Catherine Coulais

Lack of genotoxicity of bitumen fumes in transgenic mouse lung

During hot application of bitumen containing materials, e.g. in hot paving or roofing, fumes are emitted that contain polycyclic aromatic compounds. Previous studies with rodents exposed to bitumen and coal-tar fume condensates showed formation of DNA adducts. In order to clarify the genotoxicity of bitumen fumes, we designed a study by using mice carrying a reporter gene for mutagenesis analysis and exposed by nose-only to a constant and reproducible aerosol of bitumen fumes. We analyzed the genotoxic activity of inhaled bitumen fumes generated under those controlled conditions through the induction of mutation and DNA adducts in Big Blue mice. Mice were exposed to bitumen fumes (100 mg/m(3) total particulate matter) 6 h per day during 5 days by nose-only in an inhalation chamber designed in our laboratory. Following a 30-day fixation period, the experiment was terminated and lung DNA was extracted for mutant frequency and adduct determinations. The mutant frequency was determined using the cII and the lacI mutant analysis systems. In, addition, 61 and 54 mutants were sequenced in control and exposed groups, respectively. The study did not show any mutation or adduct induction in the exposed group compared to the control group: cII mutant frequencies were 11.0+/-4.5x10(-5) and 11.0+/-4.8x10(-5) in control and exposed lungs, respectively. Identically, using the lacI mutation detection system, the mutant frequencies were 6.4+/-3.1x10(-5) and 5.8+/-2.0x10(-5). The mutation spectra of both series were quite similar with regard to transition and transversion frequencies. The absence of genotoxicity in the group exposed to 100 mg/m(3) bitumen is discussed with regard to dosage of inhaled polycyclic aromatic compounds and species.

Genotoxicity of 3-methylcholanthrene in liver of transgenic big Blue mice.

Transgenic mice provide a unique tool for studying the tissue specificity and mutagenic potential of chemicals. Because 3‐methylcholanthrene (3MC) was found mutagenic in bacteria, clastogenic in bone marrow, and induces DNA adducts in animals, we were interested to determinine whether this xenobiotic provokes (1) cell proliferation, (2) transcriptional activity changes, (3) DNA adducts, and (4) hepatic mutations in transgenic Big Blue® mice carrying the λLIZ phage shuttle vector. Big Blue C57/Bl male mice were treated with a single intraperitoneal dose of 80 mg/kg 3MC for 1, 3, 6, 14, or 30 days. Cell proliferation was checked by 5‐bromo‐2‐deoxyuridine labeling and immunohistochemical detection. The maximal increase of the mitotic index was evidenced after 3 days (2.9 times the control value; P < 0.01). The relative nucleus area, reflecting the transcriptional activity, was also the highest in the treated group after 3 days: 1.86 times the control value, on average (P < 0.01). Four major DNA adducts, determined according to the [32P]‐postlabeling method, were evidenced in liver DNA of treated mice, 6 days after the treatment: the spot intensities increased in a time‐dependent manner. The mutant frequency of liver DNA was the highest after 14 days: 20.3 ± 2.9 × 10−5 in the treated vs. 7.6 ± 2.7 × 10−5 in the control mice (P < 0.01). Sequencing of the λ lacI mutant plaques showed mainly G:C → T:A and C:G → A:T transversions. In conclusion, 3MC at first induced nuclear enlargement and a slight increase of cell proliferation in liver, followed by parallel formation of DNA adducts and mutations. This study shows how transgenic models allow in vivo evaluation of mechanistically simultaneous endpoints. Environ. Mol. Mutagen. 36:266–273, 2000

Short-term crocidolite inhalation studies in mice: validation of an inhalation chamber

A nose-only inhalation chamber is described: this chamber being computer automated has been particularly designed for mice on which it was validated using a crocidolite aerosol at a nominal concentration of 13.6 mg/m3, 6 h/day during 5 days. A month later the mice showed typical inflammatory bronchoalveolar liquids with many polynucleated or activated macrophages and asbestos bodies. The burden of crocidolite fibers ranged from 345,000 to 1,300,000 fibers per mg of dried lung. This study demonstrates that during the month that followed a short-term mice exposure to crocidolite fibers, the inflammatory response was still persistent. These toxicological endpoints validate the nose-only inhalation chamber to be useful for common or transgenic mice.

In Vitro Cytotoxicity and Transforming Potential of Industrial Carbon Dust (Fibers and Particles) in Syrian Hamster Embryo (SHE) Cells

Carbon fibers have many applications, mainly in high-tech industries such as the aviation industry. Eleven carbon samples (fibers and particles) coming from an aeronautic group were tested for their cytotoxicity and carcinogenic potential using in vitro short-term assays in Syrian hamster embryo cells. These samples were taken during each important step of the process, i.e. from the initial heating of polyacrylonitrile fibers to pure carbon fibers. They were compared to an asbestos fiber, an amorphous silica, and two commercial graphite powders. Their physical-chemical characteristics and their capacity to release reactive oxygen species (ROS) were determined. This study showed that none of the carbon samples was able to generate ROS as measured by Electron Paramagnetic Resonance analysis, and in our biological assays, they demonstrated no morphological transformation potential and low cytotoxicity compared to positive control (chrysotile asbestos).

Evaluation of non-radioactive labelling and detection of deoxyribonucleic acids: Part one: chemiluminescent methods

The growth of analytical methods for the detection of nucleic acid from various biological samples reflects recent advances in biotechnology development especially in the areas of genetic, infections and cancer diagnosis. The target DNA is detected by hybridization techniques derived from Southerns blotting. However such assays, based on the use of 32P labelled DNA probes, bring with them the associated problems of handling radioactive materials. In order to overcome these difficulties, a number of chemiluminescent detection methods have recently been developed. These new, alternative probe labelling procedures and chemiluminescent detection methods are easy to use in routine assays performed in research laboratories as well as for medical applications, and can reach the level of sensitivity found in classical radiolabelling techniques. The techniques investigated include peroxydase, biotin 16-dUTP or digoxigenin 11-dUTP probe labelling. The target DNAs are transferred onto nitrocellulose or nylon membranes and further fixed by heat or UV crosslinking. Specific hybridization on the target DNA is finally revealed by the use of chemiluminescent substrates. For all these techniques the detection limit is 10 aM (attomol) of a 561 bp target DNA. However for the probes labelled with peroxydase and with digoxigenin the detection limit drops to 1.0 aM of the target DNA. In the present paper we shall compare several of these DNA labelling and detection procedures and show that the detection threshold can vary by as much as a factor of 20 from method to method. This is the first time that various chemiluminescent methods for label and detection of DNA are compared and evaluated in order to determine the best protocol.

Generation of Actinomycetes Aerosols Containing Spores and Mycelium: Performances of a Liquid Bubbling Aerosolizer

The presence of actinomycetes in many workplaces and their role in the incidence of various respiratory symptoms remains poorly understood and underestimated. A sampling and culture-independent analysis method to measure airborne actinomycetes has yet to be developed and controlled bioaerosols are needed for laboratory investigations. In this article, the performances of a single-pass bubbling aerosolizer were characterized to evaluate the feasibility of generating actinomycetes from a liquid source and to confirm that viability of aerosolized entities was preserved. Six preparation protocols for liquid Thermoactinomycetes vulgaris cultures were compared in terms of culturable flora and total spores concentrations (culture and epifluorescence microscopy) and size distributions (optical counter and cascade impactor) of the bioaerosols generated. Using the best protocol, the generators performances were then validated using three species: Thermoactinomyces vulgaris, Thermobifida fusca, and Streptomyces californicus. Bioaerosols contained a mixture of spores and mycelium and their properties were stable throughout generation (120 min) and were satisfactorily reproducible between runs. Depending on the species generated, the culturable concentrations measured were between 104 and 108 CFU.m−3, with corresponding total spore concentrations between 105 and 109 Spores.m−3. These concentrations cover the ranges measured in the workplace. The generators flexibility should make it possible to produce bioaerosols with other actinomycetes species, and use them in laboratory trials with various objectives and constraints. Copyright 2013 American Association for Aerosol Research

Evaluation of non-radioactive labelling and detection of deoxyribonucleic acids: Part two: colorigenic methods and comparison with chemiluminescent methods

The diagnosis of genetic infections and cancerous diseases is carried out more and more often at a molecular level using Southerns technique which is based on the use of 32P-labelled DNA. In order to circumvent the risks and rapid decrease in radioactivity associated with these latter techniques, new colorigenic methods have been developed. In this work, we describe the use of dTTP analogues (digoxigenin-dUTP and biotin-dUTP) for the labelling of probes and detection of target DNA. Using digoxigenin-11-dUTP, 0.1 aM of a 561 bp target DNA was detected by using a modified Southern procedure. The reliability and the high sensitivity of such methods make them a good tool for DNA investigation in research as well as in testing laboratories.