Bertrand Rihn

Selective inhibition of NF-κB activation by the flavonoid hepatoprotector silymarin in HepG2: Evidence for different activating pathways

The bioflavonoid silymarin is found to potently suppress both nuclear factor kappa‐B (NF‐κB)‐DNA binding activity and its dependent gene expression induced by okadaic acid in the hepatoma cell line HepG2. Surprisingly, tumor necrosis factor‐α‐induced NF‐κB activation was not affected by silymarin, thus demonstrating a pathway‐dependent inhibition by silymarin. Many genes encoding the proteins of the hepatic acute phase response are under the control of the transcription factor NF‐κB, a key regulator in the inflammatory and immune reactions. Thus, the inhibitory effect of silymarin on NF‐κB activation could be involved in its hepatoprotective property.

Differential gene expression in mesothelioma.

To investigate the molecular events controlling malignant transformation of human pleural cells, we compared constitutive gene expression of mesothelioma cells to that of pleural cells. Using cDNA microarray and high‐density filter array, we assessed expression levels of >6500 genes. Most of the highly expressed transcripts were common to both cell lines and included genes associated with stress response and DNA repair, outcomes consistent with the radio‐ and chemo‐resistance of mesothelioma. Interestingly, of the fewer than 300 genes that differed between cell lines, most functioned in (i) macromolecule stability, (ii) cell adhesion and recognition, (iii) cell migration (invasiveness), and (iv) extended cell division. Expression levels of several of these genes were confirmed by RT‐PCR and could be useful as diagnostic markers of human mesothelioma.

DNA-adducts in subjects exposed to urban air pollution by benzene and polycyclic aromatic hydrocarbons (PAHs) in Cotonou, Benin

Air pollution effect on humans represents a major public health problem. Exposure to genotoxic compounds in the ambient air is evaluated using different biomarkers. In the present study we assessed DNA‐adducts levels in apparently healthy people living and working in the city of Cotonou (Benin) in which exposure to air pollutants such as benzene and polycyclic aromatic hydrocarbons (PAHs) mainly benzo(a)pyrene has been evidenced. Rural inhabitants were enrolled as control group. Taxi‐motorbike drivers, street food vendors, and gasoline salesmen were recruited in Cotonou whereas suburban residents were recruited in Godomey, 12 km from Cotonou. We found that taxi‐motorbike drivers, roadside residents, street vendors, taxi‐motor‐bike drivers and gasoline sellers had significantly higher levels of DNA‐adducts than suburban and village inhabitants (P < 0.001; post hoc, LSD). Means values were 24.6 ± 6.4, 23.78 ± 6.9, 34.7 ± 9.8, and 37.2 ± 8.1 in the exposed groups versus 2.1 ± 0.6 and 3.1 ± 0.8 adducts/108 nucleotides, in the two control groups, respectively. We did not find any significant difference within the high exposuregroups and inside low exposure subgroups (namely suburban residents and villagers) because the mean individual exposure values to both PAHs and benzene were similar among subjects exposed in the city of Cotonou and those in suburban and village areas. However, there is significant interindividual variations in adducts levels that may reflect variation of genetic susceptibility factors. Ranges of adduct level/108 nucleotides were: 1–69, 1–76, 3–169, 4–124, 0–9, 0–8 adducts/108 for taxi‐motorbike drivers, roadside residents, street vendors, gasoline sellers, suburban and village inhabitants, respectively. Our study demonstrated a clear‐cut elevated level of DNA adducts in city residents than in none exposed people (or very low exposure levels people) and designate these city residents groups as people at risks for the chronic diseases possibly caused by benzene and PAHs.

Comparative study of immunological properties and cytotoxic effects of Clostridium difficile toxin B and Clostridium sordellii toxin L.

We compared the immunological properties and cytotoxic effects of Clostridium difficile toxin B and Clostridium sordellii toxin L. These two cytotoxins are immunologically related in that the cytotoxic effect of either toxin can be neutralized by the polyclonal antiserum prepared against either cytotoxin. On the other hand, polyclonal antiserum prepared against Clostridium difficile enterotoxin A did not cross-react with the cytotoxins B and L when tested by cytotoxic neutralization test nor by double immunodiffusion assay. However, despite this immunological relationship between toxins B and L, the morphological modifications observed in MacCoy cells induced by treatment with these cytotoxins are clearly distinct. We describe the first quantitative analysis of specific cellular parameters which illustrates the morphological differences induced by these cytotoxins. Moreover, immunocytochemical experiments show that, whereas disruption of microfilaments is observed with toxin B- and L-treatments, alterations of F-actin network are different in the cells treated with toxin B or L. The observation that the cellular modifications induced by toxin B- and toxin L-treatment differ suggests that the molecular mechanisms involved in the respective cytotoxicities are also likely to be different.

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

Clostridium difficile toxin B: characterization and sequence of three peptides.

The cytotoxin, also named toxin B, was isolated from a toxigenic strain of Clostridium difficile, purified to homogeneity and partially characterized. The purification procedure included ultrafiltration followed by anion-exchange chromatography. We noticed that a non-specific nucleic material eluted with the protein during the purification. The presence of these nucleic acids appeared to be important for the toxic activity of the protein. Some characteristics of the cytotoxin were examined, especially the amino acid composition and the sequence of three tryptic fragments.

Chitosan nanoparticles and quercetin modulate gene expression and prevent the genotoxicity of aflatoxin B1 in rat liver

The aims of the current study were to prepare chitosan nanoparticles (CNPs) and to evaluate its protective role alone or in combination with quercetin (Q) against AFB1-induce cytotoxicity in rats. Male Sprague-Dawley rats were divided into 12 groups and treated orally for 4 weeks as follow: the control group, the group treated with AFB1 (80 μg/kg b.w.) in corn oil, the groups treated with low (140 mg/kg b.w.) or high (280 mg/kg b.w.) dose of CNPs, the group treated with Q (50 mg/kg b.w.), the groups treated with Q plus the low or the high dose of CNPs and the groups treated with AFB1 plus Q and/or CNPs at the two tested doses. The results also revealed that administration of AFB1 resulted in a significant increase in serum cytokines, Procollagen III, Nitric Oxide, lipid peroxidation and DNA fragmentation accompanied with a significant decrease in GPx I and Cu–Zn SOD-mRNA gene expression. Q and/or CNPs at the two tested doses overcome these effects especially in the group treated with the high dose of CNPs plus Q. It could be concluded that CNPs is a promise candidate as drug delivery enhances the protective effect of Q against the cytogenetic effects of AFB1 in high endemic areas.

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.

Human monocyte response to S-nitrosoglutathione-loaded nanoparticles: uptake, viability, and transcriptome.

S-Nitrosoglutathione (GSNO) is a good candidate for nitric oxide (NO(•)) delivery, and its nanoformulation improves NO(•) stability and bioavailability. We have compared the effect of empty Eudragit nanoparticles (eENP), GSNO-loaded ENP (gENP), and free GSNO on THP-1 human monocytic cell line. We investigated cellular viability and growth by WST-1 and trypan blue tests. ENP uptake was studied using transmission electron microscopy, confocal microscopy, and flow cytometry. Transcriptomic profiles were obtained using microarray. ENP entered cells by clathrin- and caveolae-mediated endocytosis. Exposure to either free GSNO or gENP induced an activation of genes from the same clusters, in favor of intracellular delivery of GSNO by ENP. GSNO nanoformulation might be a therapeutic option for NO(•) delivery.