Toxicology of chemical mixtures: international perspective.

This paper reviews major activities outside the United States on human health issues related to chemical mixtures. In Europe an international study group on combination effects has been formed and has started by defining synergism and antagonism. Successful research programs in Europe include the development and application of statistically designed experiments combined with multivariate data analysis and modeling in vitro and in vivo studies on a wide variety of chemicals such as petroleum hydrocarbons, aldehydes, food contaminants, industrial solvents, and mycotoxins. Other major activities focus on the development of safety evaluation strategies for mixtures such as the use of toxic equivalence factors or alternatives such as the question-and-answer approach, fractionation followed by recombination of the mixture in combination with a mixture design, and quantitative structure-activity relationship analysis combined with lumping analysis and physiologically based pharmacokinetic/pharmacodynamic modeling for studying complex mixtures. A scheme for hazard identification and risk assessment of complex mixtures and a consistent way to generate total volatile organic compound values for indoor air have also been developed. Examples of other activities are carcinogenicity studies on complex mixtures (petroleum middle distillates, foundry fumes, pesticides, heterocyclic amines, diesel exhaust, solid particles), neurotoxicity studies of mixtures of solvents alone or in combination with exposure to physical factors, and toxicity studies of outdoor air pollutants, focusing on particulates. Outside the United States, toxicologists and regulators clearly have a growing interest in the toxicology and risk assessment of chemical mixtures

The biological effects of subacute inhalation of diesel exhaust following addition of cerium oxide nanoparticles in atherosclerosis-prone mice

AbstractBackgroundCerium oxide (CeO2) nanoparticles improve the burning efficiency of fuel, however, little is known about health impacts of altered emissions from the vehicles.MethodsAtherosclerosis-prone apolipoprotein E knockout (ApoE−/−) mice were exposed by inhalation to diluted exhaust (1.7mg/m3, 20, 60 or 180min, 5 day/week, for 4 weeks), from an engine using standard diesel fuel (DE) or the same diesel fuel containing 9ppm cerium oxide nanoparticles (DCeE). Changes in hematological indices, clinical chemistry, atherosclerotic burden, tissue levels of inflammatory cytokines and pathology of the major organs were assessed.ResultsAddition of CeO2 to fuel resulted in a reduction of the number (30%) and surface area (10%) of the particles in the exhaust, whereas the gaseous co-pollutants were increased (6–8%). There was, however, a trend towards an increased size and complexity of the atherosclerotic plaques following DE exposure, which was not evident in the DCeE group. There were no clear signs of altered hematological or pathological changes induced by either treatment. However, levels of proinflammatory cytokines were modulated in a brain region and liver following DCeE exposure.ConclusionsThese results imply that addition of CeO2 nanoparticles to fuel decreases the number of particles in exhaust and may reduce atherosclerotic burden associated with exposure to standard diesel fuel. From the extensive assessment of biological parameters performed, the only concerning effect of cerium addition was a slightly raised level of cytokines in a region of the central nervous system. Overall, the use of cerium as a fuel additive may be a potentially useful way to limit the health effects of vehicle exhaust. However, further testing is required to ensure that such an approach is not associated with a chronic inflammatory response which may eventually cause long-term health effects

Response of spontaneously hypertensive rats to inhalation of fine and ultrafine particles from traffic: experimental controlled study

BACKGROUND: Many epidemiological studies have shown that mass concentrations of ambient particulate matter (PM) are associated with adverse health effects in the human population. Since PM is still a very crude measure, this experimental study has explored the role of two distinct size fractions: ultrafine (<0.15 μm) and fine (0.15- 2.5 μm) PM. In a series of 2-day inhalation studies, spontaneously hypersensitive (SH) rats were exposed to fine, concentrated, ambient PM (fCAP) at a city background location or a combination of ultrafine and fine (u+fCAP) PM at a location dominated by traffic. We examined the effect on inflammation and both pathological and haematological indicators as markers of pulmonary and cardiovascular injury. Exposure concentrations ranged from 399 μg/m(3 )to 3613 μg/m(3 )for fCAP and from 269μg/m(3 )to 556 μg/m(3 )for u+fCAP. RESULTS: Ammonium, nitrate, and sulphate ions accounted for 56 ± 16% of the total fCAP mass concentrations, but only 17 ± 6% of the u+fCAP mass concentrations. Unambiguous particle uptake in alveolar macrophages was only seen after u+fCAP exposures. Neither fCAP nor u+fCAP induced significant changes of cytotoxicity or inflammation in the lung. However, markers of oxidative stress (heme oxygenase-1 and malondialdehyde) were affected by both fCAP and u+fCAP exposure, although not always significantly. Additional analysis revealed heme oxygenase-1 (HO-1) levels that followed a nonmonotonic function with an optimum at around 600 μg/m(3 )for fCAP. As a systemic response, exposure to u+fCAP and fCAP resulted in significant decreases of the white blood cell concentrations. CONCLUSION: Minor pulmonary and systemic effects are observed after both fine and ultrafine + fine PM exposure. These effects do not linearly correlate with the CAP mass. A greater component of traffic CAP and/or a larger proportion ultrafine PM does not strengthen the absolute effects

Unveiling the Toxicity of Fine and Nano-Sized Airborne Particles Generated from Industrial Thermal Spraying Processes in Human Alveolar Epithelial Cells

High-energy industrial processes have been associated with particle release into workplace air that can adversely affect workers' health. The present study assessed the toxicity of incidental fine (PGFP) and nanoparticles (PGNP) emitted from atmospheric plasma (APS) and high-velocity oxy-fuel (HVOF) thermal spraying. Lactate dehydrogenase (LDH) release, 2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1) metabolisation, intracellular reactive oxygen species (ROS) levels, cell cycle changes, histone H2AX phosphorylation (γ-H2AX) and DNA damage were evaluated in human alveolar epithelial cells at 24 h after exposure. Overall, HVOF particles were the most cytotoxic to human alveolar cells, with cell viability half-maximal inhibitory concentration (IC50) values of 20.18 μg/cm2 and 1.79 μg/cm2 for PGFP and PGNP, respectively. Only the highest tested concentration of APS-PGFP caused a slight decrease in cell viability. Particle uptake, cell cycle arrest at S + G2/M and γ-H2AX augmentation were observed after exposure to all tested particles. However, higher levels of γ-H2AX were found in cells exposed to APS-derived particles (~16%), while cells exposed to HVOF particles exhibited increased levels of oxidative damage (~17% tail intensity) and ROS (~184%). Accordingly, APS and HVOF particles seem to exert their genotoxic effects by different mechanisms, highlighting that the health risks of these process-generated particles at industrial settings should not be underestimated. © 2022 by the authors. Licensee MDPI, Basel, Switzerland.Funding text 1: transitória (Ref. DL-57/INSA-06/2018). Thanks are also due to FCT/MCTES for the financial support to EPIUnit and ITR (UIDB/04750/2020 and LA/P/0064/2020). Informed Consent Statement: Not applicable. Institutional Review Board Statement: Not applicable.; Funding text 2: Funding: This research was funded by the project CERASAFE with the support of ERA-NET SIINN (project id:16) and the Portuguese Foundation for Science and Technology (FCT; SIINN/0004/2014). This work was also supported by the project NanoBioBarriers (PTDC/MED-TOX/31162/2017), cofinanced by the Operational Program for Competitiveness and Internationalization (POCI) through European Regional Development Funds (FEDER/FNR) and FCT and the Spanish Ministry of Sci- ence and Innovation (projects PCIN-2015-173-C02-01 and CEX2018-000794-S-Severo Ochoa). M.J. Bessa (SFRH/BD/120646/2016) and F. Brandão (SFRH/BD/101060/2014) are recipients of FCT PhD scholarships under the framework of the Human Capital Operating Program (POCH) and European Union funding. The Doctoral Program in Biomedical Sciences of the ICBAS—University of Porto Int.J.Mol. Sci. 2022,of23fer, x FOR PEER REVIEWed additional funds. S. Fraga thanks FCT for funding through program DL 57/2016—Norm

Effects of particulate matter on the pulmonary and vascular system: time course in spontaneously hypertensive rats

BACKGROUND: This study was performed within the scope of two multi-center European Commission-funded projects (HEPMEAP and PAMCHAR) concerning source-composition-toxicity relationship for particulate matter (PM) sampled in Europe. The present study aimed to optimize the design for PM in vivo toxicity screening studies in terms of dose and time between a single exposure and the determination of the biological responses in a rat model mimicking human disease resulting in susceptibility to ambient PM. Dust in thoracic PM size-range (aerodynamic diameter <10 μm) was sampled nearby a road tunnel (RTD) using a high volume cascade impactor. Spontaneously hypertensive rats were exposed to urban dust collected in Ottawa, Canada (EHC-93 10 mg/kg of body weight; reference PM) or different RTD doses (0.3, 1, 3, 10 mg/kg of body weight) by intratracheal instillation. Necropsy was performed at 4, 24, or 48 hr after exposure. RESULTS: The neutrophil numbers in bronchoalveolar lavage fluid increased tremendously after exposure to the highest RTD doses or EHC-93. Furthermore, PM exposure slightly affected blood coagulation since there was a small but significant increase in the plasma fibrinogen levels (factor 1.2). Pulmonary inflammation and oxidative stress as well as changes in blood coagulation factors and circulating blood cell populations were observed within the range of 3 to 10 mg PM/kg of body weight without significant pulmonary injury. CONCLUSION: The optimal dose for determining the toxicity ranking of ambient derived PM samples in spontaneously hypertensive rats is suggested to be between 3 and 10 mg PM/kg of body weight under the conditions used in the present study. At a lower dose only some inflammatory effects were detected, which will probably be too few to be able to discriminate between PM samples while a completely different response pattern was observed with the highest dose. In addition to the dose, a 24-hr interval from exposure to sacrifice seemed appropriate to assess the relative toxic potency of PM since the majority of the health effects were observed one day after PM exposure compared to the other times examined. The aforementioned considerations provide a good basis for conducting PM toxicity screening studies in spontaneously hypertensive rats

In Vitro Toxicity of Industrially Relevant Engineered Nanoparticles in Human Alveolar Epithelial Cells: Air-Liquid Interface versus Submerged Cultures

Diverse industries have already incorporated within their production processes engineered nanoparticles (ENP), increasing the potential risk of worker inhalation exposure. In vitro models have been widely used to investigate ENP toxicity. Air–liquid interface (ALI) cell cultures have been emerging as a valuable alternative to submerged cultures as they are more representative of the inhalation exposure to airborne nano-sized particles. We compared the in vitro toxicity of four ENP used as raw materials in the advanced ceramics sector in human alveolar epithelial-like cells cultured under submerged or ALI conditions. Submerged cultures were exposed to ENP liquid suspensions or to aerosolised ENP at ALI. Toxicity was assessed by determining LDH release, WST-1 metabolisation and DNA damage. Overall, cells were more sensitive to ENP cytotoxic effects when cultured and exposed under ALI. No significant cytotoxicity was observed after 24 h exposure to ENP liquid suspensions, although aerosolised ENP clearly affected cell viability and LDH release. In general, all ENP increased primary DNA damage regardless of the exposure mode, where an increase in DNA strand-breaks was only detected under submerged conditions. Our data show that at relevant occupational concentrations, the selected ENP exert mild toxicity to alveolar epithelial cells and exposure at ALI might be the most suitable choice when assessing ENP toxicity in respiratory models under realistic exposure conditions.This research was funded by CERASAFE (www.cerasafe.eu; accessed on 26 October 2021), with the support of ERA-NET SIINN (project id:16) and the Portuguese Foundation for Science and Technology (FCT; SIINN/0004/2014). This work was also supported by the NanoBioBarriers project (PTDC/MED-TOX/31162/2017), co-financed by the Operational Program for Competitiveness and Internationalization (POCI) through European Regional Development Funds (FEDER/FNR) and FCT; Spanish Ministry of Science and Innovation (projects PCIN-2015-173-C02-01 and CEX2018-000794-S-Severo Ochoa), and by the Romanian National Authority for Scientific Research and Innovation (CCCDI-UEFISCDI, project number 29/2016 within PNCDI III). M.J. Bessa (SFRH/BD/120646/2016) and F. Brandão (SFRH/BD/101060/2014) are recipients of FCT PhD scholarships under the framework of Human Capital Operating Program (POCH) and European Union funding. The Doctoral Program in Biomedical Sciences, of the ICBAS—University of Porto, offered additional funds. S. Fraga thanks FCT for funding through program DL 57/2016–Norma transitória (Ref. DL-57/INSA-06/2018). Thanks are also due to FCT/MCTES for the financial support to EPIUnit (info:eu-repo/grantAgreement/FCT/6817 - DCRRNI ID/UIDB/04750/2020/PT)

Evaluation of neurological effects of cerium dioxide nanoparticles doped with different amounts of zirconium following inhalation exposure in mouse models of Alzheimer’s and vascular disease

Increasing evidence from toxicological and epidemiological studies indicates that the brain is an important target for ambient (ultrafine) particles. Disturbance of redox-homeostasis and inflammation in the brain are proposed as possible mechanisms that can contribute to neurotoxic and neurodegenerative effects. Whether and how engineered nanoparticles (NPs) may cause neurotoxicity and promote neurodegenerative diseases such as Alzheimer's disease (AD) is largely unstudied. We have assessed the neurological effects of subacute inhalation exposures (4 mg/m3 for 3 h/day, 5 days/week for 4 weeks) to cerium dioxide (CeO2) NPs doped with different amounts of zirconium (Zr, 0%, 27% and 78%), to address the influence of particle redox-activity in the 5xFAD transgenic mouse model of AD. Four weeks post-exposure, effects on behaviour were evaluated and brain tissues were analysed for amyloid-β plaque formation and reactive microglia (Iba-1 staining). Behaviour was also evaluated in concurrently exposed non-transgenic C57BL/6J littermates, as well as in Western diet-fed apolipoprotein E-deficient (ApoE-/-) mice as a model of vascular disease. Markers of inflammation and oxidative stress were evaluated in brain cortex. The brains of the NP-exposed 5xFAD mice revealed no accelerated amyloid-β plaque formation. No significant treatment-related behaviour impairments were observed in the healthy C57BL/6J mice. In the 5xFAD and ApoE-/- models, the NP inhalation exposures did not affect the alternation score in the X-maze indicating absence of spatial working memory deficits. However, following inhalation exposure to the 78% Zr-doped CeO2 NPs changes in forced motor performance (string suspension) and exploratory motor activity (X-maze) were observed in ApoE-/- and 5xFAD mice, respectively. Exposure to the 78% doped NPs also caused increased cortical expression of glial fibrillary acidic protein (GFAP) in the C57BL/6J mice. No significant treatment-related changes neuroinflammation and oxidative stress were observed in the 5xFAD and ApoE-/- mice. Our study findings reveal that subacute inhalation exposure to CeO2 NPs does not accelerate the AD-like phenotype of the 5xFAD model. Further investigation is warranted to unravel whether the redox-activity dependent effects on motor activity as observed in the mouse models of AD and vascular disease result from specific neurotoxic effects of these NPs

Silica encapsulation of ZnO nanoparticles reduces their toxicity for cumulus cell-oocyte-complex expansion

Background Metal oxide nanoparticles (NPs) are increasingly used in many industrial and biomedical applications, hence their impact on occupational and public health has become a concern. In recent years, interest on the effect that exposure to NPs may exert on human reproduction has grown, however data are still scant. In the present work, we investigated whether different metal oxide NPs interfere with mouse cumulus cell-oocyte complex (COC) expansion. Methods Mouse COCs from pre-ovulatory follicles were cultured in vitro in the presence of various concentrations of two types of TiO2 NPs (JRC NM-103 and NM-104) and four types of ZnO NPs (JRC NM-110, NM-111, and in-house prepared uncoated and SiO2-coated NPs) and the organization of a muco-elastic extracellular matrix by cumulus cells during the process named cumulus expansion was investigated. Results We show that COC expansion was not affected by the presence of both types of TiO2 NPs at all tested doses, while ZnO NM-110 and NM-111 induced strong toxicity and inhibited COCs expansion at relatively low concentration. Medium conditioned by these NPs showed lower toxicity, suggesting that, beside ion release, inhibition of COC expansion also depends on NPs per se. To further elucidate this, we compared COC expansion in the presence of uncoated or SiO2-coated NPs. Differently from the uncoated NPs, SiO2-coated NPs underwent slower dissolution, were not internalized by the cells, and showed an overall lower toxicity. Gene expression analysis demonstrated that ZnO NPs, but not SiO2-coated ZnO NPs, affected the expression of genes fundamental for COC expansion. Dosimetry analysis revealed that the delivered-to-cell mass fractions for both NPs was very low. Conclusions Altogether, these results suggest that chemical composition, dissolution, and cell internalization are all responsible for the adverse effects of the tested NPs and support the importance of a tailored, safer-by-design production of NPs to reduce toxicity

Comparative evaluation of the effects of short-term inhalation exposure to diesel engine exhaust on rat lung and brain

Combustion-derived nanoparticles, such as diesel engine exhaust particles, have been implicated in the adverse health effects of particulate air pollution. Recent studies suggest that inhaled nanoparticles may also reach and/or affect the brain. The aim of our study was to comparatively evaluate the effects of short-term diesel engine exhaust (DEE) inhalation exposure on rat brain and lung. After 4 or 18 h recovery from a 2 h nose-only exposure to DEE (1.9 mg/m(3)), the mRNA expressions of heme oxygenase-1 (HO-1), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and cytochrome P450 1A1 (CYP1A1) were investigated in lung as well as in pituitary gland, hypothalamus, olfactory bulb, olfactory tubercles, cerebral cortex, and cerebellum. HO-1 protein expression in brain was investigated by immunohistochemistry and ELISA. In the lung, 4 h post-exposure, CYP1A1 and iNOS mRNA levels were increased, while 18 h post-exposure HO-1 was increased. In the pituitary at 4 h post-exposure, both CYP1A1 and HO-1 were increased; HO-1 was also elevated in the olfactory tuberculum at this time point. At 18 h post-exposure, increased expression of HO-1 and COX-2 was observed in cerebral cortex and cerebellum, respectively. Induction of HO-1 protein was not observed after DEE exposure. Bronchoalveolar lavage analysis of inflammatory cell influx, TNF-α, and IL-6 indicated that the mRNA expression changes occurred in the absence of lung inflammation. Our study shows that a single, short-term inhalation exposure to DEE triggers region-specific gene expression changes in rat brain to an extent comparable to those observed in the lung

Characterizing the Chemical Profile of Incidental Ultrafine Particles for Toxicity Assessment Using an Aerosol Concentrator

Incidental ultrafine particles (UFPs) constitute a key pollutant in industrial workplaces. However, characterizing their chemical properties for exposure and toxicity assessments still remains a challenge. In this work, the performance of an aerosol concentrator (Versatile Aerosol Concentration Enrichment System, VACES) was assessed to simultaneously sample UFPs on filter substrates (for chemical analysis) and as liquid suspensions (for toxicity assessment), in a high UFP concentration scenario. An industrial case study was selected where metal-containing UFPs were emitted during thermal spraying of ceramic coatings. Results evidenced the comparability of the VACES system with online monitors in terms of UFP particle mass (for concentrations up to 95 µg UFP/m3 ) and between filters and liquid suspensions, in terms of particle composition (for concentrations up to 1000 µg/ m3). This supports the applicability of this tool for UFP collection in view of chemical and toxicological characterization for incidental UFPs. In the industrial setting evaluated, results showed that the spraying temperature was a driver of fractionation of metals between UF (<0.2 µm) and fine (0.2– 2.5 µm) particles. Potentially health hazardous metals (Ni, Cr) were enriched in UFPs and depleted in the fine particle fraction. Metals vaporized at high temperatures and concentrated in the UF fraction through nucleation processes. Results evidenced the need to understand incidental particle formation mechanisms due to their direct implications on particle composition and, thus, exposure. It is advisable that personal exposure and subsequent risk assessments in occupational settings should include dedicated metrics to monitor UFPs (especially, incidental).What’s important about this paper: Our work addresses the challenge of characterizing the bulk chemical composition of ultrafine particles in occupational settings, for exposure and toxicity assessments. We tested the performance of an aerosol concentrator (VACES) to simultaneously sample ultrafine particles (UFPs) on filter substrates and as liquid suspensions, in a high UFP concentration scenario. An industrial case study was selected where metal-bearing UFPs were emitted. We report the chemical exposures characterized in the industrial facility, and evidence the comparability of the VACES system with online monitors for UFP particle mass (up to 95 µg UFP/m3) as well as between UFP chemical composition on filters and in suspension. This supports the applicability of this tool for UFP collection in view of chemical and toxicological characterization of exposures to incidental UFPs in workplace settings.Highlights: - The VACES system is a useful tool for UFP sampling in high-concentration settings; - UFP collected simultaneously on filters and in suspension showed good comparability; - UFP chemical profiles were characterized; - Health-hazardous metals Ni and Cr accumulated in UFPs; - Understanding emission mechanisms is key to identifying exposure sources.This work was funded by SIINN ERA-NET (project id: 16), the Spanish MINECO (PCIN-2015-173-C02-01) and the French agency (Region Hauts de France). The Spanish Ministry of Science and Innovation (Project CEX2018-000794-S; Severo Ochoa) and the Generalitat de Catalunya (project number: AGAUR 2017 SGR41) provided support for the indirect costs for the Institute of Environmental Assessment and Water Research (IDAEA-CSIC). We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).info:eu-repo/semantics/publishedVersio