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Archives of Toxicology (v.77, #3)
Nickel-induced oxidative stress and effect of antioxidants in human lymphocytes by Chang-Yu Chen; Yi-Fen Wang; Yu-Hwan Lin; Shu-Fang Yen (pp. 123-130).
The purpose of this study was to evaluate the oxidative effect in human lymphocytes after acute nickel (Ni) treatment for 1 h; levels of intracellular reactive oxygen species (ROS), lipid peroxidation (LPO) and hydroxyl radicals (•OH) were examined in isolated lymphocytes. The potential effects of antioxidants were also examined. After acute treatment, NiCl2 (0–10 mM) significantly decreased the viability of lymphocytes. NiCl2 appear to increase the degree of dichlorofluorescein (DCF) fluorescence and the levels of thiobarbituric acid-reactive substances (TBARS) in human lymphocytes in vitro in a concentration-dependent manner. The level of •OH was quantified by two main hydroxylated derivates, 2,3- and 2,5-dihydroxybenzate (DHB). Levels of 2,3- and 2,5-DHB were significantly higher in the Ni-treated group than in controls. Catalase partially reduced the NiCl2-induced elevation of oxidants and TBARS, whereas superoxide dismutase (SOD) enhanced the level of oxidants and TBARS. Both NiCl2-induced fluorescence and LPO were prevented significantly by glutathione (GSH) and mannitol. NiCl2-induced increase in generation of •OH was prevented significantly by catalase, GSH and mannitol, but not by SOD. These results suggest that NiCl2-induced lymphocyte toxicity may be mediated by oxygen radical intermediates, for which the accelerated generation of •OH may plays an important role in Ni-induced oxidative damage of human lymphocytes. Catalase, GSH and mannitol each provides protection against the oxidative stress induced by Ni.
Keywords: Nickel Human lymphocyte Lipid peroxidation Hydroxyl radical Antioxidant
Decreased GSSG reductase activity enhances cellular zinc toxicity in three human lung cell lines by U. I. Walther; A. Czermak; H. Mückter; S. C. Walther; B. Fichtl (pp. 131-137).
Cellular reduced glutathione (GSH) levels have been identified as an essential determinant in zinc-induced cytotoxicity. However, cytotoxic effects of zinc have also been observed without depletion of GSH stores. In a previous study, the intracellular activity of GSSG reductase (GR) has come into focus (Walther et al. 2000, Biol Trace Elem Res 78:163–177). In the present paper we have tried to address this issue more deeply by inhibiting the activity of cellular GR without any appreciable decreases of cellular glutathione. In three pulmonary cell lines, GR activity was inhibited in a dose-dependent manner by the alkylating agent carmustine (BCNU), a known inhibitor of GR. Cells were pretreated with BCNU for 14 h, followed by exposure to various concentrations of zinc chloride. Then we determined the incorporation of radiolabelled methionine (to assess protein synthesis), and measured the GSH and oxidized glutathione (GSSG) levels. Additionally, GR activity of controls was measured. IC50 values for zinc-induced inhibition of methionine incorporation, as well as GSH contents, was strongly correlated to the decreased GR activity. These results firmly suggest that GR is an important factor in the event chain of zinc cytotoxicity. Together with the results from our previously cited study where impaired regeneration of GSH levels were accompanied by a decrease in total cellular glutathione (GSH + GSSG) we conclude that GSSG itself is an important effector in zinc cytotoxicity.
Keywords: Glutathione A549cells L2 cells 11Lu cells Zinc anions BCNU
Metabolism of tributyltin and triphenyltin by rat, hamster and human hepatic microsomes by Shuji Ohhira; Masatomo Watanabe; Hisao Matsui (pp. 138-144).
Tributyltin and triphenyltin are metabolized by cytochrome P-450 system enzymes, and their metabolic fate may contribute to the toxicity of the chemicals. In the current study, the in vitro metabolism of tributyltin and triphenyltin by rat, hamster and human hepatic microsomes was investigated to elucidate the metabolic competence for these compounds in humans. The metabolic reaction using microsome-NADPH system that is usually conducted was not applicable to in vitro metabolism of organotins, especially triphenyltin. We therefore examined the effects of dithiothreitol (DTT), one of the antioxidants for sulfhydryl groups, to determine the in vitro metabolism of tributyltin and triphenyltin. As a result, the treatment with 0.1 mM DTT in vitro increased the activity of the microsomal monooxygenase system for metabolism of tributyltin as well as triphenyltin; the total yield of tributyltin and triphenyltin metabolites as tin increased, respectively, by approximately 1.8 and 8.9 times for rat, 2.1 and 1.2 times for hamster, and 1.6 and 1.5 times for human. It is suggested that the organotins directly inactivate cytochrome P-450 because of the interaction with critical sulfhydryl groups of the hemoprotein. We confirmed the utility of this in vitro metabolic system using DTT in the hepatic microsomes of phenobarbital (PB)-pretreated and untreated hamsters. Thus, the in vitro metabolic system described here was applied to a comparative study of the metabolism of organotins in rats, hamsters and humans. Tributyltin was metabolized more readily than triphenyltin in all the species. In humans, the in vitro metabolic pattern resembled that of hamsters, which were susceptible to in vivo triphenyltin toxicity because of incompetent metabolism. It is possible that the hamster is a qualitatively and quantitatively suitable animal model for exploring the influence of tributyltin and triphenyltin in humans.
Keywords: Tributyltin Triphenyltin In vitro metabolism Hepatic microsome Human
Expression of cytochromes P450 3A in mouse lung: effects of dexamethasone and pregnenolone 16α-carbonitrile by Muriel Haag; Alain Fautrel; André Guillouzo; Nelly Frossard; Françoise Pons (pp. 145-149).
Expression of cytochromes P450 3A (CYP3A) has been reported in the lung, but its regulation has received little attention. In the present study, we assessed lung levels of Cyp3a mRNA, protein and activity in control mice and in mice treated with either dexamethasone (DEX), pregnenolone 16α-carbonitrile (PCN) or a mixture of DEX+PCN. Lung expression of the pregnane X receptor (PXR) was also investigated. Constitutive levels of Cyp3a mRNA were found in the lung from control mice by polymerase chain reaction after reverse transcription of total RNA (RT-PCR). These levels were significantly increased (2.0-fold, P<0.05) in mice treated with DEX and further enhanced (2.7-fold increase, P<0.01) in mice treated with DEX+PCN. In control mice, basal levels of Cyp3a protein and activity were also found, as assessed by western blot and measure of testosterone 6β-hydroxylation, respectively. In mice treated with DEX or DEX+PCN, changes in Cyp3a protein and activity exhibited the same pattern as those in Cyp3a mRNA. In contrast, PCN alone failed to trigger consistent increases in lung Cyp3a mRNA, protein and activity. PXR mRNA was not detected in the lung from control or PCN-treated mice by RT-PCR, but was found at significant levels in the lungs from mice treated with DEX or DEX+PCN. Our results show that expression of Cyp3a is upregulated by glucocorticoids in mouse lung, and that this effect is potentiated by antiglucocorticoids. This potentiation may involve PXR, expression of which is induced in the lung of glucocorticoid-treated mice.
Keywords: Cytochromes P450 3A Dexamethasone Pregnenolone 16α-carbonitrile Pregnane X receptor Lung
Differential expression of c-fos and c-myc protooncogenes by estrogens, xenobiotics and other growth-stimulatory agents in primary rat hepatocytes by Chow H. Lee; Anthony M. Edwards (pp. 150-159).
Mechanism(s) of tumour promotion in liver by estrogens and other xenobiotics such as α-hexachlorocyclohexane (HCH), 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (DDT) and phenobarbital (PB), are not well understood although it is clear that growth stimulation is one important element in their action. To help in characterizing mechanisms of growth control by these compounds, their effects on the expression of immediate-early protooncogenes c-fos and c-myc have been examined and compared with other compounds that stimulate DNA synthesis in primary cultures of normal rat hepatocytes. Expression of c-fos was undetectable in cultures not exposed to growth factors. Although neither epidermal growth factor (EGF) nor 17β-estradiol (E2) alone had marked effects on c-fos mRNA, the two acted synergistically to cause clear c-fos expression, maximal 1–2 h after growth factor addition and when test agents were added on the first day in culture. Neither insulin nor dexamethasone alone induced c-fos mRNA but stimulation of c-fos expression by EGF plus estradiol occurred earlier in the presence of insulin, and was augmented by preincubation of cells with dexamethasone. EGF + E2-induced c-fos mRNA was completely abolished by actinomycin D, suggesting that transcription is the major mechanism for c-fos induction by E2 + EGF. Compounds that strongly stimulate hepatocyte DNA synthesis such as norepinephrine, pyruvate, prolactin, glutethimide, monensin, ammonium chloride, and normal rat serum when in combination with EGF, all failed (when added with EGF) to affect c-fos mRNA expression. Thus, induction of c-fos expression may be a component of estradiol's growth stimulatory effect in cultured hepatocytes but this is not the case for other compounds that strongly stimulate DNA synthesis. Unlike c-fos mRNA, c-myc mRNA was detectable in hepatocyte cultures without added growth factor, was augmented within 2 h of exposure to EGF, and was further increased by adding E2, other estrogens or a variety of other stimulators of DNA synthesis in hepatocytes. This suggests that increased c-myc expression may be a common effect of many of these agents in combination with EGF.
Keywords: Primary hepatocytes c-myc mRNA c-fos mRNA Gene expression Tumour promoters
The anti-breast cancer drug tamoxifen alters Ca2+ movement in Chinese hamster ovary (CHO-K1) cells by Chung-Ren Jan; Chiang An-Jen; Hong-Tai Chang; Cherng-Jau Roan; Yih-Chau Lu; Bang-Ping Jiann; Chin-Man Ho; Jong-Khing Huang (pp. 160-166).
The anti-breast cancer drug tamoxifen has recently been shown to cause an increase in intracellular free-Ca2+ concentrations ([Ca2+]i) in renal tubular cells, breast cells and bladder cells. Because tamoxifen is known to alter ovary function in human patients and in rats, the present study was aimed at exploring whether tamoxifen could alter Ca2+ movement in Chinese hamster ovary (CHO-K1) cells. Cytosolic free-Ca2+ levels in populations of cells have been explored by using fura-2 as a fluorescent Ca2+ indicator. Tamoxifen at concentrations above 1 µM increased [Ca2+]i in a concentration-dependent manner with an EC50 value of 8 µM. The Ca2+ signal was reduced by removing extracellular Ca2+, but was not affected by nifedipine, verapamil, diltiazem or ICI 182,780 (an estrogen receptor antagonist). Pretreatment with 1 µM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor) to deplete the endoplasmic reticulum Ca2+ abolished 10 µM tamoxifen-induced Ca2+ release. Neither inhibition of phospholipase C with 2 µM U73122 nor depletion of ryanodine-sensitive Ca2+ stores with 50 µM ryanodine affected tamoxifen-induced Ca2+ release. Cell proliferation assays using ELISA revealed that overnight incubation with 5–10 µM tamoxifen inhibited cell proliferation by 20%, and 20 µM tamoxifen killed all cells. Together, the results suggest that, in CHO-K1 cells, tamoxifen induced a [Ca2+]i increase by causing store-Ca2+ release from the endoplasmic reticulum in an phospholipase C-independent manner, and by inducing Ca2+ influx. The action of tamoxifen appears to be dissociated from estrogen receptor activation. Longer incubation with tamoxifen (>5 µM) was cytotoxic.
Keywords: Tamoxifen Calcium Fura-2 Thapsigargin Ovary cells CHO-K1 cells
Nasal midazolam as a novel anticonvulsive treatment against organophosphate-induced seizure activity in the guinea pig by E. Gilat; M. Goldman; E. Lahat; A. Levy; I. Rabinovitz; G. Cohen; R. Brandeis; G. Amitai; D. Alkalai; G. Eshel (pp. 167-172).
Seizures and status epilepticus, which may contribute to brain injury, are common consequences of exposure to organophosphorus (OP) cholinesterase inhibitors. Effective management of these seizures is critical. To investigate the efficacy of nasal midazolam as an anticonvulsive treatment for OP exposure, as compared to intramuscular midazolam, guinea pigs were connected to a recording swivel for electrocorticograph (ECoG) monitoring and clinical observation. The experimental paradigm consisted of pyridostigmine pretreatment (0.1 mg/kg i.m.) 20 min prior to sarin exposure (1.2× LD50, 56 µg/kg i.m.). One minute post-exposure, atropine (3 mg/kg i.m.) and TMB-4 (1 mg/kg im) were administered. Within 3–8 min after sarin exposure all animals developed electrographic seizure activity (EGSA), with convulsive behavior. Treatment with midazolam (1 mg/kg i.m.) 10 min after the onset of EGSA abolished EGSA within 389±181 s. The same dose was not effective, in most cases, when given 30 min after onset. However, a higher dose (2 mg/kg) was found efficacious after 30 min (949±466 s). In contrast, nasal application of midazolam (1 mg/kg) was found most effective, with significant advantages, in amelioration of EGSA and convulsive behavior, when given 10 min (216±185 s) or 30 min (308±122 s) following the onset of EGSA (P<0.001). Thus, nasal midazolam could be used as a novel, rapid and convenient route of application against seizure activity induced by nerve agent poisoning.
Keywords: Organophosphate Seizures Midazolam Intranasal EEG
Comparison of hepatotoxicity and metabolism of butyltin compounds in the liver of mice, rats and guinea pigs by Shunji Ueno; Takashige Kashimoto; Nobuyuki Susa; Masamitsu Ishii; Toshikazu Chiba; Ken-ichiro Mutoh; Fumio Hoshi; Takashi Suzuki; Masayasu Sugiyama (pp. 173-181).
The hepatotoxicity of tributyltin chloride (TBTC) and dibutyltin dichloride (DBTC) was compared among mice, rats and guinea pigs in vivo. Further, the metabolism of these butyltin compounds in the liver was also investigated in these species. The oral administration of TBTC and DBTC to mice induced obvious liver injury, as demonstrated by both serodiagnosis and histopathological diagnosis. The concentrations of TBTC and DBTC that induced hepatotoxicity in mice at 24 h after oral administration were 180 and 60 µmol/kg, respectively. In the case of rats, the liver injury induced by TBTC and DBTC was detected at 24 h by the serodiagnosis, but not by histopathological diagnosis. On the other hand, in guinea pigs, TBTC and DBTC administration did not produce any clear liver injury at 24 h, as evaluated by these two diagnostic methods. Thus, the following ranking was obtained with regard to increasing order of sensitivity to liver injury caused by TBTC and DBTC: mice, rats and guinea pigs. The total butyltin contents in the liver of mice were equivalent at 3 h and 24 h after the administration of TBTC or DBTC; however, the contents in the liver of rats and guinea pigs were relatively lower at 3 h and higher at 24 h than those of mice, although there were no differences between rats and guinea pigs in the total liver butyltin content. Concerning the liver metabolism of these butyltin compounds, the main form of butyltin compounds in these animals treated with TBTC was DBTC within 3 h after oral administration, while the main metabolites at 24 h were different in each species, indicating that the liver metabolism of TBTC might vary by animal type. When the animals were treated with DBTC orally, DBTC was hardly metabolized in the livers of these animals even at 24 h, and the liver levels of DBTC were two times greater in mice and guinea pigs than in rats at 3 h and were lower in mice at 24 h than in rats and guinea pigs. The analysis of cellular distributions of DBTC in the liver at 3 h after the administration showed that the levels of DBTC in the nuclear, microsomal and mitochondrial fractions of mice hepatocytes were relatively higher than in those of rats, which were greater than in those of guinea pigs. These results suggest differences in the sensitivity of mice, rats and guinea pigs to hepatotoxicity caused by butyltin compounds and demonstrate that the difference in the sensitivity of these animals to the hepatotoxicity induced by TBTC and DBTC may be partly due to differences in hepatic metabolism of TBTC and in the distribution of DBTC within cell organelles, respectively.
Keywords: Tributyltin Dibutyltin Hepatotoxicity Metabolism In vivo