Toxicity and expression of oxidative stress genes in Tribolium castaneum induced by toluene,xylene, and thinner

Thinner is a solvent that contains toluene, xylene, and other substances. The aim of this study was to examine the toxicity of thinner, xylene, and toluene on adult red flour beetles, Tribolium castaneum (Herbst), as well as the effects of thinner and toluene on gene expression. The major compounds identified in the thinner with peak area >2% were p-xylene (6%), toluene (4%), 2,4-dimethylheptane (3%), methylcyclohexane (2.75%), 2-methylheptane (2%), cyclohexanone (2.6%), and nonane (2.1%). Insects were exposed to solvents at 0–240 µl/L air for 4–48 hr. Animals that survived after a 4 hr exposure to 80 µl/L air were employed to determine mRNA expression using real-time PCR. Xylene was highly toxic, and the majority of the beetles did not survive 4 hr exposure at 40 µl/L air. The lethal concentration 50 (LC₅₀) values for toluene, xylene and thinner at 48 hr exposure were 97.7, <40 and 99.8 µl/L air, respectively. Thinner and toluene induced lethargy and sluggish movement in treated insects, while with xylene these effects were not observed. Glutathione-S-transferase (Gst) gene expression increased after thinner and toluene exposure. Superoxide dismutase (SOD) gene expression rose after toluene exposure. These results suggest that T. castaneum provides an alternative model for the study of toxicological effects of volatile aromatic compounds.     

DNA recombination induced by aflatoxin B1 activated by cytochrome P450 1A enzymes

Mutations in tumor suppressor genes are intricately associated with the etiology of neoplasia. Often, such mutations are followed by the second, functional alleles of tumor suppressor genes, a phenomenon known as loss of heterozygosity. Loss of heterozygosity may occur by different molecular mechanisms, including mitotic recombination, and it is conceivable that these molecular events are influenced by endogenous as well as exogenous factors. To test whether mitotic recombination is induced by certain carcinogens, we genetically engineered a Saccharomyces cerevisiae tester strain so that it metabolized two important classes of carcinogens, polycyclic aromatic hydrocarbons and heterocyclic arylamines. This was accomplished by expressing human cDNA's coding for the cytochrome P450 (CYP) enzymes CYP1A1 or CYP1A2 in combination with NADPH-CYP oxidoreductase in a strain heterozygous for two mutations in the trp5 gene. Microsomes isolated form the transformed yeast strains activated various xenobiotics to powerful mutagens that were detected in the were detected in the Ames test. Of these, the mycotoxin aflatoxin B1, when activated intracellularly in the strains containing either human CYP enzyme, significantly induced mitotic recombination. These results are discussed in light of possible mechanisms that are involved in aflatoxin B₁-mediated hepatocarcinogenesis. Similarly, benzo[a]pyrene-trans-7,8-dihydrodiol and 3-amino-1-methyl-5H-pyrido [4,3-b]indole were activated to recombinagenic products, whereas benozo[a]pyrene and 2-amino-3,8-dimethylimidazo[4,5-f] quinoxaline were negative in this assay. Our results argue that the contstructed yeast strains may be a valuable tool for the investigation of drug-induced mitotic recombination. ©1994 Wiley-Liss, Inc.     

Early effects of benzene exposure in mice. Hematological versus genotoxic effects

Female BDF₁ mice were exposed to 100, 300 and 900 ppm benzene 6 h/day, 5 days/week, up to 8 weeks. Hematological studies included peripheral blood data, T4 and T8 lymphocyte counts in the blood and the spleen, hemopoietic stem and progenitor cell assays in the marrow (CFU-S, CFU-C, BFU-E, CFU-E). The single cell gel assay (“comet assay”) was applied in parallel with cells from the peripheral blood, bone marrow, spleen and liver. The results showed minor changes in the stem and progenitor cells and the development of a slight anemia at 4 and 8 weeks, in agreement with reported data. New was the increase of the T4/T8 ratio in the peripheral blood (not in the spleen) at the end of the first week of exposure to 300 and 900 ppm. The results of the “comet assay” indicate a much higher sensitivity of this test system (strand breaks and alkali labile sites of DNA). The tail moment indicative of the damage to DNA increased as early as 3 days with 300 ppm in the peripheral blood cells. Furthermore, the liver cells did react to a much higher extent than the other cells tested. With 100 ppm significant changes were seen in the liver after 5 days, but not in the blood. The repair, studied 24 and 48 h after the end of the exposure, was almost complete after 5-day exposure period in the blood and the liver, but not after 4 weeks of exposure with 300 ppm in the blood, and 100 and 300 ppm in the liver.     

Respiratory and other hazards of isocyanates

Isocyanates are increasingly being used for manufacturing polyurethane foam, elastomers, adhesives, paints, coatings, insecticides, and many other products. At present, they are regarded as one of the main causes of occupational asthma. The large number of workers who are exposed to these chemicals have a concentration-dependent risk of developing chronic airway disorders, especially bronchial asthma. Different pathophysiologic mechanisms are involved. Immunoglobulin E (IgE)-mediated sensitization and irritative effects have been clearly demonstrated in both exposed subjects and animals. Presumably, neural inflammation due to neuropeptide release of capsaicin-sensitive afferent nerves is crucial. We collected data on 1780 isocyanate workers who had been examined by our groups. Of them 1095 (including subjects from outpatient departments) had work-related symptoms, predominantly of the respiratory tract. Specific IgE antibodies were found in 14% of the 1095 subjects. The methacholine challenge test was shown to be an inadequate predictor of the results of inhalative isocyanate provocation tests in workers and in asthmatic controls. Isocyanate (toluene diisocyanate TDI) air concentrations of 10 ppb (0.07 mg/m³) and 20 ppb (0.14 mg/m³), respectively, did not cause significant bronchial obstruction in the majority of previously unexposed asthmatics with bronchial hyperreactivity. IgG-mediated allergic alveolitis, a rare disease among isocyanate workers, was found in approximately 1 % of the symptomatic subjects. Experimental studies exhibit dose-dependent toxic effects and give evidence for tachykinin-mediated bronchial hyperreactivity after exposure to isocyanates. The clinical role of genotoxic effects of isocyanates and their by-products demonstrated here in vitro and in vivo has yet to be clarified.Abbreviations ACH acetylcholine - BALF bronchoalveolar lavage fluid - CGRP calcitonin gene-related peptide - cpm counts per minute - DMSO dimethylsulfoxide - EAST enzyme allergo sorbent test - FEV ₁ forced expiratory volume in one second - HDI hexamethylene diisocyanate - HSA human serum albumin - IGV intrahoracic gas volume, measured by bodyplethysmography - IVC inspiratory vital capacity - LC ₅₀ lethal concentration for 50% of tested animals - MDI diphenylmethane diisocyanate - PD _50(100)/PC _50(100) dose/concentration causing a change of 50% (100%) of the parameter indicated - ppb (ppm) parts per billion (million) - RAST radio allergo sorbent test - Raw airway resistance, measured by bodyplethysmography - sRaw specific airway resistance, measured by bodyplethysmography - TDI toluene diisocyanate - TLV threshold limit value These studies were partly supported by the Ministry of Research and Technology, Federal Republic of Germany, grant 07 ALL 16     

Protective effects of 4-nerolidylcatechol against genotoxicity induced by cyclophosphamide.

In the present work we evaluated both the mutagenicity and antimutagenicity of the Pothomorphe umbellata root extract (PUE) and its isolated active principle, the 4-nerolidylcatechol (4-NC), in bone marrow cells of mice using the micronucleus test. Swiss male mice were orally treated for 4 days with PUE (200, 100 or 50mg/kg/day) or 4-NC (50, 25 or 12.5mg/kg/day) prior to exposition with a single dose (200mg/kg) of cyclophosphamide (CP), 24h after the end of the treatment. The results demonstrated that the PUE and 4-NC did not have any mutagenic effect on mouse bone marrow cells; quite the opposite, there was a protective effect against genotoxicity induced by cyclophosphamide. Taken together, under the conditions tested herein, mice treated with PUE and 4-NC showed, in a dose-dependent manner, protective effect against CP-induced genotoxicity. Due to their ability to prevent chromosomal damage, with apparent low toxicity and cost, PUE or pure 4-NC are likely to open a field of interest concerning their possible use in clinical applications.     

Nitropolycyclic aromatic hydrocarbons are inducers of mitotic homologous recombination in the wing-spot test of Drosophila melanogaster

In this study, the widespread environmental pollutants 1-nitronaphthalene (1NN), 1,5-dinitronaphthalene (1,5DNN), 2-nitrofluorene (2NF) and 9-nitroanthracene (9NA), were investigated for genotoxicity in the wing somatic mutation and recombination test (SMART) of Drosophila--using the high bioactivation (HB) cross. Our in vivo experiments demonstrated that all compounds assessed induced genetic toxicity, causing increased incidence of homologous somatic recombination. 2NF, 9NA and 1NN mutant clone induction is almost exclusively related to somatic recombination, although 1,5DNN-clone induction depends on both mutagenic and recombinagenic events. 1NN has the highest recombinagenic activity (approximately 100%), followed by 2NF (approximately 77%), 9NA (approximately 75%) and 1,5DNN (33%). 1NN is the compound with the strongest genotoxicity, with 9NA being approximately 40 times less potent than the former and 2NF and 1,5DNN approximately 333 times less potent than 1NN. The evidence indicating that the major effect observed in this study is an increased frequency of mitotic recombination emphasizes another hazard that could be associated to NPAHs--the increment in homologous recombination (HR).     

Review of the toxicology of carbonyl sulfide, a new grain fumigant

Carbonyl sulfide (COS) is a new grain fumigant which has been developed to replace methyl bromide, being phased out due to its ozone depletion properties, and to supplement phosphine gas which is experiencing increased insect resistance. Treatment of commodities with COS, a highly effective fumigant, results in residues that are near or indistinguishable to natural background levels of this compound. COS is a naturally occurring gas, being the predominant sulfur moiety in the atmosphere, occurs naturally in food and is a normal by-product of mammalian aerobic metabolism. COS has low acute inhalational toxicity but with a steep dose response curve; COS is neither genotoxic nor a developmental toxicant but does reversibly impair male fertility. Prolonged, repeated exposure to COS is likely to present similar neurotoxicity hazards to that of the structurally and toxicologically related compound carbon disulfide. Although the occupational risks presented by COS as a fumigant of bulk grain are significant, these are, as they have been for a considerable time for phosphine and methyl bromide, manageable by good occupational safety practices. Consideration may need to be given to scrubbing of ventilated COS and its breakdown product hydrogen sulfide, at the completion of fumigation to minimise worker and bystander exposure. In terms of classical regulatory toxicology studies, the available database for COS is deficient in many aspects and registration in most jurisdictions will depend on sound scientific argument built upon the totality of the existing scientific data as there are strong arguments supporting the registration of this compound.     

The mutagenic hazards of environmental PM2.5 in Turin

Owing to the large number of natural and anthropogenic sources, particulate matter (PM) may present several physical and chemical patterns in different areas. The finer PM2.5 fraction, which is now widely but not routinely measured in Europe, is considered to be the alveolar fraction of the ambient particles. Annual and winter mean concentrations of PM2.5 substantially vary in Europe, with higher concentrations in the South. The aims of this work were to (a) measure the PM2.5 levels in Turin over a long period, (b) evaluate mutagenic activities of organic extracts containing this collected complex mixture using the Ames test and (c) determine the level of polycyclic aromatic hydrocarbons (PAHs) in order to identify important mutagens in ambient air. Sampling was carried out from November 2001 to December 2004. The monthly mean of PM2.5 was 48.76+/-24.12 microg/m3. From the beginning to the end of the sample period there was a decrease in gravimetric levels, with annual means of 54.10+/-29.77 microg/m3 in 2002; 42.48+/-15.73 microg/m3 in 2003 and 45.89+/-24.92 microg/m3 in 2004. Samples were tested for mutagenicity using Salmonella typhimurium strains TA98 and TA100, with and without S9 mix metabolic activation. A positive genotoxic response was observed for TA98, with and without metabolic activation. The measured PAHs monthly mean level was 8.24+/-6.30 ng/m3, with values ranging from 0.20 to 21.38 ng/m3 Seasonal variation of gravimetric, mutagenic and PAH values was significant. The Salmonella assay results statistically correlated to PM2.5 and PAHs levels, but sometimes the mutagenic potencies were rather different despite an equal concentration of pollutant. The results confirm the usefulness of this biological approach to detect genotoxic properties of sampled PM2.5 and they show the variability of the mutagenic properties of the airborne mixture over time.     

Ⅰ号软胶囊遗传毒性实验研究

目的：采用生物检测技术评价Ⅰ号软胶囊是否有急性经口毒性及遗传毒性,为其应用安全性提供依据。方法依据《保健食品安全性毒理学评价程序和检验方法规范》2003年版,进行了小鼠最大耐受量（ MTD）的测定、小鼠骨髓嗜多染红细胞微核试验及小鼠精子畸形试验。结果小鼠最大耐受量（MTD）大于74.08g· kg －1,相当于临床成人用量的1111.2倍;灌胃给予该受试物2.5g· kg －1-10.g· kg －1对小鼠体细胞及雄性生殖细胞无诱变性。结论在本受试剂量范围内Ⅰ号软胶囊无毒性,也未发现有遗传毒性。     

Ochratoxin A-induced cytotoxicity,genotoxicity and reactive oxygen species in kidney cells: An integrative approach of complementary endpoints

Ochratoxin A (OTA) is a well-known nephrotoxic and potential carcinogenic agent but no consensus about the molecular mechanisms underlying its deleterious effects has been reached yet. The aim of this study is to integrate several endpoints concerning OTA-induced toxicological effects in Vero kidney cells in order to obtain additional mechanistic data, especially regarding the influence of reactive oxygen species (ROS). One innovative aspect of this work is the use of the superoxide dismutase mimic (SODm) MnTnHex-2-PyP as a mechanistic tool to clarify the involvement of oxidative stress in OTA toxicity. The results showed concentration and time-dependent cytotoxic effects of OTA (crystal violet, neutral red and LDH leakage assays). While the SODm mildly increased cell viability, trolox and ascorbic acid had no effect with regards to this endpoint. OTA induced micronuclei formation. Using the FPG modified comet assay, OTA modestly increased the % of DNA in tail, revealing the presence of oxidative DNA lesions. This mycotoxin increased apoptosis, which was attenuated by SODm. In addition, the SODm decreased the ROS accumulation observed in DHE assay. Taken together, our data suggest that ROS partially contribute to the cytotoxicity and genotoxicity of OTA, although other mechanisms may be relevant in OTA-induced deleterious effects.