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Archives of Toxicology (v.84, #5)

In vitro evaluation of selenium genotoxic, cytotoxic, and protective effects: a review by Vanessa Valdiglesias; Eduardo Pásaro; Josefina Méndez; Blanca Laffon (pp. 337-351).

Selenium is an oligoelement with essential biological functions. Diet is the most important selenium source, and intake of this element depends on its concentration in food and amount of food consumed. Among the essential human micronutrients, selenium is peculiar due to its beneficial physiological activity and toxicity. It may have anticarcinogenic effects at low concentrations, whereas at concentrations higher than those necessary for nutrition, it can be genotoxic and carcinogenic. Because of that, selenium is probably the most widely investigated of all the oligonutrients. In the last decades, there has been increasing interest in several nutritional Se compounds because of their environmental, biological, and toxicological properties, particularly for their cancer- and disease-preventing activities. This article gives an overview of the results of in vitro studies on mutagenicity, genotoxicity, cytotoxicity, and DNA repair conducted within the last decades with different organic and inorganic selenium compounds. Results from these studies provide a better knowledge on the selenium activity and help to elucidate the reasons underlying its duality in order to regulate its correct use in nutrition and clinic.

Keywords: Cytotoxicity; DNA repair; Genotoxicity; Mutagenicity; Selenium

Effects of sodium fluoride on hyperactivation and Ca²⁺ signaling pathway in sperm from mice: an in vivo study by Zilong Sun; Ruiyan Niu; Kai Su; Bin Wang; Jinming Wang; Jianhai Zhang; Jundong Wang (pp. 353-361).

Sperm hyperactivation is crucial for a successful fertilization; however, the influence of fluoride (F) to hyperactivation is still in its infancy. The purpose of this study was to investigate the effect of sodium fluoride (NaF) on sperm hyperactivation, Ca²⁺/CALM-CAMK2 signaling, and CatSper1 and CatSper2 mRNA expression in mice sperm. Adult male Kunming mice were administrated with 30, 70, and 150 mg NaF/l (corresponding to 2.84 ± 0.29, 6.28 ± 0.61, and 14.18 ± 1.00 mg F/kg body weight per day) through drinking water for 49 days. The results showed that NaF reduced the sperm hyperactivated motility in a dose-dependent manner. Compared with the controls, intracellular Ca²⁺ concentration and CAMK2 protein were significantly decreased in mice treated with 70 and 150 mg NaF/l, while no effect on CALM was determined in all treatment groups. Furthermore, decreased sperm CatSper1 mRNA expression was also observed in response to middle and higher doses of NaF (70, 150 mg/l) with comparison to the control group, whereas no change in the mRNA expression of CatSper2 was detected in NaF administrated groups. Treatment with 30 mg NaF/l exhibited slight effects on the above indexes with no statistical difference. These findings indicated that exposure to 70 and 150 mg/l NaF for 49 days could result in low hyperactivation via alteration of Ca²⁺ signaling pathway involving CatSper1 in sperm from mice.

Keywords: Fluoride; Hyperactivation; Ca²⁺ signaling pathway; Mice; Sperm

Functional characterization of human cytochrome P450 2E1 allelic variants: in vitro metabolism of benzene and toluene by recombinant enzymes expressed in yeast cells by Nobumitsu Hanioka; Maki Yamamoto; Toshiko Tanaka-Kagawa; Hideto Jinno; Shizuo Narimatsu (pp. 363-371).

Benzene and toluene are common organic solvents currently in worldwide industrial usage, which are metabolized mainly by hepatic cytochrome P450 2E1 (CYP2E1) in humans. Genetic polymorphism of CYP2E1 in 5′-flanking and coding regions has been found previously in Caucasian and Chinese populations. In this study, the effects of CYP2E1 alleles causing amino acid substitutions (CYP2E1*2, CYP2E1*3 and CYP2E1*4; wild-type, CYP2E1.1A) on benzene hydroxylation and toluene methylhydroxylation were studied using recombinant CYP2E1 enzymes of wild-type (CYP2E1.1) and variants (CYP2E1.2 having Arg76His, CYP2E1.3 having Val389Ile and CYP2E1.4 having Val179Ile) expressed in yeast cells. The K _m, V _max and CL _int values of CYP2E1.1 were 10.1 mM, 9.38 pmol/min/pmol CYP and 0.99 nL/min/pmol CYP for benzene hydroxylation, and 3.97 mM, 19.9 pmol/min/pmol CYP and 5.26 nL/min/pmol CYP for toluene methylhydroxylation, respectively. The K _m, V _max and CL _int values for benzene and toluene metabolism of CYP2E1.2, CYP2E1.3 and CYP2E1.4 were comparable to those of wild-type CYP2E1. These findings may mean that the polymorphic alleles of CYP2E1 causing amino acid substitutions are not directly associated with the metabolic activation of benzene and toluene. The information gained in this study should help to identify the variations in the toxicity of environmental pollutants.

Keywords: Benzene; Toluene; Metabolism; Cytochrome P450 2E1 (CYP2E1); Genetic polymorphism

Convulsant action of diphenyl diselenide in rat pups: measurement and correlation with plasma, liver and brain levels of compound by Marina Prigol; Simone Pinton; Ricardo Schumacher; Cristina Wayne Nogueira; Gilson Zeni (pp. 373-378).

Diphenyl diselenide [(PhSe)₂], an organoselenium compound, presents toxicological effects in rat pups, manifested by the appearance of seizure episodes. The aim of this study was to carry out the determination and quantification of (PhSe)₂ in plasma, liver and brain of rat pups after oral administration (p.o) of this compound (500 mg/kg). The second objective of this study was to correlate the latency to the appearance for the first seizure episode with (PhSe)₂ plasma, liver and brain levels. Analysis of (PhSe)₂ in plasma, liver and brain samples was performed by gas chromatography/flame ionized detector system (GC/FID). The average levels of (PhSe)₂ in plasma, liver and brain of rat pups were 3.67, 5.07 and 1.15 μg/ml, respectively, at 20.58 min post dosing, the latency media for the first seizure episode. (PhSe)₂ levels in plasma did not correlate with the latency for the first seizure episode induced by this compound. A significant negative correlation between the latency for the first seizure episode and the levels of (PhSe)₂ liver and brain of rat pups was found. It demonstrates that rat pups which had highest levels of (PhSe)₂ in liver and brain showed the shortest latency for the first seizure episode.

Keywords: Selenium; Diphenyl diselenide; Rat pups; Seizure; Liver; Brain

Pentachlorophenol and its derivatives induce oxidative damage and morphological changes in human lymphocytes (in vitro) by Jaromir Michałowicz (pp. 379-387).

In this study, the effect of environmental toxins such as pentachlorophenol (PCP), tetrachlorocatechol (TeCC) and tetrachloroguaiacol (TeCG) on human peripheral blood lymphocytes was investigated. All the compounds studied increased the size and granularity of the lymphocytes in the concentrations range from 5 to 600 ppm. The PCP caused the strongest increase in the size of the cells, whereas lymphocytes granularity was more strongly increased by TeCC and PCP than by TeCG. The PCP and its derivatives in the concentrations range from 1 to 125 ppm significantly depleted ATP level. It was also observed that PCP most strongly decreased ATP content at its highest concentration of 125 ppm. Moreover, PCP caused the highest loss of lymphocytes viability in the concentrations range from 125 to 600 ppm. The TeCC in the concentrations of 1 and 5 ppm significantly increased the level of strand breaks in DNA, whereas lower damage was noted for PCP, and particularly for TeCG. The increase in carbonyl groups content was more strongly induced by TeCG and TeCC than by PCP in the concentrations range from 0.04 to 1 ppm; however, in a concentration of 5 ppm, all the compounds studied increased this parameter to a similar degree. DNA and protein damage was the most probably induced by free radical formation, as it was observed that all the compounds examined, and TeCC, in particular, were able of oxidize a fluorescent probe 6-carboxy-2′,7′-dichlorodihydrofluorescein in the concentrations range from 0.01 to 1 ppm.

Keywords: Pentachlorophenol; Tetrachloroguaiacol; Tetrachlorocatechol; Peripheral blood lymphocytes; Cell morphology; ATP level; Protein carbonylation; DNA damage; ROS formation

Comparative effects of curcumin and resveratrol on aflatoxin B₁-induced liver injury in rats by Dina S. El-Agamy (pp. 389-396).

Aflatoxin B₁ is a potent hepatotoxic and hepatocarcinogenic mycotoxin. Lipid peroxidation and oxidative DNA damage are the principal manifestations of aflatoxin B₁-induced toxicity that could be counteracted by antioxidants. Many plant constituents have been reported to prevent liver damage associated with lipid peroxidation. In this study, curcumin (polyphenolic antioxidant purified from turmeric) and resveratrol (polyphenol obtained from grapes) were evaluated for possible protection against liver injury induced by aflatoxin B₁ in rats. Adult male Fischer rats were divided into six groups including untreated control, curcumin control (200 mg/kg BW), resveratrol control (10 mg/kg BW) and aflatoxin B₁ (25 μg/kg BW). Other two groups were administered either curcumin or resveratrol along with aflatoxin B₁. The study was carried out for 90 days. At the end of the experiment period, blood and tissue samples were collected from the animals before they were killed. Livers were collected for histopathologic studies and fixed in 10% buffered formalin solution. Serum was used for estimation of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and γ-glutamyl transferase (γ-GT) enzymes. The lipid peroxidation, reduced glutathione (GSH), superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) were estimated in liver homogenates. The results revealed that aflatoxin B₁ administration caused liver damage as indicated by statistically significant (P < 0.05) increase in serum ALT, AST and γ-GT levels. In addition, there were general statistically significant reductions in the activities of GSH, SOD, CAT, GSH-Px, and an increase in lipid peroxidation in the liver of aflatoxin B₁-treated group compared to the untreated control group. Curcumin showed a significant hepatoprotective activity by lowering the levels of serum marker enzymes, lipid peroxidation and elevating the levels of GSH, SOD, CAT and GSH-Px. However, resveratrol failed to protect from the aflatoxin B₁-induced liver injury. These findings suggest that curcumin but not resveratrol has a hepatoprotective effect against aflatoxin B₁-induced liver injury.

Keywords: Aflatoxin B₁ ; Liver injury; Curcumin; Resveratrol; Oxidative stress; Rat

Acute inflammation and immunoresponses induced by ortho-phthalaldehyde in mice by Toru Morinaga; Go Hasegawa; Satoshi Koyama; Yoko Ishihara; Toshio Nishikawa (pp. 397-404).

ortho-Phthalaldehyde (OPA) has been used as a safe alternative disinfectant instead of glutaraldehyde; however, recently some adverse effects of OPA were reported in patients and medical professions. We examined the acute toxicity of OPA in male ICR mice injected with 0.125–0.5% OPA and killed some animals 1 day after a single OPA injection, and others 1 or 13 days after two OPA injections 5 days apart. Hematology, blood cell counts, specific antibody production, organ weights, hepatic enzymes, hepatic histopathology and gene expression of cytochrome P450 (CYP) mRNA in liver were examined. Single OPA injections elevated leukocyte counts, the proportion of neutrophils, alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Two OPA injections dose-dependently increased leukocyte counts, the proportion of neutrophils, ALT and AST, and decreased alkaline phosphatase. Leukocyte counts and proportions of neutrophils normalized 13 days after the second of two injections. However, both ALT and AST remained high in mice given higher OPA doses. Significant increased liver-to-body weight ratio and mild hepatic lesions were observed. Gene expression of CYP1a1 and CYP2e1 revealed a tendency of up-regulation 1 day after two OPA injections. However, expression of these genes was then down-regulated 13 days after OPA injections. OPA induced specific IgE and IgG significantly in the sera, suggesting that OPA acts as a hapten. Overall, OPA caused acute inflammation and acted as a haptenic allergen, although it caused only mild liver injury. Such evidence suggested that careful washing and prevention of exposure were needed after OPA disinfection of medical instruments.

Keywords: Acute inflammation; Adverse effects; Disinfectant; Hapten; ortho-Phthalaldehyde

Microcystin-LR induces toxic effects in differentiated and undifferentiated Caco-2 cells by María Puerto; Silvia Pichardo; Ángeles Jos; Ana María Cameán (pp. 405-410).

Microcystins (MCs) are toxins of heptapeptidic structure produced by toxic cyanobacteria in surface eutrophic waters. MCs are known to be hepatotoxic in humans, but they are also able to induce gastrointestinal alterations, allergic reactions, irritation, and pneumonia-like symptoms. The impact of MC-LR, one of the most common cyanobacterial toxins, was studied on the Caco-2 cell line, a commonly used enterocytic model, established from a human colon carcinoma. Caco-2 cells were differentiated in order to compare the effect of MC-LR in differentiated and non-differentiated cells. They were seeded in a 96-well microtiter plate and treated with MC-LR pure standard (98% purity). The effects of different concentrations of this cyanotoxin (50, 100, 150, and 200 μM) were investigated at 24 and 48 h of exposure by morphological observation and biochemical changes (total protein content, neutral red uptake, and MTS metabolization). Differentiated Caco-2 cells were slightly more sensitive than undifferentiated cells. Moreover, toxic effects induced by MCs were higher at 48 h compared to those observed at 24 h. The most sensitive endpoint for the cell line was the reduction of total protein content. Morphological changes induced by MC-LR were reduction in the cell number and hydropic degeneration, being these alterations more evident 48 h after the exposure to MC-LR.

Keywords: Cyanobacteria; Microcystin-LR; Cell line; Caco-2; Cytotoxicity

A comparative analysis of chromosomal aberrations in cultured human lymphocytes due to fluoroquinolone drugs at different expression periods by M. Anupama; J. P. Seiler; P. B. Murthy (pp. 411-420).

Fluoroquinolones (FQ) are broad-spectrum antibacterial agents widely used for the treatment of infections with various types of gram negative and gram positive bacteria. Specifically, gatifloxacin (GFX) is under development as a component in a new antituberculosis fixed-dose drug combination. In the context of this project, GFX was also tested for genotoxic activity in human peripheral lymphocytes, and the induction of chromosomal aberrations by GFX in PHA-M stimulated cultured human lymphocytes, investigated under conditions of conventional and increased expression times, was further compared to the analogous effects induced by some other second- and third-generation FQ antibacterial agents, namely ofloxacin (OFX), ciprofloxacin (CFX) and sparfloxacin (SFX). OFX did not induce any significant chromosomal aberrations in human lymphocytes. CFX and SFX exhibited slight to moderate clastogenic potential at cytotoxic concentrations (150, 175, 200 and 225 μg/ml), and GFX, a third-generation FQ, induced a clear, concentration-dependent increase in the frequency of chromosomal aberrations at cytotoxic concentrations (150, 200 and 250 μg/ml). These effects were not apparent when metaphases were analysed at the conventionally used sampling time of 24 h, but only after prolongation of the expression time between treatment and harvesting to a sampling time of 36 h (4 h exposure and 32 h expression period). Also, an increased incidence of numerical aberrations (polyploidy and endoreduplication) was seen with GFX at non-cytotoxic concentrations (12.5, 25, 50 and 75 μg/ml). These effects can be attributed to the slight cross-reactivity of FQs between their inhibitory activity towards their intended targets, the prokaryotic type II topoisomerase enzymes DNA gyrase and topoisomerase IV, and the analogous mammalian enzyme topoisomerase II. We have also observed the formation of polycentrics, i.e., chromosomes with five to six centromeres, a rarely reported structural aberration, in GFX-treated cells. The significance of these observations with respect to the conventional conduct of such studies and to the interpretation of the effects is discussed.

Keywords: Gatifloxacin; Ciprofloxacin; Ofloxacin; Sparfloxacin; Chromosomal aberration; Human lymphocyte

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Archives of Toxicology (v.84, #5)