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Archives of Toxicology (v.73, #1)
Phospholipase A2 inhibitors p-bromophenacyl bromide and arachidonyl trifluoromethyl ketone suppressed interleukin-2 (IL-2) expression in murine primary splenocytes by Yanli Ouyang; Norbert E. Kaminski (pp. 1-6).
Phospholipase A2 (PLA2) has been postulated to play a role in the regulation of cytokine expression. Therefore, the objective of the present study was to investigate the effects of PLA2 inhibitors p-bromophenacyl bromide (BPB) and arachidonyl trifluoromethyl ketone (AACOCF3) on interleukin-2 (IL-2) expression in murine primary splenocytes. Pretreatment of the splenocytes with both BPB and AACOCF3 suppressed phorbol 12-myristate 13-acetate plus ionomycin-induced IL-2 secretion in a concentration-dependent manner. Inhibition >90% of IL-2 secretion was observed at 1 μM BPB and 10 μM AACOCF3 compared to the respective vehicle control. Likewise, IL-2 steady-state mRNA expression was inhibited by both PLA2 inhibitors in a concentration-dependent fashion with >90% inhibition at 1 μM BPB and 20 μM AACOCF3. Taken together, these data demonstrated that PLA2 inhibitors BPB and AACOCF3 are robust inhibitors of IL-2 expression at both the mRNA and protein levels in murine splenocytes. Moreover, these findings suggest that drugs and chemicals which inhibit PLA2 may have marked effects on T-cell function.
Keywords: Key words Phospholipase A2 inhibitors; p-Bromo- phenacyl bromide; Arachidonyl trifluoromethylketone; IL-2 expression
Dimethylphosphoryl-inhibited human cholinesterases: inhibition, reactivation, and aging kinetics by F. Worek; C. Diepold; P. Eyer (pp. 7-14).
Human poisoning by organophosphates bearing two methoxy groups, e.g. by malathion, paraoxon-methyl, dimethoate and oxydemeton-methyl, is generally considered to be rather resistant to oxime therapy. Since the oxime effectiveness is influenced not only by its reactivating potential but also by inhibition, aging and spontaneous reactivation kinetics, experiments were performed with human acetyl- (AChE) and butyrylcholinesterase (BChE) to determine the respective kinetic constants. The efficacy of obidoxime in reactivating dimethylphosphoryl-AChE was 40, 9 and 3 times higher than of HI 6, pralidoxime and HLö 7, respectively. Aging (t 1/2 3.7 h) and spontaneous reactivation (t 1/2 0.7 h) occurred concomitantly, with the portion of the aged enzyme being dependent on the presence of excess inhibitor. Calculation of steady-state AChE activity in the presence of inhibitor and oxime revealed that obidoxime was superior to pralidoxime. In addition, organophosphate concentrations up to 10−6 M (paraoxon-methyl) and 10−4 M (oxydemeton-methyl) could be counteracted at clinically relevant oxime concentrations (10 μM). These data indicate that oximes may effectively reactivate human dimethylphosphoryl-AChE. Failure of oximes may be attributed to megadose intoxications and to prolonged time intervals between poison uptake and oxime administration. The potency of the oximes to reactivate dimethylphosphoryl-BChE was much lower and the spontaneous reactivation slower (t 1/2 9 h), while aging proceeded at a comparable rate. Thus, BChE activity determination for diagnosis and therapeutic monitoring may give no reliable information on AChE status.
Keywords: Key words Organophosphates; Acetylcholinesterase; Oximes; Human; Reactivation
Activity of O6-methylguanine DNA methyltransferase in mononuclear blood cells of formaldehyde-exposed medical students by K. Schlink; K. Janßen; S. Nitzsche; S. Gebhard; J. G. Hengstler; S. Klein; F. Oesch (pp. 15-21).
A recent study reported that exposure of student embalmers in Cincinnati to high concentrations of formaldehyde (2 mg/m3) reduced the activity of the DNA repair protein O6-methylguanine DNA methyltransferase (MGMT). Reduction in a DNA repair enzyme may strongly increase the cancer risk not only with respect to the repair-enzyme causing agent but with respect to all carcinogens causing lesions subject to repair by the enzyme in question. Thus, we examined whether formaldehyde exposure of 57 medical students during their anatomy course at two different Universities in Germany influenced MGMT activity in mononuclear blood cells. Mean formaldehyde exposure of 41 students was 0.2 ± 0.05 mg/m3 for 6 h per week. MGMT activity was 133.2 ± 14.9 fmol MGMT/106 cells before the beginning of the formaldehyde exposure, 131.1 ± 15.8 fmol MGMT/106 cells after 50 days (P = 0.56) and 128.2 ± 19.0 fmol MGMT/106 cells after 111 days of exposure (P=0.92). Similarly, no significant influence of formaldehyde exposure was observed, when smoking habits, alcohol consumption, allergic disease and sex of students were considered. In addition no significant difference was obtained in MGMT activity between 16 students with mean formaldehyde exposure of 0.8 ± 0.6 mg/m3 and students without formaldehyde exposure (n=51; P=0.37). In conclusion, exposure of the medical students in western Europe to formaldehyde did not decrease MGMT activity in mononuclear blood cells.
Keywords: Key words O6-methylguanine DNA methyltransferase (MGMT); Formaldehyde; Mononuclear blood cells; DNA repair
Behavioral abnormalities and apoptotic changes in neurons in mice brain following a single administration of allylnitrile by Xiao-ping Zang; Hideji Tanii; Katsuji Kobayashi; Tomomi Higashi; Rie Oka; Yoshifumi Koshino; Kiyofumi Saijoh (pp. 22-32).
A single dose of allylnitrile in mice might induce persistent behavioral abnormalities, of which the mechanism is not yet known. The present study was undertaken to explore the relationship between behavioral abnormalities and pathological changes in the brain of mice following exposure to allylnitrile. Exposure to allylnitrile (63, 84, and 112 mg/kg, p.o.) resulted in dose-dependent changes in behavioral abnormalities, including increased locomotor activity, circling, retropulsion, head twitching, and alteration in reflexive behavior, which appeared at day 2 postdosing and were persistent throughout the experimental period (60 days) at the higher dose levels. Allylnitrile produced neuronal retraction including hyperchromasia of the nuclei in the raphe nuclei, cerebral cortex, hypothalamus, hippocampal CA1 and dentate gyrus later than 30 days. No gliosis was observed in these regions. Not all but a significant number of neurons in the hippocampal CA1, medial habenula and raphe nuclei were immunoreactive to CPP32 (Caspase-3) even at day 2. These neurons were also positive to Hoechst 33258 staining, indicating allylnitrile caused apoptotic changes in specific neurons when neuronal behaviors became apparent. These apoptotic changes were persistent even in the area without neuronal contraction such as medial habenula. However, almost all neurons in these areas were also positive to terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling (TUNEL). It is conceivable that allylnitrile caused apoptotic changes in neurons but did not always lead them to cell death immediately. Moreover, even when neuronal contraction resulted in retention of behavioral abnormalities, onset of these abnormalities seems to be associated with the impairment in the habenulo-raphe relay due to activation of apoptotic cascade in neurons.
Keywords: Key words Allylnitrile; Apoptosis; Behavioral abnormalities; Habenula; Mice brain
Metabolism of benzene in human liver microsomes: individual variations in relation to CYP2E1 expression by Vessela Nedelcheva; Ivan Gut; Pavel Souček; Bronislava Tichavská; Lucie Týnkova; Jaroslav Mráz; F. Peter Guengerich; Magnus Ingelman-Sundberg (pp. 33-40).
In human liver microsomes the oxidations of benzene, chlorzoxazone, aniline, dimethylformamide, and 4-nitrophenol were significantly correlated with each other and with the level of cytochrome P450 (CYP) 2E1 estimated by immunoblotting. Moreover, benzene oxidation to water-soluble metabolites was suppressed by 0.1 mM diethyldithiocarbamate, supposedly a specific inhibitor of CYP2E1 at this level. None of these metabolic rates correlated with immunochemically determined levels of CYP1A2, 2C9, and 3A4 nor oxidation of 7-ethoxyresorufin, tolbutamide, and nifedipine. Benzene oxidation to water-soluble metabolites was characterized by typical Michaelis-Menten kinetics. The different benzene K m values seen in individual human microsomal samples were not correlated with the level or activity of CYP1A2, 2C9, 2E1, and 3A4 but could be due to CYP2E1 microheterogeneity. The lowest K m for benzene oxidation could be related to C/D and/or c1/c2 polymorphism of CYP2E1 gene. Covalent binding of benzene reactive metabolites to microsomal proteins was also correlated with the CYP2E1 metabolic rates and immunochemical levels. At high concentrations of benzene covalent binding was inversely related to benzene concentrations (as well as to formation of water-soluble metabolites) in agreement with the view that secondary metabolites, mainly benzoquinone, are responsible for the covalent binding.
Keywords: Key words Benzene; Chlorzoxazone; Covalent binding; Cytochrome P450; Enzyme kinetics
Mechanism of decrease in levels of hepatic P450 isozymes induced by intracerebral endotoxin: independence from sympathetic nervous and adrenocortical systems by Yoshinori Shimamoto; Hiroshi Kitamura; Masaru Iwai; Masayuki Saito; Akio Kazusaka; Shoichi Fujita (pp. 41-49).
We previously reported that lipopolysaccharide (LPS) injected intracerebroventricularly (i.c.v.) at an ineffective dose (0.1 μg/rat) decreased the drug metabolizing activities and related cytochrome P450 (CYP) isozymes in rat liver microsomes when injected intraperitoneally (i.p.). The dose study (doses <0.1 μg intracerebrally and >0.1 μg i.p.), which was carried out to examine how much more effective is i.c.v.-LPS than i.p.-LPS, showed that the pattern of alteration of expression of CYP isozymes induced by i.c.v.-LPS was different from that caused by i.p.-LPS at an effective dose (10 μg/rat). These results indicate that the decrease in hepatic CYP isozymes caused by i.c.v.-LPS could not be explained by the LPS leaked from the brain, suggesting that the decrease in hepatic CYP isozymes by i.c.v.-LPS may be caused by a central action of LPS. In this study, the possible involvement of sympathetic nervous and adrenocortical systems in the down-regulation of CYP isozymes by i.c.v.-LPS was investigated using surgical or chemical sympathetecomized or adrenalectomized rats. The norepinephrine (NE) content in the liver in rats with surgical hepatic sympathetectomy was reduced by 88% compared with that of sham-operated rats that received i.c.v.-saline and 85% compared with that of sham-operated rats that received i.c.v.-LPS, indicating that hepatic denervation was successful. The NE content in the liver in rats chemically sympathetectomized by two i.p. injections of 6-hydroxydopamine (40 mg/kg each time) 1 and 2 days before i.c.v. injection was reduced by 82% in i.c.v.-saline-treated and by 74% in i.c.v.-LPS-treated groups compared with that in rats pretreated with i.p.-saline. These results indicate that sympathetic NE terminals were effectively removed. Intracerebroventricular LPS decreased the total P450 content and the activities of CYP dependent drug metabolizing enzymes, ethoxyresorufin O-deethylase (EROD), pentoxyresorufin O-depentylase (PROD), imipramine N-demethylase (IMND) and erythromycin N-demethylase (ERND) after 24 h in both sympathetectomized rats and non-denervated rats. Adrenalectomy (ADX) reduced the level of corticosterone in serum by 81% compared to sham-operated rats, indicating that adrenalectomy was successful. ADX did not inhibit the decrease in the total P450 content and the metabolism of drugs induced by i.c.v.-LPS, but more profoundly emphasized the inhibitory effect of i.c.v.-LPS than the sham-operation. These results suggest that the sympathetic nervous systems both directly and indirectly innervating the liver do not participate in the primary mechanism of the decrease in the activities of CYP isozymes in rat liver microsomes induced by i.c.v.-LPS. Also, the adrenal glands, especially the adrenocortical system, play a suppressive role in the decrease in CYP isozymes caused by i.c.v.-LPS.
Keywords: Key words Adrenalectomy; Cytochrome P450; Intracerebroventricular; Lipopolysaccharide; Sympathetectomy
The phytoestrogens coumoestrol and genistein induce structural chromosomal aberrations in cultured human peripheral blood lymphocytes by S. E. Kulling; B. Rosenberg; E. Jacobs; M. Metzler (pp. 50-54).
The clastogenic potential of the phytoestrogens coumoestrol (COUM), genistein (GEN) and daidzein (DAI) has been studied in human peripheral blood lymphocytes in vitro. After exposure of the cultured lymphocytes to 50 or 75 μM COUM or 25 μM GEN for 6 h, a clear induction of structural chromosomal aberrations was observed by cytogenetic analysis. The major alterations were chromatid breaks, gaps and interchanges. In contrast, DAI did not induce chromosome aberrations even at 100 μM. These results, together with previously published reports on the induction of micronuclei and DNA strand breaks in cultured Chinese hamster V79 cells by COUM and GEN, but not DAI, suggest that some but not all phytoestrogens have the potential for genetic toxicity.
Keywords: Key words Phytoestrogens; Coumoestrol; Genistein; Daidzein; Human peripheral blood lymphocytes
Effects of 2-phenoxyethanol on N-methyl-d-aspartate (NMDA) receptor-mediated ion currents by Ulrich Mußhoff; Michael Madeja; Norbert Binding; Ute Witting; Erwin-Josef Speckmann (pp. 55-59).
The actions were examined of 17 frequently used glycol ether compounds on the glutamate receptor-mediated ion currents. The receptors were expressed in Xenopus oocytes by injection of rat brain mRNA. Most of the 17 glycol ethers exerted no effects on the glutamate subreceptors activated by kainate and N-methyl-d-aspartate (NMDA), whereas 2-phenoxyethanol (ethylene glycol monophenyl ether) caused a considerable reduction of NMDA-induced membrane currents in a reversible and concentration-dependent manner. The threshold concentration of the ethylene glycol monophenyl ether effect was <10 μmol/l. The concentration for a 50% inhibition (IC50) was ∼360 μmol/l. The results indicate a neurotoxic potential for 2-phenoxyethanol.
Keywords: Key words N-methyl-d-aspartate; Glutamate receptor; Glycol ether; 2-Phenoxyethanol; Ethylene glycol monophenyl ether
Pubertal dependent effects of cadmium on episodic prolactin secretion in male rats by A. Lafuente; E. Alvarez-Demanuel; N. Márquez; A. I. Esquifino (pp. 60-63).
This work was undertaken to assess if exposure to cadmium related to puberty may affect the episodic pattern of prolactin. Male rats were submitted to cadmium exposure, from day 30 to 60 or from day 60 to 90 of life respectively, at a dose of 50 ppm in the drinking water. Control age-matched rats received cadmium-free water. Prepubertal cadmium administration decreased mean serum prolactin levels and the absolute amplitude of the prolactin pulses. Subchronic exposure to cadmium of adult rats decreased mean serum prolactin levels, the absolute amplitude of the prolactin pulses and their duration, and the mean half-life of the hormone. These results suggest that subchronic cadmium exposure changes the secretory pattern of prolactin in adult male rats in a puberty-dependent way.
Keywords: Key words Cadmium; Prolactin pulsatility; Puberty