Toxicology and Biodegradability of a Phthalate-Free and Bio-Based Novel Plasticizer.

Phthalate esters, mainly di-ethylhexylphthalate (DEHP), represent a class of chemicals primarily used as plasticizers for polyvinyl chloride in a wide range of domestic and industrial applications. These phthalate esters are low-toxicity environmental contaminants. To address these drawbacks, POLYSORB® ID 37, a blend of diesters obtained from esterification of isosorbide with plant-based fatty acids, was developed. The company can now offer PVC manufacturers a new product which competes with phthalates and other such chemicals. The market for plasticizers is very important, and ROQUETTE intends to provide a more sustainable and safer product. Isosorbide diester is bio-based (made from glucose and vegetable fatty acids). This plasticizer is registered in REACH regulation for high volumes (>1000 T/year). Risk assessment was obtained by conducting a wide range of biodegradability and toxicological protocols, using rodent models, according to established guidelines. Overall, all of the toxicological and biodegradability studies demonstrated that POLYSORB® ID 37 is nontoxic to mammalian life and is readily biodegradable.

Metal enriched quasi-ultrafine particles from stainless steel gas metal arc welding induced genetic and epigenetic alterations in BEAS-2B cells.

Recent evidence has supported welding fume (WF)-derived ultrafine particles (UFP) could be the driving force of their adverse health effects. However, UFP have not yet been extensively studied and are currently not included in present air quality standards/guidelines. Here, attention was focused on the underlying genetic and epigenetic mechanisms by which the quasi-UFP (Q-UFP, i.e., ≤ 0.25 µm) of the WF emitted by gas metal arc welding-stainless steel (GMAW-SS) exert their toxicity in human bronchial epithelial BEAS-2B cells. The Q-UFP under study showed a monomodal size distribution in number centered on 104.4 ± 52.3 nm and a zeta potential of -13.8 ± 0.3 mV. They were enriched in Fe > Cr > Mn > Si, and displayed a relatively high intrinsic oxidative potential. Dose-dependent activation of nuclear factor erythroid 2-related factor 2 and nuclear factor-kappa B signaling pathway, glutathione alteration, and DNA, protein and lipid oxidative damage were reported in BEAS-2B cells acutely (1.5 and 9 µg/cm2, 24 h) or repeatedly (0.25 and 1.5 µg/cm2, 3 × 24 h) exposed to Q-UFP (p < 0.05). Alterations of the Histone H3 acetylation were reported for any exposure (p < 0.05). Differentially regulated miRNA and mRNA indicated the activation of some critical cell signaling pathways related to oxidative stress, inflammation, and cell cycle deregulation towards apoptosis. Taken together, these results highlighted the urgent need to better evaluate the respective toxicity of the different metals and to include the Q-UFP fraction of WF in current air quality standards/guidelines relevant to the occupational settings.

In vitro and in vivo genotoxicity assessment of ferric ferrocyanide and potassium-cobalt ferrocyanide.

Ferric hexacyanoferrate(II) (Fe4[Fe(CN)6]3), i.e. Prussian blue (PB) has been used for many years to remove from the body the two toxic isotopes of cesium and thallium following irradiation. Recently, potassium cobalt hexacyanoferrate(II) (K2COFe(CN)6), which has shown a better efficacy for decontamination, is also being considered for use to enhance the elimination of cesium isotopes. In view to its preclinical and clinical development, in vitro and in vivo GLP-compliant genotoxicity studies were carried out on this product as well as on PB for comparison. Several tests dissecting the main events leading to genotoxicity, i.e. mutagenicity and chromosomal aberrations, both structural and quantitative were implemented. In vitro, no mutagenic effect was observed in the Ames test but both compounds were positive in the mouse lymphoma assay on TK locus and induced clastogenic effects in the in vitro chromosomal aberrations test on human lymphocytes, either in absence or in presence of metabolic activation. K-Co-ferrocyanide was also assayed in vivo in the mouse bone marrow micronucleus assay and PB was assessed for DNA fragmentation in the rodent Comet assay in both glandular stomach and colon. In the in vivo micronucleus mouse bone marrow, K-Co-ferrocyanide did not display any genotoxic activity up to 2000 mg/kg/d (x2) by oral route. In opposite, PB induced a significant increase in DNA fragmentation both in the glandular stomach and in the colon of rat treated 3 times with intake ranging from 2000 to 500 mg/kg. PB should be considered as an in vivo mutagen as well as Potassium cobalt hexacyanoferrate(II) since the in vitro genotoxicity profiles of both ferrocyanides are quite similar. Their use as cesium/ thallium decontamination agents in human should be assessed following a benefit/risk approach to enable a robust decision-making.

Study of in vitro and in vivo genotoxic effects of air pollution fine (PM2.5-0.18) and quasi-ultrafine (PM0.18) particles on lung models.

Air pollution and particulate matter (PM) are classified as carcinogenic to humans. Pollutants evidence for public health concern include coarse (PM10) and fine (PM2.5) particles. However, ultrafine particles (PM0.1) are assumed to be more toxic than larger particles, but data are still needed to better understand their mechanism of action. In this context, the aim of our work was to investigate the in vitro and in vivo genotoxic potential of fine (PM2.5-018) and quasi ultra-fine (PM0.18) particles from an urban-industrial area (Dunkirk, France) by using comet, micronucleus and/or gene mutation assays. In vitro assessment was performed with 2 lung immortalized cell lines (BEAS-2B and NCI-H292) and primary normal human bronchial epithelial cells (NHBE) grown at the air-liquid interface or in submerged conditions (5 µg PM/cm2). For in vivo assessment, tests were performed after acute (24 h, 100 µg PM/animal), subacute (1 month, 10 µg PM/animal) and subchronic (3 months, 10 µg PM/animal) intranasal exposure of BALB/c mice. In vitro, our results show that PM2.5-018 and PM0.18 induced primary DNA damage but no chromosomal aberrations in immortalized cells. Negative results were noted in primary cells for both endpoints. In vivo assays revealed that PM2.5-018 and PM0.18 induced no significant increases in DNA primary damage, chromosomal aberrations or gene mutations, whatever the duration of exposure. This investigation provides initial answers regarding the in vitro and in vivo genotoxic mode of action of PM2.5-018 and PM0.18 at moderate doses and highlights the need to develop standardized specific methodologies for assessing the genotoxicity of PM. Moreover, other mechanisms possibly implicated in pulmonary carcinogenesis, e.g. epigenetics, should be investigated.

Combined toxic effects and DNA damage to two plant species exposed to binary metal mixtures (Cd/Pb).

Acute and long-term (3-, 10- and 56-day exposure) laboratory toxicity tests were carried out to assess the individual and combined toxic effects of cadmium (Cd) and lead (Pb) in Brassica oleracea and Trifolium repens. In addition to morphological parameters, this work also used comet assay to address endpoints in relation to genotoxicity. Bioaccumulation was measured to demonstrate the influence of the mixture on the concentrations of each metal in the plant. The statistical method reported by Ince et al. (1999) was used to evaluate the types of interaction between Cd and Pb in each treatment and concerning their combined effect. This study concludes that the combined effects of binary metal combinations of Cd/Pb on morphological parameters are most often additive, sometimes antagonistic and more rarely synergistic, thus extending the findings of previous publications on this subject. DNA damage analysis revealed concentration- and time-dependent interactions. Synergistic effects of mixed metals (more breaks than individually applied metals) are observed in T. repens after a short exposure. Antagonistic effects are statistically significant after 10 days-exposure, suggesting competition between metals. At 56 days, the rate of DNA damage observed in plants exposed to the Cd/Pb mixture was similar to that measured in plants exposed to lead only and was significantly lower than the rate of DNA damage induced by Cd. This supports the idea that there may be competition between metals and also strengthens the hypothesis that long-term reparation mechanisms may be implemented. Cd/Pb co-exposure does not significantly influence the bioaccumulation of each metal. It is nevertheless important to note that a statistically significant 'interaction' is not necessarily biologically relevant and should therefore be considered with caution when assessing heavy metals combined effects.

Genetic and epigenetic alterations in normal and sensitive COPD-diseased human bronchial epithelial cells repeatedly exposed to air pollution-derived PM2.5.

Even though clinical, epidemiological and toxicological studies have progressively provided a better knowledge of the underlying mechanisms by which air pollution-derived particulate matter (PM) exerts its harmful health effects, further in vitro studies on relevant cell systems are still needed. Hence, aiming of getting closer to the human in vivo conditions, primary human bronchial epithelial cells derived from normal subjects (NHBE) or sensitive chronic obstructive pulmonary disease (COPD)-diseased patients (DHBE) were differentiated at the air-liquid interface. Thereafter, they were repeatedly exposed to air pollution-derived PM2.5 to study the occurrence of some relevant genetic and/or epigenetic endpoints. Concentration-, exposure- and season-dependent increases of OH-B[a]P metabolites in NHBE, and to a lesser extent, COPD-DHBE cells were reported; however, there were more tetra-OH-B[a]P and 8-OHdG DNA adducts in COPD-DHBE cells. No increase in primary DNA strand break nor chromosomal aberration was observed in repeatedly exposed cells. Telomere length and telomerase activity were modified in a concentration- and exposure-dependent manner in NHBE and particularly COPD-DHBE cells. There were a global DNA hypomethylation, a P16 gene promoter hypermethylation, and a decreasing DNA methyltransferase activity in NHBE and notably COPD-DHBE cells repeatedly exposed. Changes in site-specific methylation, acetylation, and phosphorylation of histone H3 (i.e., H3K4me3, H3K9ac, H3K27ac, and H3S10ph) and related enzyme activities occurred in a concentration- and exposure-dependent manner in all the repeatedly exposed cells. Collectively, these results highlighted the key role played by genetic and even epigenetic events in NHBE and particularly sensitive COPD-DHBE cells repeatedly exposed to air pollution-derived PM2.5 and their different responsiveness. While these specific epigenetic changes have been already described in COPD and even lung cancer phenotypes, our findings supported that, together with genetic events, these epigenetic events could dramatically contribute to the shift from healthy to diseased phenotypes following repeated exposure to relatively low doses of air pollution-derived PM2.5.

Carboxylated nanodiamonds can be used as negative reference in in vitro nanogenotoxicity studies.

Nanodiamonds (NDs) are promising nanomaterials for biomedical applications. However, a few studies highlighted an in vitro genotoxic activity for detonation NDs, which was not evidenced in one of our previous work quantifying γ-H2Ax after 20 and 100 nm high-pressure high-temperature ND exposures of several cell lines. To confirm these results, in the present work, we investigated the genotoxicity of the same 20 and 100 nm NDs and added intermediate-sized NDs of 50 nm. Conventional in vitro genotoxicity tests were used, i.e., the in vitro micronucleus and comet assays that are recommended by the French National Agency for Medicines and Health Products Safety for the toxicological evaluation of nanomedicines. In vitro micronucleus and in vitro comet assays (standard and hOGG1-modified) were therefore performed in two human cell lines, the bronchial epithelial 16HBE14o- cells and the colon carcinoma T84 cells. Our results did not show any genotoxic activity, whatever the test, the cell line or the size of carboxylated NDs. Even though these in vitro results should be confirmed in vivo, they reinforce the potential interest of carboxylated NDs for biomedical applications or even as a negative reference nanoparticle in nanotoxicology. Copyright © 2017 John Wiley & Sons, Ltd.

Antioxidant defense gene analysis in Brassica oleracea and Trifolium repens exposed to Cd and/or Pb.

This study focused on the expression analysis of antioxidant defense genes in Brassica oleracea and in Trifolium repens. Plants were exposed for 3, 10, and 56 days in microcosms to a field-collected suburban soil spiked by low concentrations of cadmium and/or lead. In both species, metal accumulations and expression levels of genes encoding proteins involved and/or related to antioxidant defense systems (glutathione transferases, peroxidases, catalases, metallothioneins) were quantified in leaves in order to better understand the detoxification processes involved following exposure to metals. It appeared that strongest gene expression variations in T. repens were observed when plants are exposed to Cd (metallothionein and ascorbate peroxidase upregulations) whereas strongest variations in B. oleracea were observed in case of Cd/Pb co-exposures (metallothionein, glutathione transferase, and peroxidase upregulations). Results also suggest that there is a benefit to use complementary species in order to better apprehend the biological effects in ecotoxicology.

Combined effect of Cd and Pb spiked field soils on bioaccumulation, DNA damage, and peroxidase activities in Trifolium repens.

The present study was designed to investigate the combined effects of Cd and Pb on accumulation and genotoxic potential in white clover (Trifolium repens). For this purpose, T. repens was exposed to contaminated soils (2.5-20 mg kg(-1) cadmium (Cd), 250-2000 mg kg(-1) lead (Pb) and a mixture of these two heavy metals) for 3, 10 and 56 days. The resulting bioaccumulation of Cd and Pb, DNA damage (comet assay) and peroxidase activities (APOX and GPOX) were determined. The exposure time is a determinant factor in experiments designed to measure the influence of heavy metal contamination. The accumulation of Cd or Pb resulting from exposure to the two-metal mixture does not appear to depend significantly on whether the white clover is exposed to soil containing one heavy metal or both. However, when T. repens is exposed to a Cd/Pb mixture, the percentage of DNA damage is lower than when the plant is exposed to monometallic Cd. DNA damage is close to that observed in the case of monometallic Pb exposure. Peroxidase activity cannot be associated with DNA damage under these experimental conditions.

Human cell line-dependent WC-Co nanoparticle cytotoxicity and genotoxicity: a key role of ROS production.

Although tungsten carbide-cobalt (WC-Co) nanoparticles (NPs) have been widely used because of their robustness, their risk to human health remains poorly studied, despite the International Agency for Research on Cancer (IARC) classifying them as "probably carcinogenic" for humans (Group 2A) in 2006. Our current study aimed at defining the cytotoxicity and genotoxicity of one set of commercially available 60-nm diameter WC-Co NPs on three human cell lines representative of potential target organs: A549 (lung), Hep3B (liver), and Caki-1 (kidney). The cytotoxicity of WC-Co NPs was determined by evaluating cell impedance (xCELLigence), cell survival/death, and cell cycle checkpoints. Flow cytometry was used to not only evaluate cell cycle checkpoints, but to also estimate reactive oxygen species (ROS) generation. In addition, γ-H2Ax foci detection (confocal microscopy), considered to be the most sensitive technique for studying DNA double-strand breaks, was utilized to evaluate genotoxicity. As a final part of this study, we assessed the cellular incorporation of WC-Co NPs, first byflow cytometry (side scatter), and then by confocal microscopy (light reflection) to ensure that the NPs had entered cells. Overall, our current findings demonstrate that WC-Co NPs induce cell mortality, DNA double-strand breaks, and cell cycle arrest in human renal (Caki-1) and liver (Hep3B) cell lines, but do not induce significant cytotoxic effects in A549 lung cells. Interestingly, although WC-Co NPs effectively entered the cells in all 3 lines tested, ROS were detected in Caki-1 and Hep3B, but not in A549. This may explain the great differences in the cytotoxic and genotoxic effects we observed between these lines.

Antioxidant responses of Annelids, Brassicaceae and Fabaceae to pollutants: a review.

Pollutants, such as Metal Trace Elements (MTEs) and organic compounds (polycyclic aromatic hydrocarbons, pesticides), can impact DNA structure of living organisms and thus generate damage. For instance, cadmium is a well-known genotoxic and mechanisms explaining its clastogenicity are mainly indirect: inhibition of DNA repair mechanisms and/or induction of Reactive Oxygen Species (ROS). Animal or vegetal cells use antioxidant defense systems to protect themselves against ROS produced during oxidative stress. Because tolerance of organisms depends, at least partially, on their ability to cope with ROS, the mechanisms of production and management of ROS were investigated a lot in Ecotoxicology as markers of biotic and abiotic stress. This was mainly done through the measurement of enzyme activities The present Review focuses on 3 test species living in close contact with soil that are often used in soil ecotoxicology: the worm Eisenia fetida, and two plant species, Trifolium repens (white clover) and Brassica oleracea (cabbage). E. fetida is a soil-dwelling organism commonly used for biomonitoring. T. repens is a symbiotic plant species which forms root nodule with soil bacteria, while B. oleracea is a non-symbiotic plant. In literature, some oxidative stress enzyme activities have already been measured in those species but such analyses do not allow distinction between individual enzyme involvements in oxidative stress. Gene expression studies would allow this distinction at the transcriptomic level. A literature review and a data search in molecular database were carried out on the basis of keywords in Scopus, in PubMed and in Genbank™ for each species. Molecular data regarding E. fetida were already available in databases, but a lack of data regarding oxidative stress related genes was observed for T. repens and B. oleracea. By exploiting the conservation observed between species and using molecular biology techniques, we partially cloned missing candidates involved in oxidative stress and in metal detoxification in E. fetida, T. repens and B. oleracea.

Genotoxic activity of chlorohydroxyfuranones in the microscale micronucleus test on mouse lymphoma cells and the unscheduled DNA synthesis assay in rat hepatocytes.

Chlorohydroxyfuranones (CHFs) are mutagenic disinfection by-products found in chlorine-treated drinking water. In the current study, the genotoxicity of four CHFs, 3,4-dichloro-5-hydroxy-2(5H)-furanone (MCA), 3-chloro-4-methyl-5-hydroxy-2(5H)-furanone (MCF), 3-chloro-4-(chloromethyl)-5-hydroxy-2(5H)-furanone (CMCF) and 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), was determined. Two in vitro assays, the microscale micronucleus assay on L5178Y mouse lymphoma cells and the unscheduled DNA synthesis assay on a hepatocyte primary culture from Fisher F344 rats, were carried out. All four CHFs demonstrated genotoxic effects in both assays. In the two systems used, CMCF was the most genotoxic compound, followed by MCA, MX and MCF, respectively. This work was the first study of the DNA damaging properties of all four CHFs in two in vitro genotoxicity tests. These new data expand the range of genetic damages induced by this group of compounds.

A micromethod for the in vitro micronucleus assay.

A micromethod for the in vitro micronucleus assay was developed using L5178Y cells to enable the rapid screening of a large number of molecules. The method is quick, simple to perform and needs very small amounts of compound, i.e. <10 mg. In this methodology, three types of treatment were carried out in parallel, enabling an optimal detection of both aneugenic and clastogenic compounds: two treatments without metabolic activation with or without a recovery period after a 24 h continuous treatment and one treatment with metabolic activation by Aroclor 1254-induced rat or hamster liver S9 mix. Seventeen known genotoxins (12 clastogens and five aneugens) and seven known non-genotoxins were tested. The in vitro micronucleus micromethod using L5178Y cells exhibited good sensitivity (16 positive/17 known genotoxins tested) and specificity (7 negative/7 known non-genotoxins tested) for the 24 test compounds studied with or without metabolic activation. Furthermore, this test showed a good correlation with other in vitro micronucleus tests performed using macromethods with various mammalian cell cultures. We conclude that the in vitro micronucleus micromethod with L5178Y cells could be used in the earliest stages of development of new molecules as a preliminary short-term screening assay before starting regulatory tests.