Antifungal climbazole alters androgenic pathways in mammalian cells.

Among antifungal agents used in pharmaceuticals and personal care products, the synthetic azole climbazole (CBZ; 1-(4-Chlorophenoxy)-1-(imidazol-1-yl)-3,3-dimethylbutan-2-one) acts on the fungus Malassezia. Despite concerns surrounding its effects on health, based on alterations to reproduction and steroidogenesis found in fish, little is known about its mechanism of action as an endocrine disrupting chemical (EDC) in mammalian cells. In this study, using OECD test guidelines, we investigated the effects of CBZ (i) in H295R cells, on the production of estradiol and testosterone, as well as intermediate metabolites in steroidogenesis pathway, and (ii) in HeLa9903 and AR-EcoScreen cell lines, on the transactivation of estrogen and androgen receptors. Our results are the first evidence in H295R cells, that CBZ treatment (from 0.3 µM) decreased secreted levels of testosterone and estradiol. This was associated with reduced 17α-hydroxypregnenolone and 17α-hydroxyprogesterone levels. The altered levels of these metabolites were associated with a decrease in cytochrome P450 17α-hydroxylase/17,20-lyase (Cyp17A1) activity without any effect on its protein level. CBZ was also found to exert antagonistic effects toward androgen and estrogen α receptors. These results give insights into the toxicological mechanism of action of CBZ. Many azoles share structural similarities; therefore, caution should be adopted due to their potential toxicity.

Hazard characterization of Alternaria toxins to identify data gaps and improve risk assessment for human health.

Fungi of the genus Alternaria are ubiquitous plant pathogens and saprophytes which are able to grow under varying temperature and moisture conditions as well as on a large range of substrates. A spectrum of structurally diverse secondary metabolites with toxic potential has been identified, but occurrence and relative proportion of the different metabolites in complex mixtures depend on strain, substrate, and growth conditions. This review compiles the available knowledge on hazard identification and characterization of Alternaria toxins. Alternariol (AOH), its monomethylether AME and the perylene quinones altertoxin I (ATX-I), ATX-II, ATX-III, alterperylenol (ALP), and stemphyltoxin III (STTX-III) showed in vitro genotoxic and mutagenic properties. Of all identified Alternaria toxins, the epoxide-bearing analogs ATX-II, ATX-III, and STTX-III show the highest cytotoxic, genotoxic, and mutagenic potential in vitro. Under hormone-sensitive conditions, AOH and AME act as moderate xenoestrogens, but in silico modeling predicts further Alternaria toxins as potential estrogenic factors. Recent studies indicate also an immunosuppressive role of AOH and ATX-II; however, no data are available for the majority of Alternaria toxins. Overall, hazard characterization of Alternaria toxins focused, so far, primarily on the commercially available dibenzo-α-pyrones AOH and AME and tenuazonic acid (TeA). Limited data sets are available for altersetin (ALS), altenuene (ALT), and tentoxin (TEN). The occurrence and toxicological relevance of perylene quinone-based Alternaria toxins still remain to be fully elucidated. We identified data gaps on hazard identification and characterization crucial to improve risk assessment of Alternaria mycotoxins for consumers and occupationally exposed workers.

Can a 12-gene expression signature predict the cell transforming potential of tumor promoting agents in Bhas 42 cells?

To date, long-term rodent carcinogenesis assays are the only assays recognized by regulators to assess non-genotoxic carcinogens, but their reliability has been questioned. In vitro cell transformation assays (CTAs) could represent an interesting alternative to animal models as it has the advantage of detecting both genotoxic and non-genotoxic transforming chemicals. Among them, Bhas 42 CTA uses a cell line that has been transfected with the oncogenic sequence v-Ha-ras. This sequence confers an "initiated" status to these cells and makes them particularly sensitive to non-genotoxic agents. In a previous work, transcriptomic analysis revealed that the treatment of Bhas 42 cells with transforming silica (nano)particles and 12-O-tetradecanoylphorbol-13-acetate (TPA) commonly modified the expression of 12 genes involved in cell proliferation and adhesion. In the present study, we assess whether this signature would be the same for four other soluble transforming agents, i.e. mezerein, methylarsonic acid, cholic acid and quercetin. The treatment of Bhas 42 cells for 48 h with mezerein modified the expression of the 12 genes of the signature according to the same profile as that of the TPA. However, methylarsonic acid and cholic acid gave an incomplete signature with changes in the expression of only 7 and 5 genes, respectively. Finally, quercetin treatment induced no change in the expression of all genes but exhibited higher cytotoxicty. These results suggest that among the transforming agents tested, some may share similar mechanisms of action leading to cell transformation while others may activate different additional pathways involved in such cellular process. More transforming and non-transforming agents and gene markers should be tested in order to try to identify a relevant gene signature to predict the transforming potential of non-genotoxic agents.

Identification of a Gene Signature Predicting (Nano)Particle-Induced Adverse Lung Outcome in Rats.

Nanoparticles are extensively used in industrial products or as food additives. However, despite their contribution to improving our quality of life, concerns have been raised regarding their potential impact on occupational and public health. To speed up research assessing nanoparticle-related hazards, this study was undertaken to identify early markers of harmful effects on the lungs. Female Sprague Dawley rats were either exposed to crystalline silica DQ-12 with instillation, or to titanium dioxide P25, carbon black Printex-90, or multi-walled carbon nanotube Mitsui-7 with nose-only inhalation. Tissues were collected at three post-exposure time points to assess short- and long-term effects. All particles induced lung inflammation. Histopathological and biochemical analyses revealed phospholipid accumulation, lipoproteinosis, and interstitial thickening with collagen deposition after exposure to DQ-12. Exposure to the highest dose of Printex-90 and Mitsui-7, but not P25, induced some phospholipid accumulation. Comparable histopathological changes were observed following exposure to P25, Printex-90, and Mitsui-7. Comparison of overall gene expression profiles identified 15 potential early markers of adverse lung outcomes induced by spherical particles. With Mitsui-7, a distinct gene expression signature was observed, suggesting that carbon nanotubes trigger different toxicity mechanisms to spherical particles.

Needlelike, short and thin multi-walled carbon nanotubes: comparison of effects on wild type and p53+/- rat lungs.

Carbon nanotubes (CNTs) are nanomaterials presenting an occupational inhalation risk during production or handling. The International Agency for Research on Cancer classified one CNT, Mitsui-7 (MWNT-7), as 'possibly carcinogenic to humans'. In recognition of their similarities, a proposal has been submitted to the risk assessment committee of ECHA to classify all fibers with 'Fibre Paradigm' (FP)-compatible dimensions as carcinogenic. However, there is a lack of clarity surrounding the toxicity of fibers that do not fit the FP criteria. In this study, we compared the effects of the FP-compatible Mitsui-7, to those of NM-403, a CNT that is too short and thin to fit the paradigm. Female Sprague Dawley rats deficient for p53 (GMO) and wild type (WT) rats were exposed to the two CNTs (0.25 mg/rat/week) by intratracheal instillation. Animals (GMO and WT) were exposed weekly for four consecutive weeks and were sacrificed 3 days or 8 months after the last instillation. Exposure to both CNTs induced acute lung inflammation. However, persistent inflammation at 8 months was only observed in the lungs of rats exposed to NM-403. In addition to the persistent inflammation, NM-403 stimulated hyperplasic changes in rat lungs, and no adenomas or carcinomas were detected. The degree and extent of hyperplasia was significantly more pronounced in GMO rats. These results suggest that CNT not meeting the FP criteria can cause persistent inflammation and hyperplasia. Consequently, their health effects should be carefully assessed.

Retained particle surface area dose drives inflammation in rat lungs following acute, subacute, and subchronic inhalation of nanomaterials.

BACKGROUND: An important aspect of nanomaterial (NM) risk assessment is establishing relationships between physicochemical properties and key events governing the toxicological pathway leading to adverse outcomes. The difficulty of NM grouping can be simplified if the most toxicologically relevant dose metric is used to assess the toxicological dose-response. Here, we thoroughly investigated the relationship between acute and chronic inflammation (based on polymorphonuclear neutrophil influx (% PMN) in lung bronchoalveolar lavage) and the retained surface area in the lung. Inhalation studies were performed in rats with three classes of NMs: titanium dioxides (TiO2) and carbon blacks (CB) as poorly soluble particles of low toxicity (PSLT), and multiwall carbon nanotubes (MWCNTs). We compared our results to published data from nearly 30 rigorously selected articles. RESULTS: This analysis combined data specially generated for this work on three benchmark materials - TiO2 P25, the CB Printex-90 and the MWCNT MWNT-7 - following subacute (4-week) inhalation with published data relating to acute (1-week) to subchronic (13-week) inhalation exposure to the classes of NMs considered. Short and long post-exposure recovery times (immediately after exposure up to more than 6 months) allowed us to examine both acute and chronic inflammation. A dose-response relationship across short-term and long-term studies was revealed linking pulmonary retained surface area dose (measured or estimated) and % PMN. This relationship takes the form of sigmoid curves, and is independent of the post-exposure time. Curve fitting equations depended on the class of NM considered, and sometimes on the duration of exposure. Based on retained surface area, long and thick MWCNTs (few hundred nm long with an aspect ratio greater than 25) had a higher inflammatory potency with 5 cm2/g lung sufficient to trigger an inflammatory response (at 6% PMN), whereas retained surfaces greater than 150 cm2/g lung were required for PSLT. CONCLUSIONS: Retained surface area is a useful metric for hazard grouping purposes. This metric would apply to both micrometric and nanometric materials, and could obviate the need for direct measurement in the lung. Indeed, it could alternatively be estimated from dosimetry models using the aerosol parameters (rigorously determined following a well-defined aerosol characterization strategy).

Continuous Long-Term Exposure to Low Concentrations of MWCNTs Induces an Epithelial-Mesenchymal Transition in BEAS-2B Cells.

In the field of nanotechnology, the use of multi-walled carbon nanotubes (MWCNTs) is growing. Pulmonary exposure during their production, use, and handling is raising concerns about their potential adverse health effects. The purpose of this study is to assess how the physical characteristics of MWCNTs, such as diameter and/or length, can play a role in cellular toxicity. Our experimental design is based on the treatment of human bronchial epithelial cells (BEAS-2B) for six weeks with low concentrations (0.125-1 µg/cm2) of MWCNTs having opposite characteristics: NM-403 and Mitsui-7. Following treatment with both MWCNTs, we observed an increase in mitotic abnormalities and micronucleus-positive cells. The cytotoxic effect was delayed in cells treated with NM-403 compared to Mitsui-7. After 4-6 weeks of treatment, a clear cellular morphological change from epithelial to fibroblast-like phenotype was noted, together with a change in the cell population composition. BEAS-2B cells underwent a conversion from the epithelial to mesenchymal state as we observed a decrease in the epithelial marker E-cadherin and an increased expression of mesenchymal markers N-cadherin, Vimentin, and Fibronectin. After four weeks of recovery, we showed that the induced epithelial-mesenchymal transition is reversible, and that the degree of reversibility depends on the MWCNT.

Exposure to TiO2 Nanostructured Aerosol Induces Specific Gene Expression Profile Modifications in the Lungs of Young and Elderly Rats.

Although aging is associated with a higher risk of developing respiratory pathologies, very few studies have assessed the impact of age on the adverse effects of inhaled nanoparticles. Using conventional and transcriptomic approaches, this study aimed to compare in young (12-13-week-old) and elderly (19-month-old) fisher F344 rats the pulmonary toxicity of an inhaled nanostructured aerosol of titanium dioxide (TiO2). Animals were nose-only exposed to this aerosol at a concentration of 10 mg/m3 for 6 h per day, 5 days per week for 4 weeks. Tissues were collected immediately (D0), and 28 days after exposure (D28). A pulmonary influx of neutrophilic granulocytes was observed in exposed rats at D0, but diminished with time while remaining significant until D28. Similarly, an increased expression of several genes involved in inflammation at the two post-exposure time-points was seen. Apart from an age-specific pulmonary influx of lymphocyte, only slight differences in physio-pathological responses following TiO2 exposure between young and elderly animals were noticed. Conversely, marked age-related differences in gene expression profiles were observed making possible to establish lists of genes specific to each age group and post-exposure times. These results highlight different signaling pathways that were disrupted in rats according to their age.

Inhaled multi-walled carbon nanotubes differently modulate global gene and protein expression in rat lungs.

Inhalation of multi-walled carbon nanotubes (MWCNTs) induces lung inflammation. Depending on industrial applications, CNTs with different physicochemical characteristics are produced and workers can potentially be exposed. This raises concerns about the long-term health effects of these nanomaterials. Because of the wide variety of MWCNTs, it is essential to study the toxicological effects of CNTs of various shapes and to better understand the impact physical and chemical properties have on their toxicity. In this study, rats were exposed by nose-only to two pristine MWCNTs with different morphologies: the long and thick NM-401 or the short and thin NM-403. After four weeks of inhalation, animals were euthanized at four different times during the recovery period: three days (short-term), 30 and 90 days (intermediate-term) and 180 days (long-term). Analyses of the transcriptome in the whole lung and the proteome in the bronchoalveolar lavage fluid of exposed animals were performed to understand the MWCNT underlying mechanisms of toxicity. Following inhalation of NM-401, we observed a dose-dependent increase in the number of differentially expressed genes and proteins, whereas there is no clear difference between the two concentrations of NM-403. After NM-403 inhalation, the number of differentially expressed genes and proteins varied less between the four post-exposure times compared to NM-401, which supports the postulation of a persistent effect of this type of CNT. Our toxicogenomics approaches give insights into the different toxicological profile following MWCNT exposure.

Quantitative measurement of carbon nanotubes in rat lung.

Despite their numerous possible applications, the potential impact of carbon engineered nanomaterials (CEN) on human health, especially after inhalation exposure, is still questioned. Quantification of CEN in the respiratory system is a recurring issue and deposition and pulmonary biopersistence data are essential for toxicological evaluation. In this context, a fully validated standard method for CEN quantification in lung tissue is therefore imperative. The present method, based on the National Institute for Occupational Safety and Health 5040 method for atmospheric elemental and organic carbon analysis as well as on previous developments on biological matrices, involves a simple thermogravimetric analysis (TGA) of lyophilized samples, possibly preceded by a step of chemical digestion of the tissues depending on the nature of CEN investigated. The analytical method was validated for 4 CEN (carbon black as well as 3 long and thick or short and thin carbon nanotubes) for selectivity, linearity, detection and quantification limits, bias, and within-batch and between-batch precision. Calibration curves show linearity in the range of 1-40 mg/g lyophilized lung. Limits of detection for the different CEN range from 6 to 18 µg in 20 mg dry test sample. On average, within-batch precision was kept below 20 and 10% for analysis with or without a prior digestion step, respectively, whereas the corresponding between-batch precision levels reached almost 20 and 15%, respectively. The method was successfully applied to toxicological investigations for the quantitative analysis of CEN contents in rat lung exposed by inhalation.

Toxicity of TiO2 Nanoparticles: Validation of Alternative Models.

There are many studies concerning titanium dioxide (TiO2) nanoparticles (NP) toxicity. Nevertheless, there are few publications comparing in vitro and in vivo exposure, and even less comparing air-liquid interface exposure (ALI) with other in vitro and in vivo exposures. The identification and validation of common markers under different exposure conditions are relevant for the development of smart and quick nanotoxicity tests. In this work, cell viability was assessed in vitro by WST-1 and LDH assays after the exposure of NR8383 cells to TiO2 NP sample. To evaluate in vitro gene expression profile, NR8383 cells were exposed to TiO2 NP during 4 h at 3 cm2 of TiO2 NP/cm2 of cells or 19 µg/mL, in two settings-submerged cultures and ALI. For the in vivo study, Fischer 344 rats were exposed by inhalation to a nanostructured aerosol at a concentration of 10 mg/m3, 6 h/day, 5 days/week for 4 weeks. This was followed immediately by gene expression analysis. The results showed a low cytotoxic potential of TiO2 NP on NR8383 cells. Despite the absence of toxicity at the doses studied, the different exposures to TiO2 NP induce 18 common differentially expressed genes (DEG) which are involved in mitosis regulation, cell proliferation and apoptosis and inflammation transport of membrane proteins. Among these genes, we noticed the upregulation of Ccl4, Osm, Ccl7 and Bcl3 genes which could be suggested as early response biomarkers after exposure to TiO2 NP. On the other hand, the comparison of the three models helped us to validate the alternative ones, namely submerged and ALI approaches.

Adverse outcome pathways as a tool for the design of testing strategies to support the safety assessment of emerging advanced materials at the nanoscale.

Toxicity testing and regulation of advanced materials at the nanoscale, i.e. nanosafety, is challenged by the growing number of nanomaterials and their property variants requiring assessment for potential human health impacts. The existing animal-reliant toxicity testing tools are onerous in terms of time and resources and are less and less in line with the international effort to reduce animal experiments. Thus, there is a need for faster, cheaper, sensitive and effective animal alternatives that are supported by mechanistic evidence. More importantly, there is an urgency for developing alternative testing strategies that help justify the strategic prioritization of testing or targeting the most apparent adverse outcomes, selection of specific endpoints and assays and identifying nanomaterials of high concern. The Adverse Outcome Pathway (AOP) framework is a systematic process that uses the available mechanistic information concerning a toxicological response and describes causal or mechanistic linkages between a molecular initiating event, a series of intermediate key events and the adverse outcome. The AOP framework provides pragmatic insights to promote the development of alternative testing strategies. This review will detail a brief overview of the AOP framework and its application to nanotoxicology, tools for developing AOPs and the role of toxicogenomics, and summarize various AOPs of relevance to inhalation toxicity of nanomaterials that are currently under various stages of development. The review also presents a network of AOPs derived from connecting all AOPs, which shows that several adverse outcomes induced by nanomaterials originate from a molecular initiating event that describes the interaction of nanomaterials with lung cells and involve similar intermediate key events. Finally, using the example of an established AOP for lung fibrosis, the review will discuss various in vitro tests available for assessing lung fibrosis and how the information can be used to support a tiered testing strategy for lung fibrosis. The AOPs and AOP network enable deeper understanding of mechanisms involved in inhalation toxicity of nanomaterials and provide a strategy for the development of alternative test methods for hazard and risk assessment of nanomaterials.

Predictive early gene signature during mouse Bhas 42 cell transformation induced by synthetic amorphous silica nanoparticles.

Synthetic amorphous silica nanoparticles (SAS) are used widely in industrial applications. These nanoparticles are not classified for their carcinogenicity in humans. However, some data still demonstrate a potential carcinogenic risk of these compounds in humans. The Bhas 42 cell line was developed to screen chemicals, as tumor-initiators or -promoters according to their ability to trigger cell-to-cell transformation, in a cell transformation assay. In the present study, we performed unsupervised transcriptomic analysis after exposure of Bhas 42 cells to NM-203 SAS as well as to positive (Min-U-Sil 5® crystalline silica microparticles, and 12-O-tetradecanoylphorbol-13-acetate) and negative (diatomaceous earth) control compounds. We identified a common gene signature for 21 genes involved in the early stage of the SAS- Min-U-Sil 5®- or TPA-induced cell transformation. These genes were related to cell proliferation (over expression) and cell adhesion (under expression). Among them, 12 were selected on the basis of their potential impact on cell transformation. RT-qPCR and western blotting were used to confirm the transcriptomic data. Moreover, similar gene alterations were found when Bhas 42 cells were treated with two other transforming SAS. In conclusion, the results obtained in the current study highlight a 12-gene signature that could be considered as a potential early "bio-marker" of cell transformation induced by SAS and perhaps other chemicals.

Intra-erythrocyte chromium as an indicator of exposure to hexavalent chromium: An in vivo evaluation in intravenous administered rat.

Hexavalent chromium (Cr(VI)) compounds are classified as carcinogenic to humans. Whereas chromium measurements in urine and plasma attest to the last few hours of total chromium exposure (all oxidation states of chromium), chromium in red blood cells (RBC) is attributable specifically to Cr(VI) exposure over the last few days. Before recommending Cr in RBC (CrIE) as a biological indicator of Cr(VI) exposure, in vivo studies must be undertaken to assess its reliability. The present study examines the kinetics of Cr(VI) in rat after a single intravenous dose of ammonium dichromate. Chromium levels were measured in plasma, red blood cells and urine. The decay of the chromium concentration in plasma is one-phase-like (with half-life time of 0.55 day) but still measurable two days post injection. The excretion of urinary chromium peaks between five and six hours after injection and shows large variations. Intra-erythrocyte chromium (CrIE) was very constant up to a minimum of 2 days and half-life time was estimated to 13.3 days. Finally, Cr(III) does not interfere with Cr(VI) incorporation in RBC. On the basis of our results, we conclude that, unlike urinary chromium, chromium levels in RBC are indicative of the amount of dichromate (Cr(VI)) in blood.

Proteomic analysis of bronchoalveolar lavage fluid in rat exposed to TiO2 nanostructured aerosol by inhalation.

The pulmonary toxicological properties of inhaled titanium dioxide were studied using bronchoalveolar lavage fluid (BALF) cytology and proteomics analyses. Fischer 344 rats were exposed to 10 mg/m3 of TiO2 nanostructured aerosol by nose-only inhalation for 6 h/day, 5 days/week for 4 weeks. Lung samples were collected up to 180 post-exposure days. As previously described, cytological analyses of BALF showed a strong inflammatory response up to 3 post-exposure days, which persisted however, at a lower intensity up to 180 days. In addition, using Multidimensional Protein Identification Technology (MudPIT), we identified a total of 107, 50 and 45 proteins (UniprotKB identifiers) differentially expressed in exposed rats immediately, 3 and 180 days after the end of exposure respectively. Increased levels of inflammatory proteins, members of proteasome, various histones, proteins involved in cytoskeleton organization, were noticed up to 3 days (short-term response). Some of these proteins were linked with Neutrophil Extracellular Trap formation (NETosis). Long-term response was also characterized by a persistent altered expression of proteins up to 180 days. Altogether, these results suggest that exposure to low toxicity low solubility nanomaterials such as TiO2 may induce long-term changes in the pulmonary protein expression pattern of which the physio-pathological consequences are unknown. SIGNIFICANCE: This paper describes in rats, at the pulmonary level, the effects of inhaled nanostructured aerosol of TiO2 on the secreted proteins found in the broncho-alveolar space by comparing the proteomic profile in broncho-alveolar lavage fluid supernatants of control and exposed animals. This work brings new insights about the early events occurring following the end of exposure and suggests the formation of Neutrophil Extracellular Traps (NETosis) that could be interpret as a potential early mechanism of defense against TiO2 nanoparticles. This work also describes the long term effects (180 post-exposure days) of such an exposure and the change in secreted protein expression in the absence of significant histopathological modifications.

Pulmonary toxicity of two different multi-walled carbon nanotubes in rat: Comparison between intratracheal instillation and inhalation exposure.

Multi-walled carbon nanotubes (MWCNTs), which vary in length, diameter, functionalization and specific surface area, are used in diverse industrial processes. Since these nanomaterials have a high aspect ratio and are biopersistant in the lung, there is a need for a rapid identification of their potential health hazard. We assessed in Sprague-Dawley rats the pulmonary toxicity of two pristine MWCNTs (the "long and thick" NM-401 and the "short and thin" NM-403) following either intratracheal instillation or 4-week inhalation in order to gain insights into the predictability and intercomparability of the two methods. The deposited doses following inhalation were lower than the instilled doses. Both types of carbon nanotube induced pulmonary neutrophil influx using both exposure methods. This influx correlated with deposited surface area across MWCNT types and means of exposure at two different time points, 1-3 days and 28-30 days post-exposure. Increased levels of DNA damage were observed across doses and time points for both exposure methods, but no dose-response relationship was observed. Intratracheal instillation of NM-401 induced fibrosis at the highest dose while lower lung deposited doses obtained by inhalation did not induce such lung pathology. No fibrosis was observed following NM-403 exposure. When the deposited dose was taken into account, sub-acute inhalation and a single instillation of NM-401 and NM-403 produced very similar inflammation and DNA damage responses. Our data suggest that the dose-dependent inflammatory responses observed after intratracheal instillation and inhalation of MWCNTs are similar and were predicted by the deposited surface area.

Short- and long-term gene expression profiles induced by inhaled TiO2 nanostructured aerosol in rat lung.

The number of workers potentially exposed to nanoparticles (NPs) during industrial processes is increasing, although the toxicological properties of these compounds still need to be fully characterized. As NPs may be aerosolized during industrial processes, inhalation represents their main route of occupational exposure. Here, the short- and long-term pulmonary toxicological properties of titanium dioxide were studied, using conventional and molecular toxicological approaches. Fischer 344 rats were exposed to 10 mg/m3 of a TiO2 nanostructured aerosol (NSA) by nose-only inhalation for 6 h/day, 5 days/week for 4 weeks. Lung samples were collected up to 180 post-exposure days. Biochemical and cytological analyses of bronchoalveolar lavage (BAL) showed a strong inflammatory response up to 3 post-exposure days, which decreased overtime. In addition, gene expression profiling revealed overexpression of genes involved in inflammation that was maintained 6 months after the end of exposure (long-term response). Genes involved in oxidative stress and vascular changes were also up-regulated. Long-term response was characterized by persistent altered expression of a number of genes up to 180 post-exposure days, despite the absence of significant histopathological changes. The physiopathological consequences of these changes are not fully understood, but they should raise concerns about the long-term pulmonary effects of inhaled biopersistent NPs such as TiO2.

Toluene and methylethylketone: effect of combined exposure on their metabolism in rat.

1. Multiple exposures are ubiquitous in industrial environments. In this article, we highlight the risks faced by workers and complete the data available on the metabolic impact of a common mixture: toluene (TOL) and methylethylketone (MEK). 2. Rats were exposed by inhalation under controlled conditions either to each solvent individually, or to mixtures of the two. How the interaction between the two solvents affected their fate in the blood and brain, their main relevant urinary metabolites (o-cresol, benzylmercapturic acid for TOL and 2,3-butanediols for MEK) and their hepatic metabolism were investigated. 3. Although the cytochrome P450 concentration was unchanged, and the activities of CYP1A2 and CYP2E1 isoforms were not additively or synergistically induced by co-exposure, TOL metabolism was inhibited by the presence of MEK (and vice versa). Depending on the relative proportions of each compound in the mixture, this sometimes resulted in a large increase in blood and brain concentrations. Apart from extreme cases (unbalanced mixtures), the amount of o-cresol and benzylmercapturic acid (and to a lesser extent 2,3-butanediols) excreted were proportional to the blood solvent concentrations. 4. In a co-exposure context, ortho-cresol and benzylmercapturic acid can be used as urinary biomarkers in biomonitoring for employees to relatively accurately assess TOL exposure.

Metabolism of inhaled methylethylketone in rats.

Methylethylketone (MEK) is widely used in industry, often in combination with other compounds. Although nontoxic, it can make other chemicals harmful. This study investigates the fate of MEK in rat blood, brain and urine as well as its hepatic metabolism following inhalation over 1 month (at 20, 200 or 1400 ppm). MEK did not significantly accumulate in the organism: blood concentrations were similar after six-hour or 1-month inhalation periods, and brain concentrations only increased slightly after 1 month's exposure. Urinary excretion, based on the major metabolites, 2,3-butanediols (± and meso forms), accounted for less than 2.4% of the amount inhaled. 2-Butanol, 3-hydroxy-2-butanone and MEK itself were only detectable in urine in the highest concentration conditions investigated, when metabolic saturation occurred. Although MEK exposure did not alter the total cytochrome P450 concentration, it induced activation of both CYP1A2 and CYP2E1 enzymes. In addition, the liver glutathione concentration (reduced and oxidized forms) decreased, as did glutathione S-transferase (GST) activity (at exposure levels over 200 ppm). These metabolic data could be useful for pharmacokinetic model development and/or verification and suggest the ability of MEK to influence the metabolism (and potentiate the toxicity) of other substances.

In vitro cell transformation induced by synthetic amorphous silica nanoparticles.

Synthetic amorphous silica nanoparticles (SAS) are among the most widely produced and used nanomaterials, but little is known about their carcinogenic potential. This study aims to evaluate the ability of four different SAS, two precipitated, NM-200 and NM-201, and two pyrogenic, NM-202 and NM-203, to induce the transformation process. For this, we used the recently developed in vitro Bhas 42 cell transformation assay (CTA). The genome of the transgenic Bhas 42 cells contains several copies of the v-Ha-ras gene, making them particularly sensitive to tumor-promoter agents. The Bhas 42 CTA, which includes an initiation assay and a promotion assay, was validated in our laboratory using known soluble carcinogenic substances. Its suitability for particle-type substances was verified by using quartz Min-U-Sil 5 (Min-U-Sil) and diatomaceous earth (DE) microparticles. As expected given their known transforming properties, Min-U-Sil responded positively in the Bhas 42 CTA and DE responded negatively. Transformation assays were performed with SAS at concentrations ranging from 2µg/cm2 to 80µg/cm2. Results showed that all SAS have the capacity to induce transformed foci, interestingly only in the promotion assay, suggesting a mode of action similar to tumor-promoter substances. NM-203 exhibited transforming activity at a lower concentration than the other SAS. In conclusion, this study showed for the first time the transforming potential of different SAS, which act as tumor-promoter substances in the Bhas 42 model of cell transformation.