Elsevier

NanoImpact

Volume 23, July 2021, 100346
NanoImpact

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

https://doi.org/10.1016/j.impact.2021.100346Get rights and content

Highlights

  • Better knowledge of the critical role of WF-derived Q-UFP-induced lung toxicity.

  • ROS overproduction triggered by Q-UFP activates NRF2-ARE signaling pathway.

  • Alterations of the histone H3 acetylation triggered by Q-UFP.

  • New insights into the miRNA and mRNA profiles induced by Q-UFP.

  • Cell signaling pathways related to oxidative, inflammatory, and apoptotic events.

Abstract

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.

Introduction

Welding is commonly defined as an industrial process where two or more metal pieces are melted together by means of heat to form a join as the parts cool, leading to generation of complex and heterogenous aerosols of metal fumes, gases, and solid particles. Worldwide, an estimated 11 million workers have a job title of welder, and around 110 million additional workers probably incur welding-related exposures. Welding generally involves the exposure to high levels of fine- and ultrafine combustion-derived particulate matter (PM), UV radiation, and electromagnetic fields, and, in some cases, welders are also co-exposed to asbestos and solvents (Guha et al., 2017). In March 2017, a working group met at the International Agency for Research on Cancer (IARC) to evaluate the carcinogenicity of welding. For a majority of the cohort and case-control studies across different countries, time periods, and occupational settings they reported an elevated risk of lung cancer for workers employed as welders or occupationally exposed to welding fume (WF). The risk for lung cancer was higher in welders that had a longer or higher cumulative exposure (i.e., both mild-steel, MS and stainless-steel, SS welding). The working group concluded that there is “sufficient evidence in humans” that WF cause lung cancer and, therefore, classified welding as Group1/carcinogenic to humans (Guha et al., 2017).

Recent works have identified combustion-derived PM emitted within WF as the driving force of the adverse health effects that occur in humans (Gliga et al., 2019, McCarrick et al., 2019, Pega et al., 2020). While most of this PM mass is found in the fine-sized particle range (i.e., PM2.5, aerodynamic diameter ≤ 2.5 μm), the largest particle number is in the ultrafine-sized particle range (i.e., PM0.1, aerodynamic diameter ≤ 0.1 μm). Current health concerns were initially focused on the coarse (i.e., PM10, aerodynamic diameter ≤ 10 μm) and fine fractions, mainly because of their ability to migrate deeply and be highly retained down towards the respiratory tract. Emphasis has since then been clearly shifted from larger to ultrafine particles (UFP) (Stone et al., 2017). Epidemiological and toxicological evidence has indeed incriminated their unique properties, compared to those of the larger size particle fractions, including their higher lung deposition, particle number concentration, and surface area per mass. However, the UFP fraction of the WF has not been extensively studied yet and is currently not included in current air quality standards and/or guidelines relevant to the occupational settings, although it probably closely participates for the most part of the human health effects induced by WF (Falcone et al., 2018).

Health effects of WF exposure are complex, as their composition is affected by the type of welding alloy used (Chang et al., 2013; Erdem et al., 2020; Falcone et al., 2018; Leonard et al., 2010; Zheng et al., 2015). Welding involves several processes (e.g., oxyfuel [gas], arc, and resistance welding) and materials (e.g., MS and SS). Arc welding, including one type known as gas metal arc welding (GMAW), in which shielding gases are used to protect against oxidation, is still the most common industrial welding process (Antonini et al., 1999, Antonini et al., 2014). This strongest method of joining metals creates a relatively high levels of WF, the composition of which largely depending on whether a MS or a SS electrode is used: GMAW-MS fume contains primarily iron (Fe), zinc (Zn), and manganese (Mn), whereas GMAW-SS fume contains largely Fe, chromium (Cr, also existing in both CrIII and CrVI oxidation states), nickel (Ni), copper (Cu), and Mn (Antonini et al., 2014; Falcone et al., 2018; Ghanem et al., 2021; Stanislawska et al., 2020).

Welding exposures being somewhat complex owing to the diversity of welding processes, a majority of welders generally using multiple welding processes and consumables throughout their lifetime, and some confounders or additional exposures being possibly regarded in the workplace, associations are still difficult to established (Falcone et al., 2017; Shoeb et al., 2017; Zeidler-Erdely et al., 2010a, Zeidler-Erdely et al., 2019). All the cellular and/or molecular underlying mechanisms responsible for the toxicity and even the carcinogenicity of WF are still not fully understood. Therefore, controlled experimental studies are crucial to better understand which WF type and which of their metal components are the most toxic and have the greatest carcinogenic potential. Series of animal studies, by which the exposure to WF was well-controlled, were undertaken to better elucidate the role of WF metal composition (e.g., SS versus MS) on lung toxicity and cancer development. Some of them reported oxidative stress conditions, inflammatory responses, and immune suppression (Falcone et al., 2018; Grigg et al., 2017; Marongiu et al., 2016; McCarrick et al., 2019; Pega et al., 2020; Stanislawska et al., 2020). In a two-stage initiation-promotion model of lung carcinogenesis, GMAW-SS fume significantly increased lung tumor multiplicity after both an oropharyngeal aspiration and inhalation exposure in A/J mice (Falcone et al., 2017; Zeidler-Erdely et al., 2010b). An in vivo study indicated that WF derived from SS welding acted as a tumor promoter and lead to lung cancer in mice initially exposed to 3-methylcholanthrene (Zeidler-Erdely et al., 2019). Further demonstration of the greater toxic effect of GMAW-SS fume than GMAW-MS fume was reported in other animal models. For example, Antonini et al., 2007, Antonini et al., 2009 found that GMAW-MS fume caused no lung inflammation or lung injury in Sprague-Dawley rats 1, 4, or 11 days post-inhalation compared to GMAW-SS fume, which caused significant lung damage. Regardless, in vivo studies investigating exposures to WF as occurring in GMAW-MS or -SS are still lacking to contribute to an entire knowledge of the metal components closely responsible for their toxicity and/or their carcinogenicity. Two main topics that need to be further evaluated in future experimental models are not only the contribution of their individual chemicals and/or the specific roles of their UFP fraction in the development of lung toxicity and tumor formation, but also the cellular and/or molecular underlying mechanisms by which they contribute to cause lung toxicity and even lung tumorigenesis.

The better knowledge of the cellular and/or molecular underlying mechanisms involved in the pathogenicity of lung toxicity and even lung cancers related to WF exposure is still essential and could notably rely on relevant lung cell culture models (Leclercq et al., 2016). Indeed, clinical and basic science applications have focused on the bronchial epithelium because of the chronic inflammatory diseases resulting from disruption of this region (Boublil et al., 2013; Leclercq et al., 2017a). Up to now, only very few in vitro studies have been undertaken to better elucidate the individual chemicals and/or UFP sized-fractions of WF and their critical roles in the harmful health effects they induced.

As supported by the current literature, the underlying mechanism of post-inflammatory effects of WF is certainly mostly launched by soluble intermediate metals through the excessive production of reactive oxygen species (ROS) (Ghanem et al., 2021; Stanislawska et al., 2020). One of the most well-described toxicological mechanisms responsible for the lung adverse effects of combustion derived-PM is the pro-inflammatory response driven by oxidative stress (Abbas et al., 2019; Badran et al., 2020a; Cazier et al., 2016; Garçon et al., 2001, Garçon et al., 2006; Leclercq et al., 2018; Saint-Georges et al., 2008; Sotty et al., 2020). Of course, ROS generation and pro-inflammatory reaction have been reported among welders who are exposed to high levels of PM emitted within WF, but other underlying mechanisms, not even fully elucidated, like critical epigenetic alterations, could probably interplay to induce the adverse health effects. New toxicological research is therefore urgently requested to improve the current knowledge about the specific role of the UFP fraction in the overall toxicity of WF.

Hence, in this work, we sought to better evaluate the toxicity of the quasi-UFP fraction (Q-UFP, i.e., PM0.25) of the WF emitted by GMAW-SS in human bronchial epithelial cells (BEAS-2B). Firstly, we aimed to physically and chemically characterize this Q-UFP fraction. Secondly, attention was focused on the ability of this Q-UFP fraction to induce cytotoxicity, pro-oxidative and/or pro-inflammatory responses, genetic and/or epigenetic alterations, and, therefore, some related cell signaling pathway activation in BEAS-2B cells acutely or repeatedly exposed. These relevant results will also contribute to new insights of the critical role played by the Q-UFP fraction in human adverse effects induced by the exposure to GMAW-SS-derived WF.

Section snippets

Chemicals

Merck-Millipore (St Quentin-en-Yvelines, France) provided BEAS-2B cells (ATCC® CRL-9609™), cOmplete™, EDTA-free Protease Inhibitor Cocktail, Phosphatase Inhibitor Cocktail, RIPA buffer, CHAPS buffer, MILLIPLEX® MAP human cytokine/chemokine magnetic bead panel-immunology multiplex assays, methyl methanesulfonate (MMS), and all the other chemicals. ThermoFisher scientific (Villebon-sur-Yvette, France) provided collagen type I from rat tail, chloromethyl derivative of

Physical and chemical characteristics of WF-derived Q-UFP

The campaign to produce WF according to GMAW-SS process using an automatic welding bench was realized between the 10th and the 13th of September 2018, with the aim of collecting the Q-UFP (PM0.25). The average mass concentration of the total particles was 40.1 ± 3.9 mg/m3. Fifty-seven welding cords were used to collect 133 mg of Q-UFP. Concentrations in numbers ranged from 6.6 to 11.7 × 106/cm3 on the entire collection. The Q-UFP represented about 87% of the total amount of the particles from

Discussion

Although the current literature supported that the combustion-derived PM emitted within WF will represent the driving force of this occupational hazard, researchers are still far from having a fully detailed mechanistic explanation for its respiratory toxicity. Using a human bronchial epithelial cell model, we also try to better decipher the cellular and molecular underlying mechanisms of toxicity triggered by the WF-derived Q-UFP fraction.

Firstly, WF were produced according to GMAW-SS process

Funding sources

This work benefited from grants or contributions from “Action Santé Travail 59-62”, “Institut National de Recherche et de Sécurité”, “Institut Mines Télécom Lille Douai”, University of Lille, and the CLIMIBIO and CaPPA projects. The CaPPA project is funded by the French National Research Agency (ANR) through the PIA (Programme d'Investissement d'Avenir) under contract “ANR-11-LABX-0005-01” and by the Regional Council “Hauts-de-France”, and the “European Funds for Regional Economic Development”

Declaration of Competing Interest

The authors declare that they have no known competing financial interest nor personal relationship that could have appeared to influence the work reported in this paper.

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    LGJM and GG contribute equally to this work

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