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Archives of Toxicology (v.85, #4)

Control of oxidative stress by the Keap1-Nrf2 pathway by J. D. Stewart; J. G. Hengstler; H. M. Bolt (pp. 239-239).

The cytoprotective role of the Keap1–Nrf2 pathway by Liam Baird; Albena T. Dinkova-Kostova (pp. 241-272).

An elaborate network of highly inducible proteins protects aerobic cells against the cumulative damaging effects of reactive oxygen intermediates and toxic electrophiles, which are the major causes of neoplastic and chronic degenerative diseases. These cytoprotective proteins share common transcriptional regulation, through the Keap1–Nrf2 pathway, which can be activated by various exogenous and endogenous small molecules (inducers). Inducers chemically react with critical cysteine residues of the sensor protein Keap1, leading to stabilisation and nuclear translocation of transcription factor Nrf2, and ultimately to coordinate enhanced expression of genes coding for cytoprotective proteins. In addition, inducers inhibit pro-inflammatory responses, and there is a linear correlation spanning more than six orders of magnitude of concentrations between inducer and anti-inflammatory activity. Genetic deletion of transcription factor Nrf2 renders cells and animals much more sensitive to the damaging effects of electrophiles, oxidants and inflammatory agents in comparison with their wild-type counterparts. Conversely, activation of the Keap1–Nrf2 pathway allows survival and adaptation under various conditions of stress and has protective effects in many animal models. Cross-talks with other signalling pathways broadens the role of the Keap1–Nrf2 pathway in determining the fate of the cell, impacting fundamental biological processes such as proliferation, apoptosis, angiogenesis and metastasis.

Keywords: Keap1; Nrf2; Cytoprotective enzymes; Phase 2 inducer

Nrf2: control of sensitivity to carcinogens by Stephen L. Slocum; Thomas W. Kensler (pp. 273-284).

Induction of enzymes that enhance the detoxication of chemical carcinogens has been a broadly effective strategy for chemoprevention of experimental carcinogenesis in rodent models. Several inducing agents are now in clinical trials to evaluate utility for prevention of cancers associated with unavoidable high exposures to environmental carcinogens. The successes of these pre-clinical and clinical interventions lead to studies to define the molecular basis for protection by these agents, which now include phenolic antioxidants, dithiolethiones, isothiocyanates, and triterpenoids. In the mid-1990s, the NF-E2-related factor 2 (Nrf2) transcription factor was identified as a key regulator of the inducible expression of enzymes such as glutathione S-transferases and NAD(P)H: quinone oxidoreductase in catalyzing the detoxication of reactive electrophiles and oxidants that contribute to the formation of mutations and ultimately cancers. Nrf2 is now recognized to regulate a broad cytoprotective, transcriptional response leading to prevention of damage to DNA, proteins and lipids; recognition, repair and removal of macromolecular damage; and tissue renewal following toxic assaults. Highlighting the importance of this pathway as a determinant of susceptibility to carcinogenesis, multiple studies now demonstrate enhanced incidence, multiplicity, and/or tumor burden in Nrf2-disrupted mice compared to wild-type in models of inflammation and colon cancer, bladder cancer, lung disease and cancer, stomach cancer, mammary cancer, skin cancer, and hepatocarcinogenesis.

Keywords: Keap1; Nrf2; Cancer; Chemoprevention

Long-term detection of methyltestosterone (ab-) use by a yeast transactivation system by Sylvi Wolf; Patrick Diel; Maria Kristina Parr; Felicitas Rataj; Willhelm Schänzer; Günter Vollmer; Oliver Zierau (pp. 285-292).

The routinely used analytical method for detecting the abuse of anabolic steroids only allows the detection of molecules with known analytical properties. In our supplementary approach to structure-independent detection, substances are identified by their biological activity. In the present study, urines excreted after oral methyltestosterone (MT) administration were analyzed by a yeast androgen screen (YAS). The aim was to trace the excretion of MT or its metabolites in human urine samples and to compare the results with those from the established analytical method. MT and its two major metabolites were tested as pure compounds in the YAS. In a second step, the ability of the YAS to detect MT and its metabolites in urine samples was analyzed. For this purpose, a human volunteer ingested of a single dose of 5 mg methyltestosterone. Urine samples were collected after different time intervals (0–307 h) and were analyzed in the YAS and in parallel by GC/MS. Whereas the YAS was able to trace MT in urine samples at least for 14 days, the detection limits of the GC/MS method allowed follow-up until day six. In conclusion, our results demonstrate that the yeast reporter gene system could detect the activity of anabolic steroids like methyltestosterone with high sensitivity even in urine. Furthermore, the YAS was able to detect MT abuse for a longer period of time than classical GC/MS. Obviously, the system responds to long-lasting metabolites yet unidentified. Therefore, the YAS can be a powerful (pre-) screening tool with the potential that to be used to identify persistent or late screening metabolites of anabolic steroids, which could be used for an enhancement of the sensitivity of GC/MS detection techniques.

Keywords: Methyltestosterone; 17β-Hydroxy-17α-methylandrost-4-ene-3-one, 17α-methyl-5α-androstane-3α,17β-diol; 17α-methyl-5β-androstane-3α,17β-diol; Urine; Reporter gene system; Doping

Ethanol-induced small heat shock protein genes in the differentiation of mouse embryonic neural stem cells by Mi Ran Choi; Kyoung Hwa Jung; Ji Hyun Park; Nando Dulal Das; Mi Kyung Chung; Ihn Geun Choi; Boung Chul Lee; Kyoung Sun Park; Young Gyu Chai (pp. 293-304).

Neural stem cells (NSCs) of the neuroepithelium differentiate into one of three central nervous system (CNS) cell lineages: neurons, astrocytes, or oligodendrocytes. In this study, the differentiation potential of NSCs from the forebrain of embryonic day 15 (E15) mouse embryos was analyzed using immunocytochemistry. NSCs were differentiated early in the presence or absence of ethanol (50 mM), and gene expression patterns among NSCs, differentiated cells and ethanol-treated differentiated cells were assessed by microarray and real-time PCR analysis. Genes that were up-regulated in differentiated cells both in the presence and in the absence of ethanol when compared to NSCs were related to the Wnt signaling pathway, including Ctnna1, Wnt5a, Wnt5b, Wnt7a, Fzd3, and Fzd2; genes related to cell adhesion, including Cadm1, Ncam1, and Ncam2; and genes encoding small heat shock proteins, including HspB2, HspB7, and HspB8. In particular, the expression levels of HspB2 and HspB7 were elevated in ethanol-treated differentiated cells compared to non-treated differentiated cells. The gene expression patterns of various heat shock transcription factors (HSFs), proteins that regulate the transcription of heat shock genes, were also analyzed. The expression levels of HSF2 and HSF5 increased in differentiated cells in the presence and absence of ethanol when compared to NSCs. Of these two genes, HSF5 demonstrated an enhanced up-regulation, particularly in ethanol-treated differentiated cells compared to cells that were differentiated in the absence of ethanol. These results imply that HspB2 and HspB7, which are small heat shock proteins with tissue-restricted expression profiles, might be up-regulated by ethanol during the short-term differentiation of NSCs.

Keywords: Differentiation; Ethanol; Heat shock transcription factor; Microarray; Neural stem cells; Small heat shock protein

Characterization of toxins from the broad-banded water snake Helicops angulatus (Linnaeus, 1758): isolation of a cysteine-rich secretory protein, Helicopsin by Amalid Estrella; Elda E. Sánchez; Jacob A. Galán; W. Andy Tao; Belsy Guerrero; Luis F. Navarrete; Alexis Rodríguez-Acosta (pp. 305-313).

Helicops angulatus (broad-banded water snake) according to recent proposals is presently cited in the family Dipsadidae, subfamily Xenodontinae, forming the tribe Hydropsini along with the genera Hydrops and Pseudoeryx. The current work characterizes the proteolytic and neurotoxic activities of H. angulatus crude toxins from salivary excretion (SE) and describes the isolation and identification of a cysteine-rich secretory protein (CRISP) called helicopsin. The SE lethal dose (LD₅₀) was 5.3 mg/kg; however, the SE did not contain hemorrhagic activity. Helicopsin was purified using activity-guided, Superose 12 10/300 GL molecular exclusion, Mono Q10 ion exchange, and Protein Pak 60 molecular exclusion. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) showed a highly purified band of approximately 20 kDa. The minimal lethal dose for helicopsin was 0.4 mg/kg. Liquid chromatography mass spectrometry (LC-MS/MS) analysis identified 2 unique peptides MEWYPEAAANAER and YTQIVWYK, representing a protein sequence (deleted homology) belonging to cysteine-rich secretory proteins, which are conserved in snake venoms (CRISPs). CRISPs are a large family of cysteine-rich secretory proteins found in various organisms and participate in diverse biological processes. Helicopsin exhibited robust neurotoxic activity as evidenced by immediate death (~8 min) due to respiratory paralysis in NIH mice. These observations for helicopsin purified from H. angulatus provide further evidence of the extensive distribution of highly potent neurotoxins in the Colubroidea superfamily of snakes than previously described.

Keywords: CRISP; Colubroidea; Dipsadidae salivary excretion; Neurotoxin; Helicops angulatus ; Broad-banded water snake

In vitro study of P-glycoprotein induction as an antidotal pathway to prevent cytotoxicity in Caco-2 cells by Renata Silva; Helena Carmo; Ricardo Dinis-Oliveira; Anabela Cordeiro-da-Silva; Sofia Costa Lima; Félix Carvalho; Maria de Lourdes Bastos; Fernando Remião (pp. 315-326).

The Caco-2 cell line is a reliable in vitro model for predicting drug intestinal absorption and P-glycoprotein (P-gp)-mediated excretion in humans. Recent in vivo studies suggested the induction of P-gp as a cellular protection tool against paraquat poisoning, through the increase in its pulmonary and intestinal excretion. Thus, the aim of the present work was to evaluate P-gp expression and activity in Caco-2 cells exposed to doxorubicin (a known P-gp inducer) and to correlate these changes with paraquat toxic effects. Cytotoxicity of doxorubicin (0–100 μM) and paraquat (0–1,000 μM) was evaluated for a maximum period of 96 h. In doxorubicin-exposed cells, P-gp expression and transport activity were evaluated by flow cytometry, using a fluorescein isothiocyanate–conjugated antibody and the P-gp fluorescent subtract rhodamine 123, respectively. A significant increase in P-gp expression was observed as soon as 6 h after exposure to 5 μM doxorubicin. P-gp activity also increased after 6 h, but only at higher doxorubicin concentrations (over 50 μM). Paraquat (0–5,000 μM) cytotoxicity was then evaluated with or without previous exposure of the cells to doxorubicin (5–100 μM, a concentration range causing both an increase in P-gp expression and activity). Under P-gp induction, a significant reduction in paraquat cytotoxicity was observed. Furthermore, when these cells were incubated with a specific P-gp inhibitor (UIC2 antibody) the doxorubicin protective effects were blocked, confirming the involvement of P-gp in the reduction in paraquat cytotoxicity. In conclusion, the human Caco-2 cell line model can be used for the study of P-gp induction as an antidotal pathway against substrates of this transporter system.

Keywords: P-glycoprotein induction; P-glycoprotein transport activity; Paraquat toxicity; Caco-2 cells

Fluoride-induced histopathology and synthesis of stress protein in liver and kidney of mice by Ansuman Chattopadhyay; Santosh Podder; Soumik Agarwal; Shelley Bhattacharya (pp. 327-335).

Selective low (15 mg sodium fluoride (NaF)/L) and relatively high (150 mg NaF/L) doses of in vivo fluoride (F) treatment to Swiss albino mice through drinking water elicited organ-specific toxicological response. All the F-exposed groups showed severe alterations in both liver and kidney architectures, but there was no significant change in the rate of water consumption and body weight. Vacuolar degeneration, micronecrotic foci in the hepatocytes, and hepatocellular hypertrophy were evident in the mice exposed to low dose (15 mg NaF/L for 30 days) while sinusoidal dilation with enlarged central vein surrounded by deep-blue erythrocytes were preponderant when treated with the same dose for a period of 90 days. Blood filled spaces, disintegration of tubular epithelium, and atrophy of glomeruli were also recorded in the kidney of the same treatment group. Change in reduced glutathione level (GSH), glutathione-s-transferase (GST) activity, malondialdehyde (MDA) production in both liver and kidney, disturbances in liver function, induction of heat shock protein 70 (Hsp 70) expression in kidney and its down regulation in liver were positively correlated with histopathological lesion.

Keywords: Sodium fluoride; Genotoxicity; Heat shock protein 70; Hepatotoxicity; Histopathology; Liver function; Nephrotoxicity

Intranasal benzo[a]pyrene alters circadian blood pressure patterns and causes lung inflammation in rats by Nicole J. Gentner; Lynn P. Weber (pp. 337-346).

Polycyclic aromatic hydrocarbons, including benzo[a]pyrene (BaP), are environmental contaminants formed during organic material combustion (e.g. burning fossil fuels and cigarette smoke). BaP toxicity is mediated, in part, by activation of the aryl hydrocarbon receptor and formation of reactive metabolites, both of which lead to increased oxidative stress. Since air pollution and cigarette smoking are known to increase cardiovascular disease in humans, the objective of this study was to determine the effects of 7-day intranasal BaP exposure on circadian blood pressure patterns, arterial stiffness, and possible sources of oxidative stress in radiotelemetry-implanted rats. Arterial pulse wave dP/dt was used an indicator of arterial stiffness and was compared to both functional (nitric oxide production and bioactivity, endothelin-1 levels) and structural (wall thickness) features of the arterial wall. In addition, histology of lung, heart, and liver were examined as well as pulmonary and hepatic cytochrome P450 1A1 (CYP1A1) activity. BaP exposure altered the circadian pattern of blood pressure, with a reduction in the normal dipping pattern during sleep. This was associated with increased neutrophil recruitment in the lungs of BaP-exposed rats. In contrast, BaP had no effect on cardiovascular tissue histology, arterial stiffness, oxidative stress or lung and liver CYP1A1 activity. Thus, the current study does not support the hypothesis that BaP reactive metabolites increase oxidative stress leading to reduced vascular NO bioactivity and increased blood pressure. Instead, the current study suggests that inflammation, detected only in the lung, is associated with altered circadian rhythm of blood pressure.

Keywords: Benzo[a]pyrene; Aryl hydrocarbon receptor; Oxidative stress; Circadian patterns; Blood pressure; Endothelium; Inflammation

Ticlopidine-induced hepatotoxicity in a GSH-depleted rat model by Shinji Shimizu; Ryo Atsumi; Tsunenori Nakazawa; Takashi Izumi; Kenichi Sudo; Osamu Okazaki; Hideo Saji (pp. 347-353).

We investigated hepatotoxicity induced by ticlopidine (TIC) in glutathione (GSH)-depleted rats by pre-treatment of a well-known GSH synthesis inhibitor, l-buthionine-S,R-sulfoxinine (BSO). Although sole administration of either TIC or BSO showed no signs of hepatotoxicity, combined administration of TIC with BSO induced hepatotoxicity, which was characterized by centrilobular necrosis of the hepatocytes and an elevation of plasma alanine aminotransferase activity. Administration of radio-labeled TIC in combination with BSO resulted in significantly higher covalent binding to rat liver proteins than that observed after sole dosing of radio-labeled TIC. Pre-treatment of 1-Aminobenzotriazole, a non-specific inhibitor of P450s, completely suppressed both hepatotoxicity and the increased hepatic covalent binding caused by TIC co-treatment with BSO. The results obtained in this animal model suggest that GSH depletion and covalent binding may be involved in hepatotoxicity induced by TIC. These observations may help to understand the risk factors and the mechanism of hepatotoxicity of TIC in humans.

Keywords: Ticlopidine; Iidiosyncratic; Hepatotoxicity; Glutathione; Reactive metabolite; Covalent binding

Ziram induces apoptosis and necrosis in human immune cells by Qing Li; Maiko Kobayashi; Tomoyuki Kawada (pp. 355-361).

Ziram as a dithiocarbamate fungicide is widely used throughout the world in agriculture and as an accelerating agent is used in latex production. In order to investigate ziram-induced apoptosis/necrosis and its underlying mechanism in human immune cells, a human monocyte-like cell line (U937) was treated with ziram at 0.0312–2 μM for 2–24 h at 37°C in a 5% CO₂ incubator. Apoptosis/necrosis induced by ziram was determined by analysis of FITC-Annexin-V/PI staining and the intracellular level of active caspase-3 by flow cytometry and DNA fragmentation analysis. We found that ziram induced apoptosis/necrosis in U937 in a time- and dose-dependent manner, as shown by FITC-Annexin-V/PI staining. DNA fragmentation was detected when cells were treated with 0.5, 1, or 2 μM ziram for 24 h. Ziram also induced an increase in intracellular active caspase-3 in U937 cells in a dose-dependent manner, and a caspase-3 inhibitor, Z-DEVD-FMK, significantly inhibited the ziram-induced apoptosis. Moreover, it was found that ziram induced mitochondrial cytochrome c release in U937 cells. These findings indicate that ziram can induce apoptosis/necrosis in U937 cells, and this effect is partially mediated by activation of intracellular caspase-3 and mitochondrial cytochrome c release.

Keywords: Annexin-V; Apoptosis; Caspase-3; Cytochrome c ; DNA fragmentation; Necrosis; U937; Ziram

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Archives of Toxicology (v.85, #4)