In Vitro Study of Mutagenesis Induced by Crocidolite-Exposed Alveolar Macrophages NR8383 in Cocultured Big Blue Rat2 Embryonic Fibroblasts.

Asbestos-induced mutagenicity in the lung may involve reactive oxygen/nitrogen species (ROS/RNS) released by alveolar macrophages. With the aim of proposing an alternative in vitro mutagenesis test, a coculture system of rat alveolar macrophages (NR8383) and transgenic Big Blue Rat2 embryonic fibroblasts was developed and tested with a crocidolite sample. Crocidolite exposure induced no detectable increase in ROS production from NR8383, contrasting with the oxidative burst that occurred following a brief exposure (1 hour) to zymosan, a known macrophage activator. In separated cocultures, crocidolite and zymosan induced different changes in the gene expressions involved in cellular inflammation in NR8383 and Big Blue. In particular, both particles induced up-regulation of iNOS expression in Big Blue, suggesting the formation of potentially genotoxic nitrogen species. However, crocidolite exposure in separated or mixed cocultures induced no mutagenic effects whereas an increase in Big Blue mutants was detected after exposure to zymosan in mixed cocultures. NR8383 activation by crocidolite is probably insufficient to induce in vitro mutagenic events. The mutagenesis assay based on the coculture of NR8383 and Big Blue cannot be used as an alternative in vitro method to assess the mutagenic properties of asbestos fibres.

Bitumen fume-induced gene expression profile in rat lung.

Exposure to bitumen fumes during paving and roofing activities may represent an occupational health risk. To date, most of the studies performed on the biological effect of asphalt fumes have been done with regard to their content in carcinogenic polycyclic aromatic hydrocarbons (PAH). In order to gain an additional insight into the mechanisms of action of bitumen fumes, we studied their pulmonary effects in rodents following inhalation using the microarray technology. Fisher 344 rats were exposed for 5 days, 6 h/day to bitumen fumes generated at road paving temperature (170 degrees C) using a nose-only exposition device. With the intention of studying the early transcriptional events induced by asphalt fumes, lung tissues were collected immediately following exposure and gene expression profiles in control and exposed rats were determined by using oligonucleotide microarrays. Data analysis revealed that genes involved in lung inflammatory response as well as genes associated with PAH metabolization and detoxification were highly expressed in bitumen-exposed animals. In addition, the expression of genes related to elastase activity and its inhibition which are associated with emphysema was also modulated. More interestingly genes coding for monoamine oxidases A and B involved in the metabolism of neurotransmitters and xenobiotics were downregulated in exposed rats. Altogether, these data give additional information concerning the bitumen fumes biological effects and would allow to better review the health effects of occupational asphalt fumes exposure.

Genotoxic effects of bitumen fumes in Big Blue transgenic rat lung.

Road paving workers are exposed to bitumen fumes (CAS No. 8052-42-4), a complex mixture of volatile compounds and particles containing carcinogenic and non-carcinogenic polycyclic aromatic hydrocarbons. However, epidemiological and experimental animal studies failed to draw unambiguous conclusions concerning their toxicity. In order to gain better insights on their genotoxic potential, we used an experimental design able to generate bitumen fumes at road paving temperature (temperature: 170 degrees C, total particulate matter: 100mg/m3) and perform a nose-only exposure of Big Blue transgenic rodents 6h/day for five consecutive days. The mutagenic properties of bitumen fumes were determined by analyzing the mutation frequency and spectrum of the neutral reporter gene cII inserted into the rodent genome. We previously observed in mouse lung, that bitumen fumes did not induce an increase of cII mutants, a modification of the mutation spectrum, nor the formation of DNA adducts. Since DNA adducts were found in the lungs of rats exposed to asphalt fumes in similar conditions, we decided to carry out an analogous experiment with Big Blue rats. A DNA adduct was detected 3 and 30 days after the end of treatment suggesting that these genetic alterations were quite steady. Thirty days after exposure, the cII mutant frequency was similar in control and exposed rats. In addition, a slight but not significant modification of the mutation spectrum associated with an increase of G:C to T:A and A:T to C:G transversions was noticeable in the treated animals. Then, these data failed to demonstrate a pulmonary mutagenic potential for bitumen fumes generated at road paving temperature in our experimental conditions despite the presence of a DNA adduct. These results may provide information concerning the pulmonary mechanism of action of this aerosol and may contribute to the occupational health hazard assessment.

Microdissection, mRNA amplification and microarray: a study of pleural mesothelial and malignant mesothelioma cells.

The studies of molecular alterations in tumor cells with microarrays are often hampered by inherent tissue heterogeneity. The emergence of Laser Capture Microdissection (LCM) allowed us to overcome this challenge since it gives selective access to cancer cells that are isolated from their native tissue environment. In this report, we microdissected mesothelial cells and malignant mesothelioma cells of ex vivo resected specimens using LCM. Amplified RNA from mesothelial and mesothelioma microdissected cells allowed us to measure global gene expression with 10 K-microarrays in four independent experiments. We screened 9850 annotated human genes, 1275 of which have satisfied our data analysis requirements. They included 302 overexpressed genes and 160 downregulated genes in mesothelioma microdissected cells as compared to mesothelial microdissected cells. Among them, the expression levels of eight genes, namely BF, FTL, IGFBP7, RARRES1, RARRES2, RBP1, SAT, and TXN according to HUGO nomenclature, were increased, whereas six: ALOX5AP, CLNS1A, EIF4A2, ELK3, REQ and SYPL, were found to be underexpressed in mesothelioma microdissected cells. The ferritin light polypeptide (FTL) gene overexpression was confirmed by real time quantitative PCR. Our approach allowed a comprehensive in situ examination of mesothelioma and provided an accurate way to find new marker genes that may be useful for diagnosis and treatment of malignant pleural mesothelioma.

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.