0.15 nmol in 25 μl to 6.0 nmol in 25 μl, s.c.) up to 51% with a maximum of effectiveness at 24 h post irradiation. The inhibitor of NO synthase $\text{N}{}^{\mathrm{<mtext>G</mtext>}}\text{-}\text{nitro}\text{-}\text{L}\text{-}\text{arginine}$ methyl ester hydrochloride (L-NAME, 25 nmol in 25 μl, s.c.) attenuated skin blood flow by 38%. Concurrent injections s.c. of CGRP-(8–37) (1.5 nmol in 12.5 μl) and L-NAME (25 nmol in 12.5 μl) demonstrated an augmentive effect in attenuating skin blood flow. The tachykinin NK₁ receptor antagonist CP-96,345 (6.0 nmol in 25 μl, s.c.) attenuated skin blood flow by 27%. $\text{N}{}^{\mathrm{<mtext>G</mtext>}}\text{-}\text{Nitro}\text{-}\text{D}\text{-}\text{arginine methyl}$ ester hydrochloride (D-NAME) and CP-96,344 showed no effects on skin blood flow after UV irradiation. CGRP-(8–37) (0.6 nmol in 10 μl) i.d. and L-NAME (10 nmol in 10 μl) i.d. had no effect of oedema formation after UV irradiation. Furthermore, post UV irradiation enhanced CGRP- and NO synthase-immunoreactivity in nerve fibres in the exposed skin area were visible. Taken these findings together we suggest the involvement of the neuropeptides CGRP and substance P and of neuronal NO on the vasodilatory component of the UV-induced inflammatory reaction of the rat skin. CGRP contributing to UV-induced vasodilation acts in an endothelial NO-independent manner.

We present in this report the characteristics of the damage induced by 6-hydroxydopamine and H₂O₂ on bovine chromaffin cells in primary culture. Cytotoxicity was quantified using catecholamine cell contents, lactate dehydrogenase (LDH) release, trypan blue exclusion and morphological appearance. An excellent correlation between these four parameters was found. The cytotoxic effects of 6-hydroxydopamine were Ca²⁺-independent. In spite of this, the Ca²⁺ channel antagonists R56865 (N-[1-(4-(fluorophenoxy)butyl)]-4-piperidinyl-N-methyl-2-benzo-thiazolamine) and lidoflazine exhibited marked cytoprotective effects against both 6-hydroxydopamine and H₂O₂. The selective dopamine uptake blocker, bupropion, increased the viability of 6-hydroxydopamine and H₂O₂-treated cells from 20% to around 80%. Catalase drastically protected against the cytotoxic effects of 6-hydroxydopamine and H₂O₂. In contrast, desferrioxamine gave better protection against H₂O₂ cytotoxicity; glutathione and N-acetylcysteine only afforded substantial protection against 6-hydroxydopamine. Three main conclusions emerge from this study. (1st) 6-Hydroxydopamine causes chromaffin cell damage via a mechanism probably related to the production of free radicals, but unrelated to Ca²⁺ ions. Cytoprotection afforded by R56865 and lidoflazine must be unrelated to their Ca²⁺ antagonist properties. This suggests a novel component in the cytoprotective mechanism of action of these drugs. (2nd) The strong cytoprotective effects of bupropion seem to be unrelated to its ability to block the plasmalemmal dopamine carrier. (3rd) Bovine adrenal chromaffin cells in primary cultures are a suitable model for adult neurons to study the basic mechanism of cell damage, and to screen new drugs with putative neuroprotective properties.

A scientific evaluation was made of functionalspects of developmental toxicity of polychlorinated biphenyls (PCBs), polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) in experimental animals and in human infants. Persistent neurobehavioral, reproductive and endocrine alterations were observed in experimental animals, following in utero and lactational exposure to PCBs, PCDDs and PCDFs. The lowest observable adverse effect levels (LOAELs) for developmental neurobehavioral and reproduction endpoints, based on bodyen of TCDD-toxic equivalents (TEQs) in animals, are within the range of current background human body burdens. Relatively subtle adverse effects on neurobehavioral development and thyroid hormone alterations have also been observed in infants and children exposed to background levels. Exclusive use of the toxic equivalency factor (TEF) approach may underestimate the risk of neurodevelopmental effects, because both Ah receptor dependent and independent mechanisms may be involved in these effects. The use of marker congeners and/or bioassays based on Ah receptor mediated mechanisms are rapid, low cost pre-screening alternatives for expensive and time consuming gas chromatographic-mass spectrometric analysis.

The effects of platelet-activating factor (PAF) on myocardial injury after 1 h global ischemia-30 min reperfusion were investigated in isolated arterially perfused interventricular septum of rabbit heart. PAF did not significantly affect developed tension, ±dT/dt_max, resting tension and the times of active state in non-ischemic septa. The recovery of developed tension was significantly reduced by PAF (100 nM), after an ischemia-reperfusion challenge, from the control value of 20.9 ± 3.5% to 10.5 ± 1.8%, without a change in the resting tension (15.7 ± 2.8 vs. 15.6 ± 1.3 g). BN 52021 (20 μM), alone did not modify either parameter of ischemic damage, but antagonized the aggravating effect of PAF. Evidence of PAF activity was not found in any of the samples of the effluent perfusate obtained from ischemic control experiments. On the basis of the present results, we suggest a direct role for PAF in aggravating the myocardial damage induced by ischemia, and discard heart cells as the source of PAF in this state.

N-oxygenation of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a dopaminergic neurotoxin, was studied using recombinant rat liver flavin-containing monooxygenase (FMO), FMO1A1, expressed in yeast cells. The mean (±S.D.) kinetic parameters of MPTP N-oxygenation were: K_m = 1.8±0.5 μM, V_max = 9.5±1.6 nmol/mg per min, and V_max/K_m = 5.6±0.5 ml/mg per min. n-Octylamine, an activator of FMO, enhanced the MPTP N-oxygenation activity by 51%, while methimazole, thiobenzamide and α-naphthylthiourea, alternate substrates of FMO, inhibited it by 27.4, 68.0 and 59.2%, respectively. The results indicate that MPTP is efficiently N-oxygenated by the recombinant rat liver FMO1A1, and that the responses to the modulators of FMO activity found in the recombinant rat liver FMO1A1 resemble those of mouse and rat liver microsomes as reported previously. The findings suggest that the recombinant FMO expressed in yeast cells is considered as a useful tool to study an involvement of FMO in the metabolism of environmental toxins or chemicals. In addition, FMO1A1 appears to be one of the predominant enzymes responsible for the N-oxygenation of MPTP at least in rat liver.

The influence of ethanol (0.5, 1.0 and 2.0 g/kg i.p.) on the Bezold-Jarisch reflex in urethane-anesthetized rats was studied. 5-Hydroxytryptamine (serotonin; 5-HT; 1, 3, 10 and 30 μg/kg i.v.) and capsaicin (1, 3 and 10 μg/kg i.v.) reflexly decreased heart rate in a dose-dependent manner. The 5-HT₃ receptor antagonist ondansetron 10 μg/kg i.v. abolished the 5-HT- but not the capsaicin-stimulated bradycardia, indicating that 5-HT and capsaicin acted via different trigger mechanisms (5-HT₃ receptor-dependent and -independent, respectively). Ethanol at 1.0 and 2.0 g/kg i.p. inhibited in a dose-dependent manner (by 20–45%) the 5-HT- but not the capsaicin-stimulated decrease in heart rate. Our results demonstrate that the inhibitory effect of ethanol on the 5-HT₃ receptor-mediated Bezold-Jarisch reflex may be related to the direct effect of ethanol on 5-HT₃ receptors on sensory vagal nerves in the heart.

An NIH/3T3 cell line, stably expressing human cytochrome P450-3A4 (CYP3A4) cDNA has been developed. This cell line was used in combination with a shuttle vector, containing the bacterial lacZ′ gene as reporter gene, to study mutagenicity. Ethylmethanesulphonate and aflatoxin B₁ were used as model agents to test this system. The mutation frequency of ethylmethanesulphonate increased concentration dependently and was the same in CYP3A4-expressing cells as in parental NIH/3T3 cells, demonstrating that CYP3A4 activity has no influence on the mutagenicity of ethylmethanesulphonate. The mutation frequency of aflatoxin B₁ increased concentration dependently only in the CYP3A4-expressing cells and not in parental nor in vector-transfected cells. This increase in mutation frequency could be completely inhibited by ketoconazole, an inhibitor of cytochrome P450 activity, demonstrating the role of CYP3A4 in the activation of aflatoxin B₁. The system described in this paper opens the possibility to study the capacity of single human cytochrome P450s to activate xenobiotics into mutagenic metabolites. Since activation, phase II metabolism, DNA repair and an endpoint for mutations are all present in one cell, this system will be useful in screening as well as in mechanistic studies.