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Archives of Toxicology (v.84, #2)

Oxidative stress and hepatic carcinogenesis: new insights and applications by Hermann M. Bolt; Jan G. Hengstler (pp. 87-88).

Comparative study of quercetin and its two glycoside derivatives quercitrin and rutin against methylmercury (MeHg)-induced ROS production in rat brain slices by Caroline Wagner; Alessandra P. Vargas; Daniel H. Roos; Ademir F. Morel; Marcelo Farina; Cristina W. Nogueira; Michael Aschner; João B. Rocha (pp. 89-97).

The hypothesis that methylmercury (MeHg) potently induces formation of reactive oxygen species (ROS) in the brain is supported by observations on the neuroprotective effects of various classes of antioxidants. Flavonoids have been reported to possess divalent metal chelating properties, antioxidant activities and to readily permeate the blood–brain barrier. They can also provide neuroprotection in a wide array of cellular and animal models of neurological diseases. Paradoxically, in vivo administration of quercetin displays unexpected synergistic neurotoxic effect with MeHg. Considering this controversy and the limited data on the interaction of MeHg with other flavonoids, the potential protective effect of quercetin and two of its glycoside analogs (i.e., rutin and quercitrin) against MeHg toxicity were evaluated in rat cortical brain slices. MeHg (100 μM) caused lipid peroxidation and ROS generation. Quercitrin (10 μg/mL) and quercetin (10 μg/mL) protected mitochondria from MeHg (5 μM)-induced changes. In contrast, rutin did not afford a significant protective effect against MeHg (100 μM)-induced lipid peroxidation and ROS production in cortical brain slices. MeHg-generated ROS in cortical slices was dependent upon an increase in intracellular Ca²⁺ levels, because the over-production of MeHg-induced H₂O₂ in mitochondria occurred with a concomitant increase in Ca²⁺ transient. Here, we have extended the characterization of mechanisms associated with the neuroprotective effects of quercetin against MeHg-induced toxicity in isolated mitochondria, by performing an array of parallel studies in brain slices. We provide novel data establishing that (1) Ca²⁺ plays a central role in MeHg toxicity and (2) in brain slices MeHg induces mitochondrial oxidative stress both via direct interaction with mitochondria (as previously reported in in vitro studies) as well as via mitochondria-independent (or indirect) mechanisms.

Keywords: MeHg toxicity; Quercetin; Quercitrin; Rutin; ROS; Mitochondria

Benzene-induced mutational pattern in the tumour suppressor gene TP53 analysed by use of a functional assay, the functional analysis of separated alleles in yeast, in human lung cells by Sylvain Billet; Vincent Paget; Guillaume Garçon; Natacha Heutte; Véronique André; Pirouz Shirali; François Sichel (pp. 99-107).

Recent concern has centred on the effects of continuous exposure to low concentrations of benzene, both occupationally and environmentally. Although benzene has for a long time been recognised as a carcinogen for humans, its mechanistic pathway remains unclear. Since mutations in the tumour suppressor gene TP53 are the most common genetic alterations involved in human cancer, our objective was to establish the first mutational pattern induced by benzene on the TP53 gene in human type II-like alveolar epithelial A549 cells by using the Functional Analysis of Separated Alleles in Yeast (FASAY). Seventeen mutations linked to benzene exposure were found: 3 one- or two-base deletions, and 14 single nucleotide substitutions (1 nonsense and 13 missense mutations). A>G and G>A transitions were the most prevalent (23.5% for both). Other mutations included A>C transversions and deletions (3/17, 17.6% for both), G>T transversions (2/17, 11.8%) and A>T transversions (1/17, 5.9%). Data arising from this benzene-induced mutational pattern affecting TP53, a critical target gene in human carcinogenesis, have been compared with those reported in human acute myeloid leukaemia, the aetiology of which is clearly linked to benzene exposure, and in experimental benzene-induced carcinoma. This comparison suggests that A>G transition could be a fingerprint of benzene exposure in tumours. Furthermore, our results demonstrate that FASAY is a promising tool for the study of the carcinogenic potency of benzene in the human lung.

Keywords: A549 cells; Benzene; FASAY; Lung; Mutagenesis; TP53

Involvement of sensory nerves and TRPV1 receptors in the rat airway inflammatory response to two environment pollutants: diesel exhaust particles (DEP) and 1,2-naphthoquinone (1,2-NQ) by Aila Mirtes Teles; Yoshito Kumagai; Susan D. Brain; Simone A. Teixeira; Ana A. Varriano; Maria Alice A. G. Barreto; Wothan Tavares de Lima; Edson Antunes; Marcelo N. Muscará; Soraia K. P. Costa (pp. 109-117).

The environmental chemical 1,2-naphthoquinone (1,2-NQ) is implicated in the exacerbation of airways diseases induced by exposure to diesel exhaust particles (DEP), which involves a neurogenic-mediated mechanism. Plasma extravasation in trachea, main bronchus and lung was measured as the local ¹²⁵I-bovine albumin accumulation. RT-PCR quantification of TRPV1 and tachykinin (NK₁ and NK₂) receptor gene expression were investigated in main bronchus. Intratracheal injection of DEP (1 and 5 mg/kg) or 1,2-NQ (35 and 100 nmol/kg) caused oedema in trachea and bronchus. 1,2-NQ markedly increased the DEP-induced responses in the rat airways in an additive rather than synergistic manner. This effect that was significantly reduced by L-732,138, an NK₁ receptor antagonist, and in a lesser extent by SR48968, an NK₂ antagonist. Neonatal capsaicin treatment also markedly reduced DEP and 1,2-NQ-induced oedema. Exposure to pollutants increased the TRPV1, NK₁ and NK₂ receptors gene expression in bronchus, an effect was partially suppressed by capsaicin treatment. In conclusion, our results are consistent with the hypothesis that DEP-induced airways oedema is highly influenced by increased ambient levels of 1,2-NQ and takes place by neurogenic mechanisms involving up-regulation of TRPV1 and tachykinin receptors.

Keywords: Neurogenic inflammation; 1,2-Naphthoquinone; Diesel exhaust particles; TRPV1 receptor; Rat airways; Capsaicin

Erratum to: Involvement of sensory nerves and TRPV1 receptors in the rat airway inflammatory response to two environment pollutants: diesel exhaust particles (DEP) and 1,2-naphthoquinone (1,2-NQ) by Aila Mirtes Teles; Yoshito Kumagai; Susan D. Brain; Simone A. Teixeira; Ana A. Varriano; Maria Alice A. G. Barreto; Wothan Tavares de Lima; Edson Antunes; Marcelo N. Muscará; Soraia K. P. Costa (pp. 119-119).

Assessment of low dose effects of acute sulphur dioxide exposure on the airways using non-invasive methods by Monika Raulf-Heimsoth; Frank Hoffmeyer; Christoph van Thriel; Meinolf Blaszkewicz; Jürgen Bünger; Thomas Brüning (pp. 121-127).

Sulphur dioxide (SO₂) is an important environmental and workplace air pollutant. Some studies demonstrate that subjects without adaptation respond to SO₂ up to 10 ppm with irritative effects on the airways. The aim of our study was to assess irritative effects of SO₂ up to 2 ppm on the airways using non-invasive methods like exhaled breath condensate (EBC), nasal lavage fluid (NALF) and exhaled nitric oxide (FeNO). Sixteen healthy volunteers were exposed for 4 h to SO₂ in concentrations of 0 (clean air), 0.5, 1.0 and 2.0 ppm in a repeated measures cross-over design. Before and after exposure, FeNO and biomarkers of airway inflammation in NALF and EBC were measured. All EBC pH values, collected after exposure, were more alkaline than before, significant only for clean air (7.05 ± 0.4 vs. 7.27 ± 0.3, P = 0.0031) and 0.5 ppm SO₂ exposure (6.85 ± 0.53 vs. 7.08 ± 0.42, P = 0.0251). No dose-dependent differences before and after exposure were measured for LTB₄, PGE₂ and 8-isoPGF_2α. Substance P in NALF collected after exposure tended to result in higher concentrations compared to pre-samples, without clear dose effect. Further cellular and soluble parameters measured were not significantly affected. Our results show that 4 h SO₂ exposure up to 2.0 ppm did not induce significant changes in the biomarker composition of the EBC and NALF when compared with clean air or with pre-samples of the same subject. Therefore our data suggest that acute low dose SO₂ exposure in not adapted subjects did not induce airway irritation or/and inflammation measured under these conditions.

Keywords: Sulphur dioxide; Non-invasive methods; Exhaled breath condensate; Nasal lavage; Airways

Effect of prenatal and perinatal acrylamide on the biochemical and morphological changes in liver of developing albino rat by Ahmed Aly Allam; Abdel Whaab El-Ghareeb; Manal Abdul-Hamid; Ahlam El Bakery; Mammoun Gad; Mohammad Sabri (pp. 129-141).

Acrylamide has been employed as an experimental probe to investigate biochemical and morphological changes in developing rat liver following toxin administration in pregnant rats. Non-anesthetized pregnant rats were given acrylamide by gastric intubation at a dose of 10 mg/kg/day. The pups were divided into three groups: Group A, mothers were treated with saline (control group); Group B, mothers were treated with acrylamide from day D7 of gestation till birth (prenatal intoxication); Group C, mothers were treated with acrylamide from D7 of gestation to D28 after birth (perinatal intoxication). Acrylamide-induced biochemical changes (in liver and serum) and morphological changes (in liver) were studied in control and acrylamide-treated developing pups. Prenatally and perinatally administered acrylamide significantly increased lipid peroxidation and reduced glutathione and total thiol levels in liver. Significant inhibition of peroxidase and superoxide dismutase activities was observed in liver tissue. Total lipids including cholesterol and triglycerides were significantly increased in the serum. Acrylamide treatment increased serum alanine aminotransferase and aspartate aminotransferase activities and inhibited alkaline phosphatase activity. Sodium and potassium concentrations were increased, but calcium, phosphorus and iron levels were significantly reduced in the serum. Acrylamide produced significant electrophoretic changes in serum proteins. The most noticeable change was splitting of β-globulin into β1- and β2-globulins. Light microscopy showed acrylamide-induced fatty deposits, congested central vein, vacuolization and chromatolysis in hepatocytes. Ultrastructural studies revealed vacuolated cytoplasm, lipid droplets of variable size and mitochondria with damaged cristae and vacuolization. The nuclei in acrylamide-treated groups showed marked decrease in the staining of nuclear DNA.

Keywords: Acrylamide; Liver development; Lipid peroxidation; Glutathione; Oxidative stress

Antioxidant enzymatically modified isoquercitrin or melatonin supplementation reduces oxidative stress-mediated hepatocellular tumor promotion of oxfendazole in rats by Jihei Nishimura; Yukie Saegusa; Yasuaki Dewa; Meilan Jin; Masaomi Kawai; Sayaka Kemmochi; Tomoaki Harada; Shim-mo Hayashi; Makoto Shibutani; Kunitoshi Mitsumori (pp. 143-153).

To clarify whether enzymatically modified isoquercitrin (EMIQ) or melatonin (MLT) supplementation reduces oxidative stress-mediated hepatocellular tumor-promoting effect of oxfendazole (OX), a benzimidazole anthelmintic, male rats were administered a single intraperitoneal injection of N-diethylnitrosamine (DEN) and were fed a diet containing OX (500 ppm) for 10 weeks with or without EMIQ (2,000 ppm) or MLT (100 ppm) in the drinking water after DEN initiation. One week after the commencement of the administration of OX, rats were subjected to two-thirds of partial hepatectomy. The number of GST-P-positive foci promoted by OX was significantly inhibited by the combined antioxidant EMIQ or MLT administration, and the area of GST-P-positive foci was inhibited by the administration of MLT. Real-time RT–PCR analysis revealed decreases in mRNA expression levels of cytochrome P450, family 2, subfamily b, polypeptide 2 (Cyp2b2) and malic enzyme 1 (Me1) in the DEN-OX-EMIQ and DEN-OX-MLT groups and decreases in mRNA expression levels of Cyp1a1 and aldo–keto reductase family 7, member A3 (Akr7a3) in the DEN-OX-MLT group compared to those in the DEN-OX group. In in vitro ROS production assay, inhibited production of NADPH-dependent ROS was observed by the treatment with EMIQ or MLT. These results suggest that coadministration of EMIQ or MLT suppresses the hepatocellular tumor-promoting activity of OX in rats through the decrease in ROS production by the activation of CYPs.

Keywords: Oxfendazole; Tumor promotion; Rat; Antioxidant; Melatonin; Enzymatically modified isoquercitrin

Elevation of cell proliferation via generation of reactive oxygen species by piperonyl butoxide contributes to its liver tumor-promoting effects in mice by Masaomi Kawai; Yukie Saegusa; Yasuaki Dewa; Jihei Nishimura; Sayaka Kemmochi; Tomoaki Harada; Yuji Ishii; Takashi Umemura; Makoto Shibutani; Kunitoshi Mitsumori (pp. 155-164).

Piperonyl butoxide (PBO) is a pesticide synergist used with pyrethroids as a domestic insecticide, and it acts as a non-genotoxic hepatocarcinogen in rats and mice. To clarify whether oxidative stress is involved in the liver tumor-promoting effect of PBO in mice, male mice were subjected to two-thirds partial hepatectomy, followed by N-diethylnitrosamine (DEN) treatment, and given a diet containing 0.6% PBO for 25 weeks. The incidences of cytokeratin (CK) 8/18-positive foci, adenomas, and carcinomas significantly increased in the DEN + PBO group compared with the DEN-alone group. The PCNA-positive ratio significantly increased in non-tumor hepatocytes, CK8/18-positive foci and adenomas in the DEN + PBO group compared with the DEN-alone group. PBO increased reactive oxygen species (ROS) production in microsomes but did not change oxidative DNA damage as assessed by 8-hydroxydeoxyguanosine (8-OHdG). In real-time RT–PCR, PBO upregulated the expression of genes related to metabolism, such as Cytochrome P450 1a1, 2a5, and 2b10, and metabolic stress, such as Por and Nqo1, but downregulated Egfr and Ogg1. PBO also increased early response genes downstream of mitogen-activated protein kinase (MAPK), such as c-Myc that is induced by excessive ROS production, and G1/S transition-related genes, such as E2f1 and Ccnd1. Thus, PBO can generate ROS via the metabolic pathway without any induction of oxidative DNA damage, activate cell growth, increase c-Myc- and E2F1-related pathways, and act as a liver tumor promoter of DEN-induced hepatocarcinogenesis in mice.

Keywords: Piperonyl butoxide; Oxidative stress; Mitogen-activated protein kinase; 8-hydroxydeoxyguanosine; Hepatocarcinogenesis

Potential effects of the herbicide Diuron on mammary and urinary bladder two-stage carcinogenesis in a female Swiss mouse model by Nelci Antunes de Moura; Tony Fernando Grassi; Maria Aparecida Marchesan Rodrigues; Luís Fernando Barbisan (pp. 165-173).

The potential promoting effect of Diuron was investigated in a mouse model of mammary and urinary bladder carcinogenesis induced by 7,12-dimethylbenz(a)anthracene (DMBA) and N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN). Four-week old female Swiss mice were allocated to five groups: Groups G1-G3 received DMBA (5 × 1.5 mg/mouse) and BBN (8 × 7.5 mg/mouse) and G4 and G5 groups received only vehicles during the first 6 weeks. At week 7, G1 and G5 groups received basal diet and G2, G3 and G4 groups were fed a diet containing Diuron at 1,250, 2,500 and 2,500 ppm, respectively, during 13 weeks. At week 20, the animals were euthanized and the gross tumors were registered. Mammary glands and urinary bladder were processed for histopathological analysis. Samples from non-tumor areas were evaluated for cell proliferation by 5-bromodeoxyuridine labeling index (BrdU-LI%) and apoptosis. Dietary treatment with Diuron at 1,250 and 2,500 ppm significantly increased BrdU-LI% (P < 0.05) and the incidence of simple/nodular urothelial hyperplasia in the urinary bladder from DMBA/BBN-initiated groups (G2 and G3 vs. G1, P < 0.02) and in the non-initiated group (G4 vs. G5, P = 0.042). Two transitional cell carcinomas were observed in the group initiated and fed Diuron 2,500 ppm (G3). In contrast, in the mammary gland, Diuron feeding for 13 weeks did not significantly alter cell proliferation and apoptosis indexes or the incidence of hyperplastic lesions or neoplasms in the DMBA/BBN-initiated groups. These findings suggest that Diuron is a promoting agent to the urinary bladder but not to the mammary gland in female Swiss mice submitted to a medium-term two-stage carcinogenesis bioassay.

Keywords: Pesticides; Diuron; Mammary and urinary bladder carcinogenesis; Female Swiss mouse

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Archives of Toxicology (v.84, #2)