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Archives of Toxicology (v.81, #11)
Uterotrophic assay, Hershberger assay, and subacute oral toxicity study of 4,4´-[1-[4-[1-(4-hydroxyphenyl)-1-methylethyl]phenyl]ethylidene]bis[phenol] based on the OECD draft protocols by Kanji Yamasaki; Katumi Miyata; Takako Muroi; Hatsune Ehara; Nobuhiko Higashihara; Satsuki Houshuyama; Hiroshi Oshima; Yasushi Minobe (pp. 749-757).
We performed an uterotrophic assay, the Hershberger assay, and a 28-day repeated-dose toxicity study (enhanced OECD test guideline No. 407) of 4,4´-[1-[4-[1-(4-hydroxyphenyl)-1-methylethyl]phenyl]ethylidene]bis[phenol] based on the OECD draft protocols. In the uterotrophic assay, female SD rats were subcutaneously injected with the chemical at doses of 0, 100, 300, and 1,000 mg/kg on each of 3 days from postnatal day 20 to day 22, and the uterine weight of rats given the 1,000 mg/kg dose of the test chemical plus ethinyl estradiol decreased. In the Hershberger assay, the test chemical was orally administered at doses of 0, 100, 300, and 1,000 mg/kg day to castrated male SD rats for ten consecutive days beginning on postnatal day 56, and no changes were observed. On the other hand, when the test chemical was orally administered at doses 0, 100, 300, and 1,000 mg/kg day for at least 28 days, a decrease in LH values in rats of both sexes and a decrease in FSH and estradiol values in female rats were detected in the 1,000 mg/kg group, and abnormal estrous cycles, uterine glandular atrophy, persistence of ovarian corpora lutea, vaginal epithelial mucification, and mammary glandular hyperplasia were also observed in one female rat in the 1,000 mg/kg group. Therefore, the uterotrophic assay used in this study showed that the chemical has the estrogen–antagonist properties, and some potentially endocrine-mediated effects were detected in growing rats based on the results of the enhanced OECD test guideline No. 407. However, the changes were observed in rats given a high dose of the chemical, 1,000 mg/kg day.
Keywords: 4,4´-[1-[4-[1-(4-Hydroxyphenyl)-1-methylethyl]phenyl]ethylidene]bis[phenol]; Uterotrophic assay; Hershberger assay; Enhanced test guideline 407; Rat; Endocrine effects
Mercuric dichloride induces DNA damage in human salivary gland tissue cells and lymphocytes by Katharina Schmid; Andrea Sassen; Rainer Staudenmaier; Susanne Kroemer; Franz-Xaver Reichl; Ulrich Harréus; Rudolf Hagen; Norbert Kleinsasser (pp. 759-767).
Amalgam is still one of the most frequently used dental filling materials. However, the possible adverse effects especially that of the mercuric component have led to continued controversy. Considering that mercury may be released from amalgam fillings into the oral cavity and also reach the circulating blood after absorption and resorption, it eventually may contribute to tumorigenesis in a variety of target cells. The present investigation focuses on genotoxic effects below a cytotoxic dose level of mercuric dichloride (HgCl2) in human samples of salivary glands and lymphocytes to elucidate a possible role in tumor initiation. DNA migration due to single strand breaks, alkali labile sites and incomplete excision repair was quantified with the aid of the single cell microgel electrophoresis (Comet) assay. The concepts of Olive Tail Moment, percentage of DNA in the Tail and Tail Length were used as measures of DNA damage. To control for cytotoxic effects, the trypan blue exclusion test was applied. Human samples of the parotid salivary gland and lymphocytes of ten donors were exposed to HgCl2 concentrations from 1 to 50 μM. N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and dimethyl sulfoxide (DMSO) served as controls. Increasing dose-dependent DNA migration could be demonstrated after exposure to HgCl2 in cells of the salivary glands and lymphocytes. In both cell types a significant increase in DNA migration could be shown starting from HgCl2 concentrations of 5 μM in comparison to the negative control. The viability of the cell systems was not affected except at the highest concentration (50 μM) tested. These data indicate genotoxic effects of mercuric dichloride in human salivary glands and lymphocytes at concentrations not leading to cytotoxic effects or cell death. Consequently, a contributory role in oral salivary gland tumor initiation warrants further investigation.
Keywords: Mercuric dichloride; Genotoxicity; Salivary glands; Human study; Comet assay
Comparative study of activities in reactive oxygen species production/defense system in mitochondria of rat brain and liver, and their susceptibility to methylmercury toxicity by N. Mori; A. Yasutake; K. Hirayama (pp. 769-776).
The involvement of oxidative stress has been suggested as a mechanism for neurotoxicity caused by methylmercury (MeHg), but the mechanism for MeHg selective toxicity in the central nervous system is still unclear. In this research, to clarify the mechanism of selective neurotoxicity caused by MeHg, the oxygen consumption levels, the reactive oxygen species (ROS) production rates and several antioxidant levels in mitochondria were compared among the cerebrum, cerebellum and liver of male Wistar rats. In addition, the alterations of these indexes were examined in MeHg-intoxicated rats (oral administration of 10 mg/kg day, for 5 days). Although the cerebrum and cerebellum in intact rats showed higher mitochondrial oxygen consumption levels and ROS production rates than the liver, glutathione peroxidase (GPX) and superoxide dismutase (SOD) activities were much lower in the cerebrum and cerebellum than in the liver. Especially, the cerebellum showed the highest oxygen consumption and ROS production rate and the lowest mitochondrial glutathione (GSH) levels among the tissues examined. In the MeHg-treated rats, decrease in the oxygen consumption and increase in the ROS generation were found only in the cerebellum mitochondria, despite a lower Hg accumulation in the mitochondrial fraction compared to the liver. Since MeHg treatment produced an enhancement of ROS generation in cerebellum mitochondria supplemented with succinate substrates, MeHg-induced oxidative stress might affect the complex II–III mediated pathway in the electron transfer chain in the cerebellum mitochondria. Our study suggested that inborn factors, high production system activity and low defense system activity of ROS in the brain, would relate to the high susceptibility of the central nervous system to MeHg toxicity.
Keywords: Methylmercury; Mitochondria; Oxidative stress; Neurotoxicity
Isozyme- and gender-specific induction of glutathione S-transferases by flavonoids by Alyson E. Mitchell; Stephanie A. Burns; Jaime L. Rudolf (pp. 777-784).
Most dietary flavonoids have antioxidant activity in vitro however, secondary mechanisms such as the ability to influence gene expression with the consequent modulation of specific enzymatic activities involved in the intracellular response against oxidative stress, are being realized. In the following study, we examined the ability of the flavonoids: flavone, morin, naringenin, (+)-catechin, and quercetin to modulate the activity of glutathione S-transferases (GSTs) mGSTA, mGSTP and mGSTM in hepatic tissues of male and female Swiss Webster mice. Subchronic dietary exposure to morin, naringenin, (+)-catechin, and quercetin (2,500 mg/kg diet for 20 days) did not produce statistically significant changes in GST activity. Conversely, gender-, and isozyme-specific induction of mGSTs were observed in animals fed flavone. A sevenfold increase in total mGST activity was observed in female animals whereas a fourfold increase was observed in male animals. Enzyme specific assays indicate that there were greater increases of both mGSTM (eightfold) and mGSTP (fourfold) activities in females as compared to males (sixfold and twofold, respectively). As testosterone is involved in the regulation of GSTs in mice, castrated males were fed flavone for 5 days (2,500 mg/kg diet). In this case, dietary flavone resulted in similar fourfold increases in total GST activity in inact and castrated animals. Isozyme specific studies indicate that increases could be attributed to an induction of mGSTM and mGSTP.
Keywords: GSTs; Flavonoids; Flavone; Morin; Naringenin; (+)-Catechin; Quercetin; Gender
Hydrolysis of acetylthiocholine iodide and reactivation of phoxim-inhibited acetylcholinesterase by pralidoxime chloride, obidoxime chloride and trimedoxime by Yi Hui Zhang; Tadashi Miyata; Zhu Jian Wu; Gang Wu; Lian Hui Xie (pp. 785-792).
The hydrolysis of acetylthiocholine iodide (ATCh) by pralidoxime chloride (2-PAM Cl), trimedoxime (TMB4) and obidoxime chlpride (LÜH6) was studied at pH 5.8–8.0 and incubation temperature from 5 to 40°C in vitro. Significant ATCh hydrolysis by 2-PAM Cl, TMB4 and LÜH6 was found, with the exceptions of those at pH 7.0, 6.2 and 5.8 at 5°C and those at pH 6.2 and 5.8 at 15°C. The hydrolysis by TMB4 and LÜH6 was significantly stronger than that by 2-PAM Cl. The hydrolysis increased with increasing pH, incubation temperature and three oxime or ATCh concentration. Significant hydrolysis of ATCh by the three oximes could be found when the terminal concentration of oxime was higher than 0.01 mM at pH 7.0 and 7.4 at 30 and 37°C. However, no hydrolysis of natural substrate (acetylcholine iodide) by the three oximes was found when very high terminal concentrations of oximes were used. In addition, the three oximes displayed an extraordinary efficiency in the reactivation of phoxim-inhibited acetylcholinesterase (AChE) from fish (Carassius auratus) or rabbit (Oryctolagus cuniculus domestic) brain in vitro. Parallel to the level of ATCh hydrolysis by the oximes, TMB4 and LÜH6 displayed significantly higher reactivation efficiency than 2-PAM Cl to phoxim-inhibited AChE. And, the extent of reactivation by 2-PAM Cl was also lower than the other two. Plausible antidotal actions of the oximes against organophosphate poisoning AChE and erroneously high estimation of AChE activity by the Ellman method were discussed.
Keywords: Oximes; Acetylthiocholine iodide hydrolysis; Acetylcholinesterase reactivation; Oximes; Phoxim
Comparative CYP-dependent binding of the adrenocortical toxicants 3-methylsulfonyl–DDE and o,p′-DDD in Y-1 adrenal cells by Veronica Hermansson; Vendela Asp; Åke Bergman; Ulrika Bergström; Ingvar Brandt (pp. 793-801).
The environmental pollutant 3-MeSO2–DDE [2-(3-methylsulfonyl-4-chlorophenyl)-2-(4-chlorophenyl)-1,1-dichloroethene] is an adrenocortical toxicant in mice, specifically in the glucocorticoid-producing zona fasciculata, due to a cytochrome P450 11B1 (CYP11B1)-catalysed bioactivation and formation of covalently bound protein adducts. o,p′-DDD [2-(2-chlorophenyl)-2-(4-chlorophenyl)-1,1-dichloroethane] is toxic and inhibits steroidogenesis in the human adrenal cortex after bioactivation by unidentified CYPs, but does not exert any toxic effects on the mouse adrenal. As a step towards determining in vitro/in vivo relationships for the CYP-catalysed binding and toxicity of 3-MeSO2–DDE and o,p′-DDD, we have investigated the irreversible protein binding of these two toxicants in the murine adrenocortical cell line Y-1. The irreversible binding of 3-MeSO2–DDE previously demonstrated in vivo was successfully reproduced and could be inhibited by the CYP-inhibitors etomidate, ketoconazole and metyrapone. Surprisingly, o,p′-DDD reached similar levels of binding as 3-MeSO2–DDE. The binding of o,p′-DDD was sensitive to etomidate and ketoconazole, but not to metyrapone. Moreover, GSH depletion increased the binding of 3-MeSO2–DDE, but not of o,p′-DDD, indicating an important role of GSH conjugation in the detoxification of the 3-MeSO2–DDE-derived reactive metabolite. In addition, the specificity of CYP11B1 in activating 3-MeSO2–DDE was investigated using structurally analogous compounds. None of the analogues produced histopathological lesions in the mouse adrenal in vivo following a single i.p. injection of 100 mg/kg body weight, but two of the compounds were able to decrease the irreversible binding of 3-MeSO2–DDE to Y-1 cells. These results indicate that the bioactivation of 3-MeSO2–DDE by CYP11B1 is highly structure-dependent. In conclusion, both 3-MeSO2–DDE and o,p′-DDD bind irreversibly to Y-1 cells despite differences in binding and adrenotoxicity in mice in vivo. This reveals a notable in vitro/in vivo discrepancy, the contributing factors of which remain unexplained. We consider the Y-1 cell line as appropriate for studies of the cellular mechanisms behind the adrenocortical toxicity of these substances.
Keywords: Adrenal cortex; Y-1; Metabolic activation; Toxicity; Cytochrome P450
Analysis of cell death inducing compounds by Jeppe S. Spicker; Henrik Toft Pedersen; Henrik Bjørn Nielsen; Søren Brunak (pp. 803-811).
Biomarkers for early detection of toxicity hold the promise of improving the failure rates in drug development. In the present study, gene expression levels were measured using full-genome RAE230 version 2 Affymetrix GeneChips on rat liver tissue 48 h after administration of six different compounds, three toxins (ANIT, DMN and NMF) and three non-toxins (Caeruelein, Dinitrophenol and Rosiglitazone). We identified three gene transcripts with exceptional predictive performance towards liver toxicity and/or changes in histopathology. The three genes were: glucokinase regulatory protein (GCKR), ornithine aminotransferase (OAT) and Cytochrome P450, subfamily IIC (mephenytoin 4-hydroxylase) (Cyp2C29). RT-PCR for these three genes was performed and four additional compounds were included for validation. The quantitative RT-PCR analysis confirmed the findings based on the microarray data and using the three genes a classification rate of 55 of 57 samples was achieved for the classification of not toxic versus toxic. The single most promising biomarker (OAT) alone resulted in a surprisingly 100% correctly classified samples. OAT has not previously been linked to toxicity and cell death in the literature and the novel finding represents a putative hepatotoxicity biomarker.
Keywords: Toxicogenomics; Cell death; Ornithine aminotransferase (OAT); Glucokinase regulatory protein (GCKR); Cytochrome P450—subfamily IIC (mephenytoin 4-hydroxylase) (Cyp2C29)
Protective effect of vitamin E against carbendazim-induced testicular toxicity–histopathological evidences and reduced residue levels in testis and serum by Sivasankaran Rajeswary; Nisha Mathew; Mohammad Abdulkader Akbarsha; Muthuswamy Kalyanasundram; Bassouvalingam Kumaran (pp. 813-821).
The fungicide Carbendazim Methyl-2-benzimidazole carbamate (MBC) is known to produce male reproductive toxicity. The present study has been undertaken to investigate the impact of vitamin E, an antioxidant against the testicular toxicity induced by MBC. HPLC analysis showed that the amount of MBC in testis and serum was 57.40 ± 3.38 nmol/g and 14.10 ± 0.84 nmol/ml, respectively, in rats treated with carbendazim + vitamin-E, which were significantly lower than that of rats treated with carbendazim alone (240 ± 15.60 nmol/g and 318.70 ± 22.52 nmol/ml, respectively). MBC treatment significantly decreased the testicular weight while co-administration of vitamin-E registered normal testicular weight. Histomorphometric analysis revealed a significant decrease (P < 0.05) in the diameter of the seminiferous tubules and lumen in MBC-treated rats compared to control whereas they remained normal in vitamin E + MBC-treated rats. Leydig cells appeared dispersed and hypertrophic after MBC treatment. Various histopathological changes were observed in testis of rats treated with MBC whereas these changes were absent in vitamin-E + MBC-treated rat testis. In conclusion protection against MBC-induced toxicity was observed with co-administration of vitamin E with MBC.
Keywords: Carbendazim; Testis; Serum; Vitamin E; HPLC; Histopathology