Intracellular and extracellular factors influencing Cr(VI) and Cr(III) genotoxicity

Cr(VI) is a human and animal carcinogen. Cr(VI) does not interact directly with DNA and thus its genotoxicity is attributed to its intracellular reduction to Cr(III) via reactive intermediates. The resulting types of DNA damage can be grouped into two categories: (1) oxidative DNA damage and (2) Cr(III)-DNA interactions. This study examines the molecular mechanism of Cr(VI) and Cr(III) genotoxicity in an intact cell. A system screening for DNA deletions (DEL assay) was used to compare induction of chromosomal rearrangements in the yeast Saccharomyces cerevisiae following Cr(VI) and Cr(III) exposure. Both forms of chromium induced DNA deletions albeit with different dose-response curves. N-acetylcysteine had a protective effect against Cr(VI) genotoxicity at high exposure doses but had no protective effect at lower doses or against Cr(III). An oxidative DNA damage repair mutant was hypersensitive to Cr(VI) only at high exposure and the mutant was not hypersensitive to Cr(III) exposure. These data imply that oxidative stress is involved in Cr(VI) genotoxicity at high exposure concentrations and not so in Cr(III). The Cr(III)-DNA interaction appears to be an important genotoxic lesion following Cr(VI) exposure at low-exposure concentrations. The CAN forward mutation assay revealed that within the concentration ranges used for this study, Cr(III) does not cause point mutations and Cr(VI) causes a mild but statistically significant increase in point mutation only at the highest concentration tested. This study reveals that DNA deletions occurring as a result of intrachromosomal homologous recombination are a useful endpoint for studying chromium genotoxicity.     

Peroxynitrite-mediated DNA strand breakage activates poly (ADP-ribose) synthetase and causes cellular energy depletion in carrageenan-induced pleurisy.

The aim of the present study was to investigate the role of poly (ADP-ribose) synthetase in acute local inflammation (carrageenan-induced pleurisy), where oxyradicals, nitric oxide and peroxynitrite are known to play a crucial role in the inflammatory process. DNA single-strand breakage and activation of the nuclear enzyme poly (ADP-ribose) synthetase (PARS) triggers an energy-consuming, inefficient repair cycle, which contributes to peroxynitrite-induced cellular injury. Here we investigated whether peroxynitrite production and PARS activation are involved in cytotoxicity in macrophages collected from rats subjected to carrageenan-induced pleurisy. Macrophages harvested from the pleural cavity exhibited a significant production of peroxynitrite, as measured by the oxidation of the fluorescent dye dihydrorhodamine 123, and by nitrotyrosine Western blotting at 4 hr after carrageenan injection. Furthermore, carrageenan-induced pleurisy caused a suppression of macrophage mitochondrial respiration, DNA strand breakage, activation of PARS and reduction of NAD+ cellular levels. In vivo treatment with 3-aminobenzamide (10 mg/kg intraperitoneally, 1 hr after carrageenin injection) significantly inhibited the decrease in mitochondrial respiration and the activation of PARS and partially restored the cellular level of NAD+. In a separate group of experiments, in vivo pretreatment with NG-nitro-L-arginine methyl ester, a non-selective inhibitor of nitric oxide (NO) synthesis (10 mg/kg intraperitoneally, 15 min before carrageenan administration), reduced peroxynitrite formation and prevented the appearance of DNA damage, the decrease in mitochondrial respiration and the loss of cellular levels of NAD+. Our study suggests that formation of peroxynitrite and subsequent activation of PARS may alter macrophage function in inflammatory processes and inhibition of NO and PARS may be a novel pharmacological approach to prevent cell injury in inflammation.     

Induction of spindle inhibition and abnormal mitotic figures by Cr(II), Cr(III) and Cr(VI) ions

A differential staining technique for chromosomes and spindles was used to compare the effects of chromium compounds of different oxidation states with either no, or very low (Cr(II) and Cr(III) or high (Cr(VI) carcinogenic properties. Neither CrCl2, nor CrCl3 (except at 10(-4) M) had any effect on spindle formation and/or chromosome segregation after exposure up to 10(-7) and 10(-5) M, respectively. None of them significantly influenced the mitotic index. In contrast, the Cr(VI) products (Na2CrO4 and CaCrO4) induced a dose-dependent increase of aberrant division patterns up to 10(-4) and 10(-5) M, respectively. Moreover, with CaCrO4, a high frequency of cells with dislocated metaphase chromosomes or malformed spindles with clustered chromosomes were observed. Since these cell types are potential sources of aneuploid daughter cells, they represent a potential risk for cancer induction. These experiments clearly demonstrate that the differential staining methods allows discrimination between spindle modifiers which induce lethal toxicity and those which may lead to possible tumour induction.     

Effect of diet and vitamin C on DNA strand breakage in freshly-isolated human white blood cells

We have measured DNa strand breaks induced by ionising radiation in nucleated cells from freshly isolated whole blood from normal human subjects. Samples werer taken after subjects had fasted overnight and again 1 h after they had eaten breakfast in combination with approximately 35 mg/kg vitamin C. Damage was measured by single cell gel electrophoresis (the ‘comet’ assay), in which DNA single strand breaks generate a comet tail streaming from the nucleus. In repeat experiments on 6 subjects a reduction in DNA damage, as indicated by a highly significant decrease in overall comet length, was observed following vitamin C ingestion, both in the unirradiated control blood samples and in the dose response to ionising radiation damage. In addition, consistent differences in dose response between individual subjects were found. The peak effect was 4 h after intake of food and vitamin C. An effect was also seen with vitamin C alone and after breakfast without additional vitamin C. Protection against strand breakage was also seen in Ficoll-separated mononucleasr cells but evidence was not obtained from protection of separated, mitogen stimulated T-lymphocytes either against ionising radiation cell killing in a clonal assay, or against clastogenicity assessed by micronucleus formation following one cell division. Exposure of separated lymphocytes in vitro to vitamin C, at doses greater than 200 μM, did not offer protection but induced strand breakage. Our results raise the possibility in normal diet may not only affect susceptibility to endogenous oxidative damage, but may affect some responses of the individual to radiation.     

Effects of selenium on chromium (VI)-induced hepatotoxicity in adult rats

Chromium, a major environmental pollutant, is known for its wide toxic manifestations. The present experiment pertains to the protective role of selenium (Se) against K₂Cr₂O₇-induced hepatotoxicity. Female Wistar rats were divided into four groups of six each: group I served as controls which received standard diet; group II received in drinking water K₂Cr₂O₇ alone (700 ppm); group III received both K₂Cr₂O₇ and Se (0.5 Na₂SeO₃ mg/kg of diet); group IV received Se (0.5 mg/kg of diet) for 3 weeks. Exposure of rats to chromium promoted oxidative stress with an increase in malondialdehyde (MDA) and a decrease in glutathione (GSH) levels. A decrease in glutathione peroxidase (GPx) and an increase in superoxide dismutase (SOD) and catalase (CAT) activities were observed. Se supplementation to the diet of group III improved all the parameters cited above. Yet, plasma transaminases (AST and ALT), lactate dehydrogenase (LDH) activities, cholesterol, triglycerides (TG) and low density lipoprotein-cholesterol (LDL-C) levels increased, while high density lipoprotein-cholesterol (HDL-C) decreased. Co-administration of Se to the diet of group III restored hepatic markers to near-normal values. The biochemical results confirmed the histopathological findings. Therefore, our investigation revealed that Se was effective in preventing K₂Cr₂O₇-induced hepatotoxicity.     

In vivo protection by the xanthate tricyclodecan-9-yl-xanthogenate against amyloid beta-peptide (1-42)-induced oxidative stress

Considerable evidence supports the role of oxidative stress in the pathogenesis of Alzheimer's disease. One hallmark of Alzheimer's disease is the accumulation of amyloid beta-peptide, which invokes a cascade of oxidative damage to neurons that can eventually result in neuronal death. Amyloid beta-peptide is the main component of senile plaques and generates free radicals ultimately leading to neuronal damage of membrane lipids, proteins and nucleic acids. Therefore, interest in the protective role of different antioxidant compounds has been growing for treatment of Alzheimer's disease and other oxidative stress-related disorders. Among different antioxidant drugs, much interest has been devoted to "thiol-delivering" compounds. Tricyclodecan-9-yl-xanthogenate is an inhibitor of phosphatidylcholine specific phospholipase C, and recent studies reported its ability to act as a glutathione-mimetic compound. In the present study, we investigate the in vivo ability of tricyclodecan-9-yl-xanthogenate to protect synaptosomes against amyloid beta-peptide-induced oxidative stress. Gerbils were injected i.p. with tricyclodecan-9-yl-xanthogenate or with saline solution, and synaptosomes were isolated from the brain. Synaptosomal preparations isolated from tricyclodecan-9-yl-xanthogenate injected gerbils and treated ex vivo with amyloid beta-peptide (1-42) showed a significant decrease of oxidative stress parameters: reactive oxygen species levels, protein oxidation (protein carbonyl and 3-nitrotyrosine levels) and lipid peroxidation (4-hydroxy-2-nonenal levels). Our results are consistent with the hypothesis that modulation of free radicals generated by amyloid beta-peptide might represent an efficient therapeutic strategy for treatment of Alzheimer's disease and other oxidative-stress related disorders. Based on the above data, we suggest that tricyclodecan-9-yl-xanthogenate is a potent antioxidant and could be of importance for the treatment of Alzheimer's disease and other oxidative stress-related disorders.     

Induction of mutagenic DNA damage by chromium(VI) and glutathione

Certain chromium (Cr) compounds are known to be carcinogenic in humans and mutagenic in cell culture. However, the mechanism of Cr mutagenesis is not well understood. It appears that intracellular reduction of Cr by agents such as glutathione plays a role in the induction of DNA damage. We have used a simian virus 40-based shuttle vector to investigate the relationship between chromium-induced DNA damage and Cr mutagenicity. The treatment of the plasmid pZ189 with Cr(VI) plus glutathione (GSH) induced DNA strand breaks and reduced the plasmid biological activity, whereas Cr(III) treatment with or without GSH did not give rise to such DNA damage. When Cr(VI)/GSH- or Cr(III)/GSH-treated pZ189 was replicated in mammalian cells, a dose-dependent increase in mutant frequency was observed with Cr(VI)/GSH-treated pZ189, but not with Cr(III)/GSH-treated plasmid. About 43% of the mutants from Cr(VI)/GSH-treated pZ189 were deletion mutants. The remainder were base substitution mutants, mostly GC → AT transitions and GC → TA transversions. This pattern of mutagenesis is similar to that observed with other agents that cause oxidative DNA damage such as ionizing radiation and H₂O₂. These results support the hypothesis that Cr mutagenesis can be induced by the generation of reactive oxygen intermediates during the reduction of Cr(VI) by glutathione. © 1996 Wiley-Liss, Inc.     

Minocycline provides protection against beta-amyloid(25-35)-induced alterations of the somatostatin signaling pathway in the rat temporal cortex

Minocycline is a semi-synthetic second-generation tetracycline known to improve cognition in amyloid precursor protein transgenic mice. Whether it can protect the somatostatin (SRIF) receptor-effector system, also involved in learning and memory, from alterations induced by chronic i.c.v. infusion of beta-amyloid peptide (Abeta)(25-35) is presently unknown. Hence, in the present study, we tested the effects of minocycline on the SRIF signaling pathway in the rat temporal cortex. To this end, male Wistar rats were injected with minocycline (45 mg/kg body weight) i.p. twice on the first day of treatment. On the following day and during 14 days, Abeta(25-35) was administered i.c.v. via an osmotic minipump connected to a cannula implanted in the left lateral ventricle (300 pmol/day). Minocycline (22.5 mg/kg, i.p.) was injected once again the last 2 days of the Abeta(25-35) infusion. The animals were killed by decapitation 24 h after the last drug injection. Our results show that minocycline prevents the decrease in SRIF receptor density and somatostatin receptor (sst) 2 expression and the attenuated capacity of SRIF to inhibit adenylyl cyclase (AC) activity, alterations present in the temporal cortex of Abeta(25-35)-treated rats. Furthermore, minocycline blocks the Abeta(25-35)-induced decrease in phosphorylated cyclic AMP (cAMP) response element binding protein (p-CREB) content and G-protein-coupled receptor kinase 2 (GRK) protein expression in this brain area. Altogether, the present data demonstrate that minocycline in vivo provides protection against Abeta-induced impairment of the SRIF signal transduction pathway in the rat temporal cortex and suggest that it may have a potential as a therapeutic agent in human Alzheimer's disease, although further studies are warranted.     

DNA breakage in asbestos-treated normal and transformed (TSV40) rat pleural mesothelial cells

Asbestos has been shown to induce cell cycle arrest, DNA repair and some abnormalities consistent with DNA damage but not DNA breakage. The purpose of the study was to investigate DNA breakage in asbestos-exposed rat pleural mesothelial cells (RPMC). RPMC were compared with their transformed counterparts, RPMC-TSV40 (i.e. p53-inactivated by infection with a retroviral recombinant encoding the SV40 large T antigen), as in the latter cells the cell cycle does not arrest and DNA repair is deficient due to ineffective p53-dependent cell cycle control. RPMC and RPMC-TSV40 were exposed to chrysotile and crocidolite asbestos and also to camptothecin for comparison. The presence of DNA breakage was determined using the single cell gel (Comet) assay with alkaline electrophoresis and quantified by measuring comet tail length (TL) and the percentage of total DNA in the tail and calculating tail moment (TM). We found that comets were generated by both types of asbestos in RPMC and in RPMC-TSV40 as well as by camptothecin in RPMC. On a per weight basis, chrysotile induced more abnormalities in comet parameters than did crocidolite. The comet TL and TM increased with fibre concentration, although less so with crocidolite than with chrysotile. When exposed to chrysotile at similar concentrations, RPMC consistently showed more abnormal comet parameters than did RPMC-TSV40. We concluded that asbestos causes DNA breakage and suggest that some of the DNA breakage measured was due to repair mechanisms in the normal RPMC.     

Effect of age on endogenous DNA single-strand breakage,strand break induction and repair in the adult housefly,Musca domestica

It has been suggested that genomic alterations involving DNA damage and the ability to repair such damage play an important role in cellular sensecence. In this study, endogenous DNA single-strand breaks, the susceptibility of DNA to induced strand breakage and the capacity to repair these breaks were compared in postmitotic cells from young (3-day-old) and old (23-day-old) houseflies. DNA single-strand breaks did not accumulate during normal aging in the housefly. However, cells of the old flies exhibited a greater sensitivity to single-strand breakage induced by γ-radiation and UV light. The capacity to repair these exogenously induced single-strand breaks declined with age. Results fo not support the view that DNA single-strand breaks are casual factor in aging in the housefly. An age-related increase in the susceptibility to undergo single-strand breakage suggests alterations in chromatin during the aging process.     

Iron-Porphyrin-Based Covalent Assembly with Peroxidase-Like Activity and High Efficiency for Cr(VI) Colorimetric Detection

Efficient synthesis of functional nanomaterials for simple, sensitive, and accurate colorimetric detection of chromate [Cr(VI)] has attracted great interest due to the toxicity of Cr(VI). Here, novel covalently assembled iron porphyrins (CAIPs) are fabricated through covalently cross-linking reaction between iron-tetra-(4-hydroxy-phenyl) porphyrin and three different cross-linkers based on 3,3´-dithiodipropionic acid. The results show that CAIPs have a spherical vesicle framework with favorable dimensional structure and enzymatic activity. In the presence of H₂O₂, CAIPs exhibit high peroxidase-like activity toward 3,3´,5,5´-tetramethylbenzidine. Based on these findings, CAIPs are used as an efficient colorimetric catalyst for the sensitive and selective detection of Cr(VI) with a detection limit about 23 nm . The feasibility of this method for real-water samples is verified by a standard addition experiment. This system further facilitates the developments of nanomaterial, and its application in colorimetric detection fields.     

Induced cross-protection responses against Cr(III) and Fe(III) ions in Saccharomyces cerevisiae

Stress tolerance of yeast Saccharomyces cerevisiae was examined after exposure to iron and chromium, which are essential minerals in low concentrations but can be toxic if present in high concentrations. Induction of possible cross-protection responses was performed with the yeast pre-treatment at the start of cultivation with low concentrations of Fe(III) or Cr(III) ions, which slightly inhibit the growth and the subsequent exposure to sub-lethal concentrations of Fe(III) or Cr(III) ions in the mid-exponential phase. No cross-protection was found if yeasts were pre-treated with 0.1 mM Cr(III) and subsequent exposure to 2.5 mM Fe(III) ions took place. If pre-treated with 0.1 mM Fe(III) Saccharomyces cerevisiae conferred protection to subsequent challenges with a sub-lethal concentration of 2.5 mM Cr(III) ions resulting in higher biomass formation and higher relative cell viability in comparison to cells without pre-treatment. It is shown for the first time that iron pre-treatment enhanced yeast condition against chromium related stress via cross-protection mechanism.     

Singlet oxygen is the major species participating in the induction of DNA strand breakage and 8-hydroxydeoxyguanosine adduct by lead acetate.

To investigate DNA damage induced by Pb2+ and its prevention by scavengers, we determined DNA strand breakage and the formation of 8-hydroxydeoxyguanosine (8-OHdG) in DNA using plasmid relaxation assay and HPLC with electrochemical detection, respectively. Lead acetate induced DNA strand breakage in 10 mM of Hepes buffer, pH 6.8, in a time- and dose-dependent manner. Compared with lead, zinc acetate did not significantly induce DNA breakage. The singlet oxygen scavengers NaN3 and 2,2,6,6-tetramethyl-4-piperidone (TEMP) inhibited lead-induced DNA breakage more efficiently than the hydroxyl radical scavengers mannitol and DMPO. Deuterium oxide (D2O), a singlet oxygen enhancer, potentiated lead-induced DNA breakage. At low ratios to Pb2+, NADPH, glutathione, and 2-mercaptoethanol enhanced lead-induced DNA breakage, whereas high ratios of these agents protected it. Catalase and superoxide dismutase (SOD) did not protect DNA breaks induced by Pb2+. Lead-induced DNA breakage was markedly enhanced by H2O2, and this induction was inhibited by NaN3, TEMP, EDTA, catalase, BSA, and glutathione. In contrast, mannitol and SOD potentiated Pb2+/H2O2-induced DNA breaks. The results indicate that singlet oxygen, lead, and H2O2 are all involved in the reaction system, whereas hydroxyl radical and superoxide did not. Lead could cause a small amount of 8-OHdG formation in calf thymus DNA and dose-dependently induced the formation of this adduct in the presence of H2O2. Singlet oxygen scavengers were more effective than hydroxyl radical scavengers in protection from lead/H2O2-induced 8-OHdG adducts. Taken together, these results suggest that lead may induce DNA damage through a Fenton-like reaction and that singlet oxygen is the principal species involved.     

Antioxidant role of glutathione S-transferases: protection against oxidant toxicity and regulation of stress-mediated apoptosis

It has been known that glutathione S-transferases (GSTs) can reduce lipid hydroperoxides through their Se-independent glutathione peroxidase activity and that these enzymes can also detoxify lipid peroxidation end products such as 4-hydroxynonenal (4-HNE). In this article, recent studies suggesting that the Alpha class GSTs provide a formidable defense against oxidative stress are critically evaluated and the role of these enzymes in the regulation of oxidative stress-mediated signaling is reviewed. Available evidence from earlier studies together with results of recent studies in our laboratories strongly suggests that lipid peroxidation products, particularly hydroperoxides and 4-HNE, are involved in the mechanisms of stress-mediated signaling and that it can be modulated by the Alpha class GSTs through the regulation of the intracellular concentrations of 4-HNE.     

NF-κB抑制剂PDTC抑制UHMWPE磨损颗粒诱导界膜组织产生TNF-α的实验研究

目的 探讨NF-κB抑制剂PDTC对高分子量聚乙烯磨损颗粒(UHMWPE)诱导的假体周围界膜组织产生TNF-α的影响,寻找防治假体周围骨溶解的方法.方法 用昆明小鼠24只构建air-pouch模型,随机分成两组,每组8只,阳性对照组(囊内注入UHMWPE颗粒悬液,腹腔注射生理盐水);实验组(囊内注入UHMWPE颗粒悬液,腹腔注射PDTC),阴性对照组(囊内注入生理盐水,腹腔注射PDTC),7天后处死动物,ELISA法检测囊壁组织中TNF-α的含量.结果 阳性对照组囊壁组织TNF-α含量为6.342910.7475(OD/克);实验组囊壁组织中TNF-α含量为5.0428±0.8105;阴性对照组囊壁中TNF-α含量为3.5010±0.7248.其中阳性对照组与实验组、阳性对照组与阴性对照组、实验组与阴性对照组之间两两对比均有显著统计学差异(P＜0.01).结论 NF-κB抑制剂PDTC能够抑制UHMWPE颗粒诱导的界膜组织中TNF-α的产生,对假体周围骨溶解有防治作用.