# Evidence linking cadmium and/or lead exposure to immunomodulatory effects in mammals based upon an adverse outcome pathways approach, and research perspectives

Cloe Hadjadji, Quentin Devalloir, Colette Gaillard, Nico W van den Brink, Renaud Scheifler

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Cloe Hadjadji, Quentin Devalloir, Colette Gaillard, Nico W van den Brink, Renaud Scheifler. Evidence linking cadmium and/or lead exposure to immunomodulatory effects in mammals based upon an adverse outcome pathways approach, and research perspectives. Chemosphere, 2025, 371, pp.144056. 10.1016/j.chemosphere.2024.144056. hal-05008154

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# Evidence linking cadmium and/or lead exposure to immunomodulatory effects in mammals based upon an adverse outcome pathways approach, and research perspectives

Cloe Hadjadji<sup>a,b,*</sup> <sup>1D</sup>, Quentin Devalloir<sup>b</sup>, Colette Gaillard<sup>b,D</sup>, Nico W. van den Brink<sup>c</sup>, Renaud Scheifler<sup>b</sup>

## HIGHLIGHTS

* We built AOPs relevant to the effects of Cd and/or Pb on mammalian immune system.

* MIEs involve entrance in cells through Ca²⁺ channels or binding to cell surfaces.

* KEs involve oxidative stress, NF-κB signalling pathways, changes in CD4+/CD8+ ratio.

* AOs include immunodeficiency, autoimmunity, and potential increased infection rate.

* Gaps in current knowledge and detailed research perspectives are provided.

## GRAPHICAL ABSTRACT

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## ARTICLE INFO

Handling editor: Magali Houde

## ABSTRACT

For decades, studies have shown how exposure to non-essential trace metals such as lead (Pb) and cadmium (Cd) largely impact global wildlife. Ecoimmunotoxicology has emerged in the past two decades and focuses on the effects of pollutants on the immune system of free-ranging organisms. Adverse outcome pathways (AOPs) represent a conceptual approach to explore the mechanistic linkage between a molecular initiating event and adverse outcomes, potentially at all biological levels of organisation. The present paper proposes putative AOPs related to the effects of Cd, Pb, and the mixture Cd-Pb, on the immune system of mammals to address future questions in ecoimmunotoxicology. Molecular Initiating Events for both metals relate to entrance in cells through Ca²⁺ channels or bond to cell surfaces. Exposure to Cd, Pb and Cd-Pb share several similar Key Events (KEs), primarily an increase of oxidative stress (OS) in immune cells through production of reactive oxygen species. For both metals and the mixture, OS affects mitochondrial membranes, and induces apoptosis, ultimately decreasing immune cell number. Both metals affect innate immune system through nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) inflammatory signalling pathways, leading to an upregulation of inflammatory markers and mediators. Adaptive immune system is also affected by the exposure to both metals though a decrease of CD4+/CD8+ ratio, a decrease of MHCII, an inactivation of T<sub>H1</sub> and T<sub>H2</sub> response, and an inhibition of the humoral response mediated by various Ig. Mixture effects of Cd-Pb are less documented

<footnote><sup>a</sup> Swiss Ornithological Institute, Seerose 1, CH-6204, Sempach, Switzerland</footnote>

<footnote><sup>b</sup> Laboratoire Chrono-Environnement, UMR 6249, CNRS/Université de Franche-Comté, 16 route de Gray, 25000, Besançon, France</footnote>

<footnote><sup>c</sup> Division of Toxicology, Wageningen University, Box 8000, 6700 EA, Wageningen, the Netherlands</footnote>

<footnote>* Corresponding author. Swiss Ornithological Institute, Seerose 1, CH-6204, Sempach, Switzerland.
E-mail address: cloe.hadjadji@vogelwarte.ch (C. Hadjadji).</footnote>
resulting in a more speculative AOP, but potential synergic and antagonistic effects were identified. According to our AOPs, further research in ecoimmunotoxicology of metals in free-ranging mammals should focus on KEs related to NF-κB/MAPK inflammatory signalling pathways, changes in CD4+/CD8+ ratio and MHCI complexes, and on AOs related to auto-immune disorders and on the effective increase of infection rate, particularly in case of exposure to metal mixtures.

# 1. Introduction

Wildlife has been threatened for decades by several stressors from anthropogenic activities, which leads to the sixth mass extinction (Birnie-Gauvin et al., 2017; Ceballos et al., 2017) and makes conservation of the biodiversity one of the main issues of the century. Stressors are coming from many sources, including the exposure to different kinds of pollutants such as non-essential trace metals (TMs) (for readability purpose, this acronym and other used in the present paper are presented in Table 1). Contrary to essential elements that have important physiological functions in organisms, metals such as cadmium (Cd) or lead (Pb) are non-essential elements to vertebrates since they are not known to have any biological function (Aras and Ataman, 2007). Transfer of these toxic metals from the environment to the organisms depends on several parameters regulating their bioavailability (Lanno et al., 2004), defined as the proportion of the total concentration of a chemical that is actually taken up from the environment (Naidu et al., 2008). Toxicological bioavailability refers to the proportion of the concentration taken up that reaches toxicological targets (e.g., cells, tissues, organs) through the circulatory system, subsequently causing a biological response (Lanno et al., 2004). Historically, studies on TMs have mainly focused on accumulation in tissues and effects on classical ecotoxicological endpoints (mortality, reproduction outputs, histopathology, biochemical, physiological disorders ...) while unconventional endpoints, such as impacts on the immune system or behaviour, have attracted more attention only recently. Ecoimmunotoxicology refers to the understanding of the interactions between exposure to pollutants, immune cells and the "delicate interactions associated with homeostasis mediated by the immune system" (Tryphonas, 2005; Gallucci et al., 2020).

Exposure to non-essential TMs at toxic levels can affect the immune system through an up-regulation (immunoactivation) or a down-regulation (immunodeficiency), both referring to immunomodulation (Zelikoff et al., 1994; Devalloir et al., 2023). Immunodeficiency is defined as the decrease in effectiveness of the immune response (Gallucci et al., 2020) while immunostimulation refers to the enhancement of the defensive response, that can lead to auto-immune dysfunction or overall immune hypersensitivity (Luster and Gerberick, 2010; McKee and Fontenot, 2016; Gallucci et al., 2020). These immunomodulation processes mediated by toxic metals impact both innate and adaptive responses by unsettling the balanced mechanisms of immune cell regulation (Tersago et al., 2004; Ebrahimi et al., 2020).

<table><caption>**Table 1**
Abbreviations used in the present article.</caption><thead><tr><th></th><th></th><th></th><th></th></tr></thead><tbody><tr><td>AO(s)</td><td>Adverse Outcome(s)</td><td>MHCII</td><td>Major Histocompatibility Complex II</td></tr><tr><td>AOP (s)</td><td>Adverse Outcome Pathway (s)</td><td>MIE(s)</td><td>Molecular Initiating Event(s)</td></tr><tr><td>Ab</td><td>Antibody</td><td>MIP-2</td><td>Macrophage Inflammatory Protein 2</td></tr><tr><td>Ca</td><td>Calcium</td><td>NF-κB</td><td>Nuclear Factor Kappa B</td></tr><tr><td>Cd</td><td>Cadmium</td><td>Pb</td><td>Lead</td></tr><tr><td>GSH</td><td>Glutathione</td><td>MAPK</td><td>Mitogen-Activated Protein Kinase</td></tr><tr><td>IFN-γ</td><td>Interferon-γ</td><td>ROS</td><td>Reactive Oxygen Species</td></tr><tr><td>Ig</td><td>Immunoglobulin</td><td>TNF-α</td><td>Tumour Necrosis Factor-α</td></tr><tr><td>IL</td><td>Interleukin</td><td>TM(s)</td><td>Trace Metal(s)</td></tr><tr><td>KE(s)</td><td>Key Event(s)</td><td>δ-ALA</td><td>δ-AminoLevulinic Acid</td></tr><tr><td>LPS</td><td>Lipopolysaccharide</td><td>δ-ALA-D</td><td>δ-Aminolevulinic Acid Dehydratase</td></tr></tbody></table>

Immunomodulation is also linked to a myriad of external factors and stressors such as food shortage, life stage, or exposure to pathogens (Jackson et al., 2009; Poulsen and Escher, 2012). Toxic effects also vary with species, sex, type (chronic or acute) and route (ingestion, inhalation, dermal contact) of exposure (Gallucci et al., 2020). Immune cell dysfunction can occur through ionic substitution of toxic metals due to similar affinity to receptors on organic compounds, such as Cd²⁺ crossing Ca²⁺ channels (Choong et al., 2014). The main mechanism responsible for immune cell damages is related to the toxicological impact of reactive oxygen species (ROS) including immune cell membrane degradation (e.g., lipopolyperoxydation, Gera et al., 2015). Cadmium and Pb also have the ability to up- or down-regulate inflammatory mediators and markers (cytokines, chemokines ...), which can directly modify the immune response (Ebrahimi et al., 2020; Devalloir et al., 2023). Cadmium and Pb can thus induce differential effects on cytokine production through different signalling pathways (Thévenod and Lee, 2015). For example, an increase of the pro-inflammatory response (TNF-α) has been shown in wood mice living near a former smelter site contaminated by high Cd and Pb levels (Devalloir et al., 2023).

Since the emergence and the development of ecotoxicology in the 60's, the discipline has evolved from a relatively descriptive field to a much more mechanistic, hypothesis-based science. It aims at providing both a better understanding of how pollutants can affect organisms at various spatial, temporal, and biological organisation scales, and an improvement of risk assessment of chemicals released into the environment. Among the approaches used in ecotoxicology to study the cascade (from molecular to population or community levels) toxic effects of pollutants, adverse outcome pathways (AOPs) appeared as an innovative and useful concept that could improve mechanistic approaches, here in ecoimmunotoxicology (Tryphonas, 2005; Baudifier et al., 2024). Adverse outcome pathways represent a conceptual framework that highlights existing knowledge linking a molecular initiating event (MIE) induced by a given toxic chemical to an adverse outcome (AO) at multiple levels of biological organisation (Ankley et al., 2010). An AOP represents a succession of key events (KEs) that can take various forms and depend on the information available. Molecular initiating events are located at the molecular level and can be defined as "the initial points of chemical-biological interaction within the organism" (Fig. 1, Vinken et al., 2017). The relationship(s) between the different events can be causal, mechanistic, inferential or correlation-based (Ankley et al., 2010).

The present paper focusses on the effects of Cd and Pb on the immune system of mammals to propose putative AOPs to test more mechanistic hypotheses in ecoimmunotoxicology of metals and to address future questions in stress ecology (as other stressors than metals act on immunity) and ecotoxicology of free-ranging mammals. Here, we focus on terrestrial mammals because reviews have recently been published on immune toxicity of metals (or contaminants including metals) in fish (Lee et al., 2019, 2023), birds (Vallverdú-Coll et al., 2019) and marine mammals (Desforges et al., 2016). All arsenic (As), Cd, Pb, and mercury (Hg) belong to the 10 chemicals or groups of chemicals of major public health concern at a global scale (World Health Organization, 2024). We however focus here on Cd and Pb only as they are more relevant than As or Hg to terrestrial mammals in terms of exposure, and to keep the present article at a reasonable size. We based the construction of our AOPs on reviewing the literature in the area, our own experience in ecoimmunotoxicology, and different interactive databases related to AOPs. These databases provide open-source interfaces to deposit AOPs,
which facilitates the sharing of AOP knowledge and collaborative work (reviewed in Vinken et al., 2017).

## 2. Materials and methods

The articles presented in this paper have been selected according to the PRISMA statement (Moher et al., 2009). Literature search for study cases on terrestrial mammals was performed with Web of Sciences (June 2023) by using the following keywords, alone or in combination, full or truncated: immune system, immunity, oxidative stress, AOPs/Adverse outcomes pathways, biomarkers, mammals (wild or captive), metals/heavy metals, Pb/Lead, Cd/Cadmium, mixture effects. We also included other review articles about AOPs, immunology and immunotoxicity in mammals. We ended up with 123 articles after the identification phase and then selected the most pertinent articles based on their title and abstract (screening phase). After the eligibility phase, we finally kept 51 articles, that were included in our synthesis. After reviewing the literature and building pre-AOPs, we used the AOP-wiki interactive databases (https://aopwiki.org/, Vinken et al., 2017) to expand our AOPs and link them to the existing knowledge. Each KE identified in the literature was associated with a specific KE from AOP-wiki when it existed (Table 2). The Event ID of each KE was then added in our AOPs (see Figures). General knowledge (i.e., introduction, information about TMs) and perspectives were based on other bibliographic searches and on our experience.

## 3. Evidence of the effects of Cd on the immune system of mammals based on an AOP framework

Cadmium in the environment comes from both natural and anthropogenic sources. Primary natural source of Cd is related to geological processes, such as volcanic activity (Dølsch et al., 2006). Its occurrence is natural in all types of soils and minerals, e.g., sulfate, sulfide, carbonate ... (Kabata-Pendias, 2010). Anthropogenic sources of Cd include mining, smelting activity, transport, burial of nickel-Cd batteries, manufacturing, and pesticides spreading (Hossein-Khannazer et al., 2020). Main routes of exposure to Cd in mammals are through air, water, and food intake. Cadmium enters the body via the respiratory and the gastrointestinal tract (Mirkov et al., 2021). It is then transported into the blood, bound to some proteins such as albumin (Mirkov et al., 2021) and brought to the organs where it accumulates. Overall, all the organs are targeted by Cd but its accumulation is always higher in the liver and kidneys (Sarkar et al., 2013). Cells and tissues exposed to Cd are mainly damaged by oxidative stress, even if reactive oxygen species (ROS) are not directly produced by Cd. Reactive oxygen species are naturally produced during oxygen metabolism and are essential for cell signalling and homeostasis. However, high amounts of ROS disrupt cell functioning, including immune cells, and produce several adverse effects. Cadmium is considered as an immunotoxic inhibitor, influencing both innate and adaptive immune responses, which can alter immune cells functions (Mirkov et al., 2021). Major effects of Cd on the immune system reported in the literature are: (1) Cd-dependent apoptosis, (2) variations in immune cells activation and differentiation, and (3) enhancement of inflammatory responses in immune cells by up- and down-regulation of cytokines production (Genchi et al., 2020; Wang et al., 2021).

<caption>**Table 2**</caption>

Key-events found in the AOP-wiki database in relation to the AOPs developed in the present article. Individual Event ID are indicated in each associated box of the AOPs (see Figures). The last column contains the ID of the other AOPs from the AOP-wiki database that include the different key events of our own AOPs.

<table><thead><tr><th>Event ID</th><th>KE name</th><th>Biological level</th><th>Other AOPs</th></tr></thead><tbody><tr><td>1238</td><td>Activation of oxidative stress pathway</td><td>Molecular</td><td></td></tr><tr><td>1969</td><td>Increase, Oxidative Stress</td><td>Molecular</td><td>437/457/459/507/509/510/511</td></tr><tr><td>1770</td><td>Decrease, Mitochondrial membrane potential</td><td>Cellular</td><td>328/387/447</td></tr><tr><td>1262</td><td>Apoptosis</td><td>Cellular</td><td>205/207/212/285/419/439/452/393/476/460/500/491/502</td></tr><tr><td>1365</td><td>Increase, Apoptosis</td><td>Cellular</td><td>322/331/330/325/326/441/444/509</td></tr><tr><td>1172</td><td>Increased activation, Nuclear factor kappa B (NF-κB)</td><td>Cellular</td><td>382/377/319/443</td></tr><tr><td>130</td><td>Depletion, GSH</td><td>Cellular</td><td>492</td></tr><tr><td>151</td><td>Activation, Inflammatory cytokines, chemokines, cytoprotective gene pathways</td><td>Molecular</td><td></td></tr><tr><td>1020</td><td>Suppression, IL-2 and IL-4 production</td><td>Cellular</td><td>154</td></tr><tr><td>1225</td><td>Immune system inflammation</td><td>Tissue</td><td></td></tr><tr><td>1750</td><td>Increased inflammatory immune responses</td><td>Tissue</td><td>320/426</td></tr><tr><td>149</td><td>Increase, Inflammation</td><td>Cellular/Tissue</td><td>27/115/206/280/439/505</td></tr><tr><td>1569</td><td>Impaired T cell activation</td><td>Cellular</td><td></td></tr><tr><td>1702</td><td>Suppression of T cell activation</td><td>Cellular</td><td>227</td></tr><tr><td>1644</td><td>Impaired Ab production</td><td>Cellular</td><td></td></tr><tr><td>403</td><td>Suppression, Immune system</td><td>Individual</td><td>14/84/85/432</td></tr></tbody></table>

We identified two main MIEs for Cd, which result in significant AOs on the immune system (Fig. 2). The first one is linked to the chemical properties of Cd as, under its ionic form Cd²⁺, it is known to interfere with essential metal ions involved in biological functions of organisms (Thévenod and Lee, 2015). Thus, Cd²⁺ can block Ca²⁺ channels because of their thiol group affinity. It induces a major disturbance of cellular homeostasis and affects protein structure. The second MIE is the binding of Cd on the cell surface (Choong et al., 2014). Both MIEs are activating a reaction cascade of several KEs linked to immune dysfunctions.

The first step of this cascade is the induction of ROS production. Increase in oxidative stress affects mitochondrial membranes, which plays a major role in mitochondrial ROS formation, and leads to apoptosis (Genchi et al., 2020; Wang et al., 2021). Reactive oxygen species increase is responsible for the activation of the NF-κB pathway, which is in turn linked to the increase of the production of the pro-inflammatory cytokines IL-1b, IL-6 and TNF-α (Wang et al., 2021) and of some chemokines (IL-8, MIP-2). Enhancement of both IL-6 and TNF-α in neutrophils (i.e., the most abundant type of granulocytes) leads to potential indirect effects of Cd on haematopoiesis because these cytokines are regulators of this mechanism (Ulich et al., 1989). It can indicate a hypersensitive reaction. Bioassays using macrophages from murine cells line RAW 264.7 exposed to Cd concentrations showed an increase in the pro-inflammatory cytokine IL-1β associated with an

<figure><img src="image_5.png" /><figcaption>**Fig. 1.** Generic AOPs structure for a single chemical, that can be applied to any organism and spread for different biological organisations (adapted from Vinken et al., 2017). MIE stands for molecular initiating events, KE for key events and AO for adverse outcome.</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>**Fig. 2.** Representation of an adverse outcome pathway for cadmium toxicity on the mammal immune system. As in Fig. 1, the chemical initiator is represented in orange, molecular initiating events in green, key events in pink and adverse outcomes in red. Bright pink (also indicated with a black star) key events are potential biomarkers that could be used in future applied studies. The M and N abbreviations above some boxes stand for macrophages and neutrophils, respectively. Numerical Event ID next to the different components of the AOP are from https://aopwiki.org/, and are also presented in Table 1. *The modulation of TNF-α vary depending on cadmium exposure time. Acute exposure result in an increase of its production while a longer exposure time resulted in a decrease of TNF-α.</figcaption></figure>

inhibition of IL-6 and IL-10 (Riemschneider et al., 2015). Because of this modulation, a potential hyper-reactivity of macrophages can occur, which could lead to autoimmune disorder in mammals. In addition, Jung and Oh (2019) reported an increase of TNF-α 6 h after an exposure to Cd, which would indicate a response from macrophages to Cd toxicity. However, 18 h after the exposure, a decrease in TNF-α was associated with apoptosis in RAW 264.7 (So et al., 2018). These results suggest that Cd immunotoxicity varies according to exposure time, resulting in a hyperactivation of the immune system during short-term exposure while long-term exposure results in immunodeficiency.

During the regulation of oxidative stress balance, the increase of glutathione (GSH) can cause a decrease in CD4+/CD8+ ratio (CD: cluster of differentiation), with a decrease of CD4+ (helper T-cells) and an increase of CD8+ (cytotoxic T-cells; Devalloir et al., submitted). Moreover, a decrease in CD4+ can be linked to a diminution of IL-2 and IFN-γ production (Pathak and Khandelwal, 2009). A decrease in IL-2, the main factor for proliferation of activated T-cells, has been observed in mouse splenic cells after Cd exposure (Pathak and Khandelwal, 2009). These results show that Cd participates to the overall inactivation and immunodeficiency of the adaptive immune response in mammals. In addition, IFN-γ can be both enhanced or inhibited by Cd exposure (Krocova et al., 2000; Turley et al., 2019), depending on the dose and time-exposure. Turley et al. (2019) reported an enhancement of IFN-γ during low-dose Cd exposure (32 ppm in drinking water for 10 days) without significantly affecting T-cell polarization in rats. On the contrary, a decrease of IFN-γ has been reported in T-cells when exposed to higher Cd concentrations (Krocova et al., 2000).

Reactive oxygen species production also promotes the expression of some pro-inflammatory cytokines such as IL-1b, IL-6 and TNF-α and inhibits anti-inflammatory cytokines such as IL-10 in macrophages (Wang et al., 2021). The overexpression of IL-1b, IL-6 and TNF-α as well as the inhibition of IL-10 can result in hyper-inflammation, auto immune disorders and in rare cases, cancer (Thévenod and Lee, 2015). Overall, a dysregulation in cytokine production can affect the activation of TH1, Th2 or TH17 (Devalloir, 2023). In addition, the binding of Cd on the cell surface induces a decrease of the expression of the major histocompatibility complex class 2 (MHCII) on B-cells, which could affect the

presentation of pathogens to antigen presenting cells and decrease the effectiveness of the immune system in response to pathogens. Antibody production is also directly affected by Cd exposure. In the spleen, B-cell antibody responses differ with dose, class of antigen (IgG, IgM), antigen type (T-cell independent, T-cell dependent) and exposure duration (Fujimaki et al., 1982). A decrease in IgE production has been highlighted after Cd exposure in humans (Wang et al., 2021). Cadmium exposure also induces the transformation of IgE antibodies into IgG produced by B lymphocytes in humans (Marth et al., 2001), indicating an impairment of the cell activation initiation during cytotoxic signals (Wang et al., 2021).

## 4. Evidence of the effects of Pb on the immune system of mammals based on an AOP framework

Lead is a naturally occurring but potentially harmful element, present at high concentrations in the environment mainly because of anthropogenic sources. This element is naturally present in the earth crust at very low concentration and small amount are released in the environment due to natural processes as rocks weathering (Pattee and Pain, 2002). Regarding anthropogenic sources, Pb is used in paints, pesticides, gasoline, batteries, and other emissions that can come from smelting activity and industry (Wani et al., 2015). Main routes of exposure in mammals are through air and food intake, where it can be absorbed by respiratory and digestive tracts (Balali-Mood et al., 2021). Accumulation in several organs generate multiple adverse effects such as nervous system disorders (e.g., saturnism), kidney and liver alteration, anaemia, or learning disorders in children (Ebrahimi et al., 2020). The oxidant-antioxidant balance system can be disturbed by Pb exposure, which can lead to inflammatory responses in organs (Balali-Mood et al., 2021). Lead also is considered as an immunotoxic inhibitor, influencing both innate and adaptive immune responses that can alter all immune cells (Metryka et al., 2018). The major effects of Pb on the immune system reported in the literature are (1) the enhancement of anti- and pro-inflammatory responses, (2) depression of humoral immunity, and (3) immune cell damage at high Pb concentration.

Lead exposure, either chronic or acute, can induce two potential
MIEs, that result in severe AOs on the functioning of the immune system. First, the inhibition of δ-aminolaevulinic acid dehydratase (δ-ALA-D) by Pb can lead to a rapid oxidizing of δ-ALA. Quantification of δ-ALA-D has long been used as a biomarker of Pb exposure (Rocha et al., 2012). However, its use as a biomarker of Pb exposure has to be cautious because modulation of δ-ALA-D can occur after an exposure to other metals. When δ-ALA-D oxidation occurs, it induces a production of free radicals including ROS, leading to oxidative stress (Lopes et al., 2016). Second, Pb can bind to the cell surface to oxygen compounds due to its chemical properties. Both MIEs are activating a reaction cascade of several KEs linked to immune responses dysfunctions (Fig. 3).

Innate and adaptive immune responses are both impacted by a chronic or acute Pb exposure, by producing ROS, modulating cytokines production, and T<sub>H1</sub>/T<sub>H2</sub> polarization. The induction of ROS production promotes or suppresses the expression of a few cytokines. Iavicoli et al. (2006) reported an increase of IL-4 and a decrease of IL-2 and IFN-γ production in mice exposed to high Pb level. In comparison, low-dose Pb exposure induces a decrease in IL-4, and an increase of IL-2 and IFN-γ, meaning that immune response modulation might be not linear (Iavicoli et al., 2006). These responses differently affect T<sub>H1</sub> and T<sub>H2</sub> polarization and humoral responses. High Pb levels could cause an impairment of cytotoxic signalling (T<sub>H1</sub>) while low-dose Pb affects the coordination of helper T-cell response (T<sub>H2</sub>).

Studies also highlighted an increase of three pro-inflammatory cytokines, IL-1β, TNF-α and IL-6, after an exposure to Pb (Fenga et al., 2017), which could promote auto-immune damage. However, antioxidant activation associated with Pb exposure, in response to oxidative stress, modified pro-inflammatory responses by decreasing IL-1β, TNF-α and IL-6. Furthermore, synergistic effect between Pb and lipopolysaccharides (i.e., LPS; an inducer of inflammatory response in organisms) has been demonstrated in the literature (Cheng et al., 2006). LPS-induced mortality under Pb exposure has been observed in animal studies (Dentener et al., 1989) and could be due to the tremendous release of TNF-α by different cells including both T and B lymphocytes, macrophages, and monocytes. In human macrophages, in vitro stimulation by LPS showed a surface TNF-α expression on over 50% of the cells after 24 h. IL-8 gene expression is also enhanced by Pb exposure when

associated with p42/44 MAPK in human (Lin et al., 2015). Indeed, p42/44 MAPK has been observed to activate IL-8 gene expression (Metryka et al., 2018).

Lead also causes a decline in humoral immunity by affecting both T<sub>H1</sub> and T<sub>H2</sub>, which may increase host vulnerability to bacterial and virus infections (Hemphill et al., 1971; Gainer, 1974; Luster et al., 1978). Lead affects humoral immune response by reducing IgA and IgG production, which can induce inflammatory diseases and cancers as long-term AOs (Anetor et al., 2008). Naïve CD4<sup>+</sup> exposed to Pb will preferentially differentiate in T<sub>H2</sub> and lead to the inhibition of T<sub>H1</sub> cell growth. This results in a decrease of IFN-γ and IgG<sub>2</sub> with an increase of IL-4, IgE, and IgG<sub>4</sub>. Mice exposed to Pb and infected by *Listeria mono-cytogenes* showed significant reduction in the CD4<sup>+</sup> and CD8<sup>+</sup> production (Dyatlov and Lawrence, 2002).

**5. Similarities and differences between Cd and Pb effects on the immune system of mammals based on an AOP framework**

Overall, Cd and Pb produce similar effects in vertebrates with outcomes resulting either immunostimulation or immunodeficiency. Here, we propose a single AOP (Fig. 4) presenting mechanisms and pathways common to the exposure to both trace metals. To our knowledge, no experiments have been conducted with the very same conditions to directly compare the immunotoxicological effect of Cd and Pb. This means that we cannot be sure whether the differences in the immunomodulatory effects of the exposure to Cd or Pb are actual differences in the underlying mechanisms or if this is related to a lack of research.

Concerning the innate system, common mechanisms include tissue
inflammation related to the increased production of pro-inflammatory
cytokines within the NF-κB signalling pathway and a reduction of
phagocytosis activity. In both cases, cytokine concentrations are
modulated either up or down, leading to an increased inflammation or
an impaired signalling of the immune response. Concerning the adaptive
response, both trace metals affect the expression of MHCII and produce a
decrease of the CD4+/CD8+ ratio, leading to an inhibition of immu-
noglobulin production and an impairment of Th1 and Th2 response.
These various common pathways all lead to three main adverse effects

<figure><img src="image_7.png" /><figcaption>**Fig. 3.** Representation of an adverse outcome pathway for lead toxicity on the mammal immune system. The chemical initiator is represented in orange, molecular initiating events in green, key events in pink and adverse outcomes in red. Bright pink (also indicated with a black star) key events are potential biomarkers that could be used in future applied studies. The M and N abbreviations above some boxes stand for macrophages and neutrophils, respectively. The dotted arrows indicate that the link between two events is not precisely detailed in the literature. Numerical Event ID next to the different components of the AOP are from https://aopwiki.org/, and are also presented in Table 1.</figcaption></figure>
<figure><img src="image_8.png" /><figcaption>**Fig. 4.** Representation of an adverse outcome pathway for both lead and cadmium toxicity on the mammal immune system. The molecular initiating events are represented in green, key events in pink and adverse outcomes in red. Bright pink (also indicated with a black star) key events are potential biomarkers that could be used in future applied studies. The dotted arrows indicate that the link between two events is not precisely detailed in the literature. Numerical Event ID next to the different components of the AOP are from https://aopwiki.org/, and are also presented in Table 1.</figcaption></figure>

such as immunostimulation, potential auto-immune disorder/damage, immunodeficiency leading to potential increase infection rate, and in some cases even cancer.

Differences in immunomodulatory effects of the exposure to Cd or Pb rely mainly on one feature specific to Pb (Inhibition of the δ amino-laevulinic acid dehydratase, ALA-D) and differences in cytokines mobilised through exposure to Cd or Pb (see Figs. 2 and 3, and text below). The inhibition of ALA-D increases oxidative stress in cells (Rocha et al., 2012) but we did not find to what extend this contributes to the overall increase of oxidative stress related to the exposure to Pb. Cytokines and chemokines are modulated by the exposure to Cd or Pb but according to previous studies, most of them are different for each metal indicating that different pathways of the immune response are involved. For example, the extra activation of NF-κB leads to increased IL-8 and MIP-2 in the case of Cd exposure while an increase of IL-4 is reported in the case of Pb exposure (Iavicoli et al., 2006; Wang et al., 2021). In the case of immunoglobulin production, studies showed an inhibition of IgE and an inhibition of IgA and IgG in the case of Cd and Pb, respectively (Anetor et al., 2008; Wang et al., 2021).

## 6. Evidence of the effects of a Cd-Pb mixture on the immune system of mammals based on an AOP framework

In the environment, organisms are generally exposed to a mixture of contaminants rather than to a single trace metal. These multiple exposures can be detrimental to wild vertebrates even if contaminants are present at low concentrations. This concept is called “cocktail effect” or “mixture toxicity” (Beyer et al., 2014). Ecotoxicological studies have mainly been performed through a chemical-by-chemical approach of exposure, while wildlife are exposed to “dynamic chemical mixtures” through their life (Morrissey et al., 2023). It exists only a few papers that relate mixture effects of trace metal elements (Cd, Pb) on the immune system. Thus, little is known about their additive, synergetic and/or antagonistic effects on the immune system of vertebrates. Synergetic and antagonistic effects are the results of the competition among metals for organic and inorganic ligands during accumulation and uptake (Altenburger et al., 2003; Belden et al., 2007; Balistrieri and Mebane, 2014). It has been shown that Cd-Pb mixture can enhance the

production of white blood cells in mice, indicating the activation of the immune system in response to the exposure (Cobbina et al., 2015). In addition, these authors found that the exposure to a mixture result in a global antagonistic effect, in comparison to their individual toxic effect. One hypothesis could be that Cd and Pb both produce thiols such as GSH, enabling a protection of the cell from their toxicity. However, other antioxidant markers (superoxide dismutase, glutathione peroxidase) were lower when mice were exposed to a Cd and Pb mixture, potentially creating higher oxidative stress in immune cells. Another study in male rat showed that the exposure to a mixture of eight trace metals, including Cd and Pb, induces a suppression of both humoral and cell-mediated immune response (Jadhav et al., 2007). Fortier et al. (2008) depicted a non-significant effect of the Cd-Pb mixture in human leucocytes (viability and apoptosis proportion). These few studies provide different conclusions, suggesting that a variety of pathways could be involved in the response to Cd-Pb mixture. However, we still lack information about how cytokines signalling, $T_H1/T_H2$ polarization of B-cells and antibody production could be modulated by this mixture. Here, we propose a potential AOP for Cd-Pb mixture on the immune system of mammals with potential additive effects or synergetic and antagonistic interactions (Fig. 5), based on the few studies presented earlier as well as on hypotheses and deductions from our single-chemical AOPs (Figs. 2–4).

## 7. Gaps in current knowledge and research perspectives

In the present article, we built putative AOPs depicting the cascade effects of Cd and Pb alone, and in combination, on the immune system of mammals. We identified several pathways that could lead to immune dysfunctions in mammals. However, despite identifying links between contaminants, MIEs, KEs and AOs using the existing knowledge, how the different cascades of reactions occur in wild organisms, and what adverse outcomes arise at various levels of biological organisation remain uncertain in wildlife since most of the work has been done in lab animals. To fill this gap, we identified several biomarkers indicated in bright pink boxes (and indicated by an asterisk) in Figs. 2 to 6, and develop hereinafter the research priority identified from these putative AOPs, the literature and our own experience in immunotoxicity of
<figure><img src="image_9.png" /><figcaption>**Fig. 5.** Representation of an adverse outcome pathway of the mixture of cadmium and lead toxicity on the mammal immune system. The chemical initiator is represented in orange, molecular initiating events in green, key events in pink and adverse outcomes in red. Bright pink key events (also indicated with a black star) are potential biomarkers that could be used in future applied studies. (>=) are for additive interaction, (+) are for synergetic interaction, and (-) are for antagonistic interaction. Numerical Event ID next to the different components of the AOP are from https://aopwiki.org/, and are also presented in Table 1.</figcaption></figure>

metals in free-ranging small mammals (Devalloir et al., 2023; Powolny et al., 2023; ongoing research).

One of the primary aspects to consider, common to both Cd and Pb exposure, relies upon their impacts through NF-κB. This family of inducible transcription factors regulates multiple aspects of innate and adaptive immune functions and is a pivotal mediator of inflammatory responses (Liu et al., 2017). NF-κB induces several functions in innate immune cells, including maturation of dendritic cells, activation and differentiation of inflammatory T cells, polarization of macrophages and their production of pro-inflammatory cytokines/chemokines, and recruitment of and anti-apoptotic effect on neutrophils. These cells express pattern-recognition receptors that detect various microbial components (so-called pathogen-associated molecules patterns, PAMPs) as well as molecules released by necrotic cells and damaged tissues (co-called damage-associated molecular patterns, DAMPs). Thus, a disruption of NF-κB by contaminants (here Cd or Pb) could alter these functions and might lead to a lower ability of the innate immune system to fight against pathogens. NF-κB also exerts a central role in mediating T cell receptors signalling and naive T-cell activation, which is an important component of the adaptive immune system. Deregulation of NF-κB activation can cause aberrant T-cell activation, which might lead to associated autoimmune and inflammatory responses. Inflammation is a protective response of the host to infections and tissue damages and is normally beneficial and resolved in a timely manner. Given that wild rodents have been shown to be particularly exposed to infections, deregulation of NF-κB activation by metals might be particularly harmful to wild animals, both by lowering their ability to control pathogenic infections and through excessive or long-lasting tissue damages, ultimately leading to acute or chronic inflammatory diseases (Liu et al., 2017). Studying this pathway, especially in free-ranging wildlife, is however difficult due to the complexity of the activation of NF-κB, which involves two major pathways (canonical and non-canonical) related to different signalling mechanisms. The first pathway responds to diverse stimuli including ligands of various cytokine receptors, pattern-recognition receptors (PRRs), TNF receptor (TNFR) superfamily, B and T cell receptors. In contrast, the non-canonical pathway selectively responds to a specific group of stimuli, including ligands of a subset of TNFR superfamily members (Liu et al., 2017). Furthermore, the activity of NF-κB depends on three layers of regulation. A first layer refers to the dynamic nature of the NF-κB system, which is never static and is schematically under 4 forms of activity (OFF, low- and high-ON, and constitutively active). In “OFF” state, the NF-κB activity is usually too low to be reliably detected

(Meier-Soelch et al., 2021). Constitutively active state refers to significant NF-κB activity found in specific cells such as Sertoli cells, B cells, hair follicle cells and neurons, making it difficult to be studied in wildlife. “Low ON” state refers to a continuous low-grade activation that occurs in situations of smoldering inflammation or in tumour microenvironment, and “high ON” state is a fast and transient activation in response to proinflammatory triggers. Another layer of regulation is related to the gene specific recruitment of NF-κB and a third one involves a remarkable cell to cell variability, with cells having low NF-κB activity while adjacent cells exhibit high activity and strong activation of NF-κB target genes (Meier-Soelch et al., 2021). These authors therefore warn that (cell) population-based assays can be potentially misleading. The NF-κB activation rarely occurs in isolation and frequently other stress pathways such as p38 mitogen-activated protein kinases (p38 MAPKs), c-Jun N-terminal kinases (JNKs) or JAK-STAT are activated in parallel. As all have been shown to be involved in metal-related toxicity in human or lab-animal models (but not in AOP framework to the best of our knowledge), they are also good candidate models to be further studied in metal immunotoxicology.

Studying the responses of the exposure to Cd and/or Pb on the innate immune system involving NF-κB pathways would rely upon (i) an estimate of the exposure to the above-mentioned metals, (ii) the potential related increase of ROS production, (iii) the activation of the NF-κB pathway, (iv) the increase of some cytokines, (v) the modulation of more general mechanisms (increase of haematopoiesis, increase of neutrophils, decrease of phagocytosis activity ...), and (vi) ultimately adverse outcomes such as the increase of infection rate. Estimating the exposure of wildlife to contaminants in all its complexity (contaminant mixtures, spatio-temporal variations, species- or stage-specificity, ecological context) is still a hard task recently discussed (Morrissey et al., 2023). However, quantifying the exposure to metals and metalloids using e.g. ICP methods upon invasive (internal organs such as liver or kidneys) or less invasive (blood, hair ...) of a species of interest along a pollution gradient is relatively efficient. Similarly, measuring ROS production (free radicals such as superoxide anion or nitric oxide, non-radical ROS such as hydrogen peroxide), oxidative damage (dROMS, TBARS, protein carbonyls) and enzymatic (SOD, GPx, catalase, GST, GR) and non-enzymatic (OXY, GSH) anti-oxidant defences is now allowed by a number of assays that can be relatively easily performed on tissues or less invasive fluids like blood (Costantini, 2022; Tanabe et al., 2022). The study of the NF-κB pathway and its three layers of regulation (activity, gene specific recruitment, cell to cell variability) is far more complex in wildlife. Meier-Soelch et al. recently reviewed the concepts
and the methods related to the monitoring of levels of cellular NF-κB activation states (Meier-Soelch et al., 2021). Most of these techniques require high biochemical skills and appropriate lab equipment, and furthermore need cell isolation and/or cell culture to take into account the high variability of NF-κB activation state among cell types and induction kinetics. Most of this might be out of reach to wildlife toxicologists, at least on a short term. The relatively recent development of transcriptomic approaches in ecology and ecotoxicology, however, could bring important insights into the involvement and the regulation of NF-κB pathways in wildlife exposed to pollutants. An example, if not of a true ecotoxicological study but at least of a comparison of environments differing by pollution level (among other parameters), is a recent analysis of the transcriptome of rural and urban great tit (Parus major) populations in both blood and the liver. The study revealed that the NFKBID gene (coding for the protein called nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, delta; IκBNS) was one of the 20 most significant annotated genes that were differentially expressed between urban and rural birds in whole blood transcriptomes (Watson et al., 2017). Similar RNA-sequencing analyses could be performed in wildlife populations inhabiting habitats with contrasted levels of Cd and/or Pb, or in individuals exposed under semi-natural controlled conditions in mesocosms. Complementarily to the random search of differential expression of certain genes among exposed and non- or less-exposed populations or individuals from the global transcriptomes, the present AOPs allow to specifically look for possible differential expression of genes related to NF-κB pathways. Such approach of course requires that the genes and RNAs of interest have been sequenced and identified in databases, but the number of fully sequenced genomes increases fast. The full genome of 1,000 species is available within the Tree of Life Programme (Wellcome Sanger Institute, 2023) and overall it is estimated that the genome of more than 3,000 species has been fully sequenced, with however large disparities in genomic representation (e. g. overrepresentation of vertebrates over arthropods) and assembly quality (gene annotations available for 34% of the taxa, Hotaling et al., 2021). To go further into the key events of the present AOPs, the potential increase of some cytokines under Cd and/or Pb exposure would be required but the lack of species-specific monoclonal antibodies has historically been a barrier to integrating free-ranging mammals, beyond the well-known lab model species such as lab mice (Mus musculus) and rats (Rattus norvegicus). Even if the availability of reagents developed in lab mice has interestingly been used to study the immunity of their wild counterparts (Abolins et al., 2011, 2017, 2018), the immune system of most of wild species cannot be studied with that resolution because monoclonal antibodies developed in lab mice do not (or hardly) cross-react with other rodent species, even phylogenetically closed (e.g. Apodemus sylvaticus; Devalloir, 2023). Some cytokines and other immune molecules such as immunoglobulins can however be quantified through the use of e.g. Elisa kits that sometimes give satisfactory results among species (Devalloir et al., 2023). However, here again, the development of full genome and transcriptome sequencing is a promising perspective as it offers the opportunity to study genes or their expression rather than the proteins themselves, or produce recombinant proteins for protein-based immunology in species of interest (Flies and Wild Comparative Immunology Consortium, 2020). Flies et al. argue for “a systematic effort to develop and characterize antibodies, nanobodies, or aptamers that bind to conserved protein motifs across taxonomic orders” to overcome the funding constraints limiting the development of specific reagents for all species of interest (Flies and Wild Comparative Immunology Consortium, 2020). The study of other key events described in the present AOPs such as the increase of neutrophils or the decrease of phagocytosis activity can be performed by relatively classical -but usually fluid- and time-consuming- techniques such as blood smears and phagocytosis assays, microbial killing assays (MKA) or whole blood microbial killing assay (WBMKA), respectively (Devalloir, 2023).

Regarding the adaptive components of the immune system, exposure

to Cd or Pb has been shown to decrease CD4+/CD8+ ratio and MHCII molecules expression, affecting the activation of T<sub>H1</sub> and T<sub>H2</sub> cells and reducing humoral responses. T lymphocytes (T cells) are divided into two major cell types: T helper (T<sub>H</sub>) and T cytotoxic (T<sub>C</sub>) cells. They can be distinguished by the presence of CD4 (for T<sub>H</sub>) and CD8 (for T<sub>C</sub>) membrane glycoproteins on their surfaces. The ratio of CD4<sup>+</sup> to CD8<sup>+</sup> T cells is approximately 2:1 in peripheral blood of healthy human and mouse, while a decreased ratio is usually an indication of immunodeficiency or autoimmune disease, aging or inflammation. T cells express a unique antigen-binding receptor (T-cell receptor, TCR), which recognizes processed pieces of antigen (usually peptides) bound to cell membrane proteins, the MHC. Naïve CD4<sup>+</sup> T<sub>H</sub> cells, when they bind to an MHCII-peptide complex, become activated, proliferate and differentiate into various effector T-cell subsets, mainly T helper cells (type 1 (T<sub>H1</sub>) and type 17 (T<sub>H17</sub>)), and T helper type 2 (T<sub>H2</sub>) and T follicular (T<sub>FH</sub>) cells. T<sub>H1</sub> and 17 regulate responses to intracellular pathogens while T<sub>H2</sub> and T<sub>FH</sub> regulate responses to extracellular pathogens such as bacteria and parasitic worms. Naïve CD8<sup>+</sup> T<sub>C</sub> cells, when they bind to an MHCII-peptide complex, become activated, proliferate and differentiate into cytotoxic T lymphocyte (CTL), which have a vital function in recognizing and eliminating cells displaying non-self antigen-MHCII complex (such as virus infected or tumour cells). Further research should thus also focus on cell populations, and more precisely the CD4+/CD8+ ratio, and associated major histocompatibility complex (MHC) molecules (Punt, 2019). Such immunophenotyping can be done through flow cytometry but this would require species-specific antibodies as anti-mouse antibodies do not or hardly cross-react with immune cells from other rodent species (Devalloir, 2023; García-Mendoza et al., 2021). As for some of the perspectives mentioned above, the development of antibodies that bind to conserved protein motifs across taxonomic orders, if not the development of specific reagents for some species of interest, is required, as we do not see how the CD4+/CD8+ ratio could be measured otherwise. Complementarily, a transcriptomic approach can also be developed as it has recently been done in sheep inoculated with a virulent *Anaplasma phagocytophilum* to study the temporal patterns of gene expression in response to the inoculation (Eskeland et al., 2023). In the latter, however, the transcriptomic approach was completed by flow-cytometry of peripheral blood mononuclear cells to further study the changes of CD4+/CD8+ ratio.

The putative AOPs proposed in the present articles focus mainly on sub-individual levels of organisation, and perspectives of research aiming at better understanding immunotoxicological mechanisms at these levels of organisation have been presented above. The AOP approach, in ecotoxicology and by extension here in ecoimmunotoxicology, aims to also address effects of pollutants at higher levels of organisation. A deficient immune system at the sub-individual level could lead to severe impacts on individual fitness and population dynamics. The hypothesis that reduced immune function and/or increased oxidative stress in wildlife chronically exposed to pollutants might increase parasite prevalence and/or loads and impact fitness has poorly been investigated. It has been shown that wild rodents exhibited significant differences in the functioning of their immune system compared to lab animals. (Viney and Riley, 2017) recently reviewed this issue and stated that wild rodents compared to laboratory animals exhibit (i) much higher humoral (antibody) responses, (ii) extensive antigenic exposure as revealed by cellular immune system, (iii) depressed proliferative and cytokine responses in *ex vivo*-stimulated immune cells. Studying exposure of wildlife to pathogens is increasingly accessible through e.g. high-throughput DNA sequencing, allowing for the identification of a number of zoonotic or non-zoonotic, emerging or well-known, external or internal pathogens and parasites (e.g., Diagne et al., 2016; Villette et al., 2020). For assessing bacterial communities, a mix of organs allows to get a better view of bacterial assemblages that single organs (Villette et al., 2017) but this unfortunately requires the sacrifice of (some) individuals, which might be ethically questionable when working on protected and/or endangered wildlife. The use of
fluids like blood or faeces is less invasive but would give a partial view of the bacterial assemblages or even false negatives as their presence in the fluids depends on their distribution between organs and fluids, or on their excretion kinetics. Viruses also are increasingly studied in free-ranging vertebrates (Van Brussel and Holmes, 2022; Wu et al., 2018) and non- or little-invasive techniques based on faecal and oropharyngeal swabs have recently been developed (Bergner et al., 2019).

Ecotoxicological studies often face a lack of environmental and ecological representativeness regarding the overall exposure of wildlife, and how pollutants are reacting in the environmental matrix. *Morrissey et al.* (2023) recently address this question to improve wildlife risk assessment. Indeed, the AOP framework answers some of the specific guidelines they proposed (Morrissey et al., 2023). First, building AOPs allows a consideration of the general chemical complexity (i.e., mixture effects, chemical properties, metabolites) of pollutants. Each compound has its own mode(s) of action and pathways that lead to one or several toxic effects at different levels of biological organisation. Identifying molecular initiating event(s) and key event(s) that lead to adverse outcome(s) participates to understand how complex are the interactions between pollutants, here Cd and Pb, in organisms. Second, once AOPs are built and the main pathways identified, it is possible to make it species- or life-stages specific (i.e., considering organismal complexity; *Morrissey et al.*, 2023). This will guide the formulation of hypothesis and the choice of biomarkers. This (new) framework participates in answering new questions and guide researchers into new directions in ecotoxicology, stress ecology and ecoimmunotoxicology. Building AOPs before starting an experiment appears as something that should become more common in these field of research to better apprehend mechanisms underlying toxic effects and identify the research gaps (Baudiffier et al., 2024). In addition, developing the knowledge of the database presented by Vinken et al. (2017) would encourage data sharing and literature findings to better direct further research. To date, the most important limitations in making AOPs about a specific subject is the lack of literature that can restrict the whole process.

## 8. Conclusion

Adverse outcome pathways are an efficient method to identify the mechanisms underlying the effects of pollutants at various levels of biological organisation and thus, to identify research gaps in ecotoxicology and risk assessment. Here, we identified potential MIEs and KEs that lead to AOs related to immune disorders in mammals in response to Cd and/or Pb exposure. Cadmium and Pb may induce immunostimulation and immunodeficiency through different pathways of the immune system, impacting both innate and adaptive immune responses. Our findings could guide future research in ecoimmunotoxicology of metals. There are still large open questions about how organisms, at different levels of organisation, react to pathogens and infections in a context where exposure to pollutants affects immunity. Mixture effects of trace metal are also still largely understudied. It is now of major importance to understand how these pollutants interact with wildlife to improve conservation policies. We identified potential synergetic and antagonistic interactions produced by the co-exposure to Cd and Pb, which also brings questions about the potential adaptation of wild populations to those mixtures, in comparison to single-pollutant exposure.

### CRediT authorship contribution statement

**Cloe Hadjadji:** Writing – review & editing, Writing – original draft, Visualization, Validation, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. **Quentin Devalloir:** Validation, Supervision, Conceptualization. **Colette Gaillard:** Writing – review & editing, Validation. **Nico W. van den Brink:** Writing – review & editing, Validation, Supervision. **Renaud Scheifler:** Writing – review & editing,

Writing – original draft, Validation, Supervision, Project administration, Funding acquisition, Conceptualization.

### Funding information

This study is funded by the CHRONIC project (No. 956009) ‘Chronic exposure scenarios driving environmental risks of Chemicals’, an Innovative Training Network (ITN) funded by the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie Actions (MSCA).

### Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

### Acknowledgements

The authors thank Michaël Coeurdassier, Clémentine Fritsch, and Francis Raoul, for fruitful discussion about the present article.

### Data availability

No data was used for the research described in the article.

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