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Archives of Toxicology (v.76, #10)

A repeated 28-day oral dose toxicity study of genistein in rats, based on the 'Enhanced OECD Test Guideline 407' for screening endocrine-disrupting chemicals by Kazushi Okazaki; Shuzo Okazaki; Hideaki Nakamura; Yasuki Kitamura; Kazuhisa Hatayama; Sachiko Wakabayashi; Toshiharu Tsuda; Tomoyoshi Katsumata; Akiyoshi Nishikawa; Masao Hirose (pp. 553-559).

In association with the international validation project to establish an OECD Enhanced Test Guideline 407, we performed a 28-day repeated-dose toxicity study of genistein, which is known as a phytoestrogen. Attention was paid to the sensitivity of certain additional parameters, such as histopathology observations and organ weights of endocrine related organs, sperm characteristics, serum hormone levels and estrous cycle, for detecting endocrine-related effects of endocrine-disrupting chemicals based on the existing TG 407. Seven-week-old Crj:CD(SD)IGS rats were assigned to one of four groups, each consisting of ten males and ten females, and genistein was administered once daily by gavage at doses of 0 (control), 120, 400 or 1000 mg/kg body weight per day. Male rats were killed on the day after the 28th administration. Female rats were killed on the day of the diestrus stage during the 4 days after the 28th administration. Endocrine-disrupting effects of genistein were detected in females by histopathology. The changes included vacuolation and mucinification of the vaginal epithelium in the 400 and 1000 mg/kg groups; however, the incidences of the lesion were very low. Although increased serum prolactin levels were recorded in the males of the 1000 mg/kg group, we could not determine whether this was indeed induced by genistein. General toxicological effects of genistein were detected in blood chemistry, such as increased triglycerides and total protein and a decreased albumin/globulin ratio, as well as increased liver weight and glycogen deposition in the periportal hepatocytes. Based on these results, the no-observed-adverse-effect level (NOAEL) in the present study was estimated to be 120 mg/kg per day. In particular, endocrine-related effects were most sensitively detected by histopathology examination of sexual organs. However, the findings indicate that chemicals with weak endocrine-disrupting potential like genistein must be evaluated taking into consideration the results of other test systems.

Keywords: Genistein Phytoestrogen Rat Enhanced OECD Test Guideline 407 Endocrine disrupter

Simultaneous determination of styrene, toluene, and xylene metabolites in urine by gas chromatography/mass spectrometry by Sándor Szűcs; László Tóth; József Legoza; Attila Sárváry; Róza Ádány (pp. 560-569).

Exposure to styrene, toluene, and xylene (STX) frequently occurs in various industrial settings leading to several adverse health effects. Therefore, the biological monitoring by determination of urinary mandelic acid (MA) and phenylglyoxylic acid (PGA), hippuric acid (HA), and 2-, 3-, and 4-methylhippuric acids (2-, 3-, and 4-MHAs), the metabolites of STX, is required or at least recommended in case of workers exposed by these agents. Considering the fact that co-exposure to STX frequently occurs, methods that have been described for the separate analysis of these compounds in urine samples cannot be used effectively for monitoring. Therefore, a reliable gas chromatographic/mass spectrometric (GC/MS) method was developed for the simultaneous identification and quantification of these metabolites. Following solid phase extraction of the urine samples, the extracts were silylated and analyzed by GC/MS using a HP-5MS capillary column. The method was evaluated for linearity, limits of detection and quantification, and specificity, as well as for precision, extraction efficiency, and stability at three different concentrations prepared in urine. The assay was linear up to 0.16 mg/ml for MA, and 0.32 mg/ml for PGA, HA, and 2-, 3- and 4-MHAs. The limits of detection and quantification of STX metabolites varied between 0.001 and 0.02 mg/ml, and from 0.01 to 0.04 mg/ml, respectively. The within-day and between-day precision, determined at low, medium, and high concentrations, ranged from 2 to 12% and 2 to 19%, respectively. The extraction efficiency was 70–80%. No degradation of the metabolites occurred in the urine samples under the possible working conditions. The method was applied for the analysis of the urine samples from exposed workers. The cost- and time-effectiveness, the technical advantages and validity parameters of this GC/MS analysis make it suitable for biological monitoring of mixed exposure to styrene, toluene and xylene.

Keywords: Styrene Toluene Xylene Metabolites Gas chromatography/mass spectrometry

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) induces Fas-dependent activation-induced cell death in superantigen-primed T cells by Iris A. Camacho; Mitzi Nagarkatti; Prakash S. Nagarkatti (pp. 570-580).

Immune response against a foreign antigen is characterized by a growth phase, in which antigen-specific T cells clonally expand, followed by a decline phase in which the activated T cells undergo apoptosis, a process termed activation-induced cell death (AICD). In the current study, we have investigated the phase at which 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) acts to downregulate the antigen-specific T cell response. To this end, C57BL/6 +/+ mice were injected with staphylococcal enterotoxin A (SEA) into the footpads (10 µg/footpad), and simultaneously treated with TCDD (10 or 50 µg/kg intraperitoneally). At various time points, the draining lymph node (LN) cells were analyzed for SEA-activated T cells. The data demonstrated that in C57BL/6 +/+ mice, TCDD treatment did not alter the growth phase but facilitated the decline phase of SEA-reactive T cells. TCDD caused a significant decrease in the percentage and absolute numbers of CD4⁺ and CD8⁺ SEA-responsive T cells expressing Vβ3⁺ and Vβ11⁺ but did not affect SEA-nonresponsive Vβ8⁺ T cells. Upon in vitro culture, TCDD-exposed SEA-immunized LN cells exhibited increased levels of apoptosis when compared with the vehicle controls. When Fas-deficient (C57BL/6 lpr/lpr) or Fas ligand defective (C57BL/6 gld/gld) mice were treated with TCDD, they failed to exhibit a decrease in percentage and cellularity of SEA-reactive T cells, thereby suggesting a role of Fas-Fas ligand interactions in the TCDD-induced downregulation of SEA-reactive T cell response. The resistance to TCDD-induced decrease in T cell responsiveness to SEA seen in Fas- and FasL-mutant mice was neither due to decreased aryl hydrocabon receptor (AhR) expression nor to altered T cell responsiveness to SEA. The current study demonstrates that TCDD does not prevent T cell activation, but prematurely induces Fas-based AICD, which may contribute to the deletion of antigen-primed T cells.

Keywords: 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) T cell activation Superantigen Apoptosis Fas

Role of metabolites in MDMA (ecstasy)-induced nephrotoxicity: an in vitro study using rat and human renal proximal tubular cells by Márcia Carvalho; Gabrielle Hawksworth; Nuno Milhazes; Fernanda Borges; Terrence J. Monks; Eduarda Fernandes; Félix Carvalho; Maria Bastos (pp. 581-588).

The metabolism of 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) has recently been implicated in the mechanisms underlying ecstasy-induced neurotoxicity and hepatotoxicity. However, its potential role in ecstasy-induced kidney toxicity has yet to be investigated. Thus, primary cultures of rat and human renal proximal tubular cells (PTCs) were used to investigate the cytotoxicity induced by MDMA and its metabolites methylenedioxyamphetamine (MDA), α-methyldopamine (α-MeDA), and the glutathione (GSH) conjugates 5-(glutathion-S-yl)-α-MeDA and 2,5-bis(glutathion-S-yl)-α-MeDA. Cell viability was evaluated using the mitochondrial MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. MDMA and MDA were not found to be toxic to either rat or human PTCs at any concentration tested (100–800 µM). In contrast, 800 µM α-MeDA caused 60% and 40% cell death in rat and human PTCs, respectively. Conjugation of α-MeDA with GSH resulted in the formation of even more potent nephrotoxicants. Thus, exposure of rat and human PTC monolayers to 400 µM 5-(glutathion-S-yl)-α-MeDA caused approximately 80% and 70% cell death, respectively. 5-(Glutathion-S-yl)-α-MeDA (400 µM) was more toxic than 2,5-bis(glutathion-S-yl)-α-MeDA to rat renal PTCs but equally potent in human renal PTCs. Pre-incubation of rat PTCs with either acivicin, an inhibitor of γ-glutamyl transpeptidase (γ-GT), or bestatin, an inhibitor of aminopeptidase M, resulted in increased toxicity of 5-(glutathion-S-yl)-α-MeDA but had no effect on 2,5-bis(glutathion-S-yl)-α-MeDA-mediated cytotoxicity. The present data provide evidence that metabolism is required for the expression of MDMA-induced renal toxicity in vitro. In addition, metabolism of 5-(glutathion-S-yl)-α-MeDA by γ-GT and aminopeptidase M to the corresponding cystein-S-yl-glycine and/or cystein-S-yl conjugates is likely to be associated with detoxication of this compound. Thus, it appears that toxicity induced by thioether metabolites of ecstasy at the apical membrane of renal proximal tubular cells is the result of extracellular events, presumably redox cycling.

Keywords: MDMA Metabolites, Glutathione conjugate Rat Human Renal proximal tubular cells

Equipotent cholinesterase reactivation in vitro by the nerve agent antidotes HI 6 dichloride and HI 6 dimethanesulfonate by S. Krummer; H. Thiermann; F. Worek; P. Eyer (pp. 589-595).

The well-documented efficacy of HI 6 dichloride in reactivating acetylcholinesterase (AChE) inhibited by nerve agents is curtailed by its poor water-solubility at temperatures below 10°C. This drawback can be circumvented by using HI 6 dimethanesulfonate, which has been developed in our laboratory. Since investigations on the efficacy of this new entity are lacking, it has been proposed that some bridging experiments be performed, aimed at demonstrating reactivator equivalence in vitro. The reactivating properties of the two salts were compared on human erythrocyte AChE inhibited with paraoxon, sarin, cyclosarin and agent VX. The comparison was extended to cynomolgus erythrocytes exposed in vitro for sarin and VX. Finally, mouse diaphragm preparations were circumfused with sarin, cyclosarin and VX and reactivation by each of the HI 6 salts was examined in muscle homogenates. AChE activity in erythrocyte suspensions was monitored by a modified Ellman procedure, muscle AChE by a radiometric assay. In all models tested no differences between the HI 6 salts could be detected (P=0.05). From these data, equipotency in AChE reactivation by the two HI 6 salts can be anticipated.

Keywords: Nerve agents Acetylcholinesterase Erythrocytes Diaphragm HI 6 Oximes Reactivation

Modification of the striatal dopaminergic neuron system by carbon monoxide exposure in free-moving rats, as determined by in vivo brain microdialysis by Shuichi Hara; Toshiji Mukai; Kunihiko Kurosaki; Fumi Kuriiwa; Takahiko Endo (pp. 596-605).

Acute carbon monoxide (CO) intoxication in humans results in motor deficits, which resemble those in Parkinson's disease, suggesting possible disturbance of the central dopaminergic (DAergic) neuronal system by CO exposure. In the present study, therefore, we explored the effects of CO exposure on the DAergic neuronal system in the striatum of freely moving rats by means of in vivo brain microdialysis. Exposure of rats to CO (up to 0.3%) for 40 min caused an increase in extracellular dopamine (DA) levels and a decrease in extracellular levels of its major metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), in the striatum depending on the CO concentration. Reoxygenation following termination of the CO exposure resulted in a decline of DA to the control level and an overshoot in the recovery of DOPAC and HVA to levels higher than the control. A monoamine oxidase type A (MAO-A) inhibitor, clorgyline, significantly potentiated the CO-induced increase in DA and completely abolished the subsequent overshoot in the recovery of DOPAC and HVA. Tetrodotoxin, a Na⁺ channel blocker, completely abolished both the CO-induced increase in DA and the overshoot of DOPAC and HVA. A DA uptake inhibitor, nomifensine, strongly potentiated the CO-induced increase in DA without affecting the subsequent overshoot of DOPAC and HVA. Clorgyline further potentiated the effect of nomifensine on the CO-induced increase in DA, although a slight overshoot of DOPAC and HVA appeared. These findings suggest that (1) CO exposure may stimulate Na⁺-dependent DA release in addition to suppressing DA metabolism, resulting in a marked increase in extracellular DA in rat striatum, and (2) CO withdrawal and subsequent reoxygenation may enhance the oxidative metabolism, preferentially mediated by MAO-A, of the increased extracellular DA. In the light of the neurotoxicity of DA per se and reactive substances, such as quinones and activated oxygen species, generated via DA oxidation, the significant modification of the striatal DAergic neuronal system by CO exposure might participate in the neurological outcome following acute CO intoxication.

Keywords: Carbon monoxide Dopamine 3,4-Dihydroxyphenylacetic acid Homovanillic acid Rat striatum

In ovo carcinogenicity assay (IOCA): evaluation of mannitol, caprolactam and nitrosoproline by Klaus D. Brunnemann; Harald G. Enzmann; Carmen E. Perrone; Michael J. Iatropoulos; Gary M. Williams (pp. 606-612).

The in ovo carcinogenicity assay (IOCA) was used to examine whether the noncarcinogens ε-caprolactam (CAP), D-mannitol (MAN) and nitrosoproline (NPRO) induce toxicity and subsequently morphological changes in embryonic turkey livers compared with the carcinogen diethylnitrosamine (DEN). Various doses of the test compounds were injected into fertilized turkey or quail eggs prior to incubation. Embryonic livers were collected 3–4 days before hatching and processed for histology. The positive control DEN induced hepatocyte altered foci (HAF) and karyomegalic hepatocytes, whereas histological analysis of livers from embryos exposed to CAP, MAN and NPRO did not show such histological changes. The effects of the tested compounds on liver were further examined in hepatocytes cultured from exposed turkey and quail embryos. As observed in ovo, megalocytes as well as karyomegalic hepatocytes were present in hepatocyte cultures established from DEN-exposed turkey embryos, but not from embryos exposed to CAP, MAN or NPRO. It is concluded that CAP, MAN and NPRO do not induce histological changes in embryonic liver of the type produced by the carcinogen DEN, correlating with findings for these compounds in rodent studies.

Keywords: In ovo carcinogenicity assay Carcinogenicity bioassay Caprolactam Mannitol

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Archives of Toxicology (v.76, #10)