AOP ID and Title:

Short Title: Interaction with lung cells leading to atherosclerosis

Authors

Claudia Torero Gutierrez¹, Sarah Søs Poulsen¹, Jorid Birkelund Sørli¹,  Håkan Wallin², Sabina Halappanavar³, Carole L. Yauk⁴, Ulla Vogel^1,*

¹The National Research Centre for the Working Environment, Denmark

²Statens Arbeidsmiljøinstitutt, Norway

³Health Canada, Canada

⁴University of Ottawa, Canada

*Corresponding author: Ulla Vogel (ubv@nfa.dk)

Status

Abstract

AOP237 describes key events initiated with the interaction of substances with the membrane components of the pulmonary cells, and leading to atherosclerosis in humans. Atherosclerosis is defined as the thickening of the wall of an artery due to plaque deposition, and this condition can lead to severe events as myocardial infarction and stroke. This AOP presents the induction of acute phase response as a pathway for atherosclerosis progression. The interaction between a substance and the lung resident cell membrane components is the molecular initiating event (MIE; Event 1495) for this AOP; this interaction leads to an increased secretion of proinflammatory mediators [Key event (KE)1; Event 1496]. The release of proinflammatory factors triggers an increase in transcription of genes encoding acute phase proteins (KE2; Event 1438), leading to systemic acute phase response (KE3; Event 1439) once the acute phase proteins are translated and released into the systemic circulation. A continuous acute phase response leads to atherosclerosis, the adverse outcome (AO) of this AOP (Event 1443).

AOP 237 mainly focus on particles or particulate matter as stressors, however other compounds or inflammatory conditions that induce acute phase response, can be consider stressors and lead to atherosclerosis. In addition, most of the evidence is based on animal studies (mice) as a model for the human system, however the adverse outcome of the present AOP, atherosclerosis, is only applicable to humans. The AOP presents the biological plausibility, evidence and quantitative understanding for the relationship between KEs. In addition, evidence that KE2, KE3 and KE4 occur after the MIE is presented as non-adjacent relationships. This AOP presents a mechanism of substance-induced acute phase response leading to atherosclerosis, and it can be used for regulatory purposes and health-based risk assessments of inhalable materials.

Background

Cardiovascular disease (CVD) is the leading cause of death worldwide, being responsible for 32% of all deaths in 2019 (WHO; http://www.who.int). The term CVD covers all diseases of the cardiovascular system, including atherosclerosis, which is manifested as increased plaque deposition or build-up in the arteries. Although, atherosclerosis is not a cause of death, it can lead to fatal conditions as stroke and myocardial infarction. Atherosclerosis is normally an asymptotic disease and is initiated by a biological, chemical or physical insult to the artery walls. This leads to the expression of cell adhesion molecules on the endothelial lining of the arteries, which facilitates the activation, recruitment, and migration of monocytes through the endothelial monolayer (Cybulsky et al., 2001; Hansson & Libby, 2006). Inside the intima layer, the monocytes differentiate into macrophages and internalize fatty deposits (mainly oxidized low-density lipoprotein). This results in them transforming into foam cells, which is a major component of the atherosclerotic fatty streaks. The fatty streaks reduce the elasticity of the artery walls and the foam cells promote a pro-inflammatory environment by secretion of cytokines and reactive oxidative species. In addition, foam cells also induce the recruitment of smooth muscle cells to the intima. Added together, these changes lead to the formation of plaques on the artery walls. A fibrous cap of collagen and vascular smooth muscle cells protects the necrotic core and stabilizes the plaque (Libby, 2012; Virmani et al., 2005). However, blood clots can be formed if the plaque ruptures. These may travel with the bloodstream and obstruct the blood flow of smaller vessels, e.g. the coronary arteries, which ultimately can lead to myocardial infarction.

Inhalation of particulate matter, chemicals and pathogens have been related to increased pulmonary inflammation. Whereas a normal immune reaction is crucial for effective elimination of threats to the body, chronic and unresolved inflammation has been linked to both adverse pulmonary and adverse systemic effects in humans. In concordance with this, various retrospective and prospective epidemiological studies have linked pulmonary exposure to respirable air particulates with increased the risk of developing CVD (Clancy, Goodman, Sinclair, & Dockery, 2002; Dockery et al., 1993; Pope et al., 2004; Pope et al., 1995). Inhalation of particles has been proposed to affect the cardiovascular system in several different ways, including through disruption of vasomotor function and through acceleration of plaque progression in atherosclerosis (Cao et al., 2014; Moller et al., 2016).

Acute phase response is characterized by the change in plasma concentration of acute phase proteins (APP), along with other physiological changes during inflammatory conditions (Gabay & Kushner, 1999; Mantovani & Garlanda, 2023). Serum amyloid A (SAA) and C-reactive protein (CRP) are the major acute phase proteins in humans and are considered risk factors for CVDs (Table 1 presents acute phase response characteristics in humans and mice). In particular, SAA restricts the transport of cholesterol to the liver, allowing the accumulation of cholesterol in arteries and the formation of foam cells.

Table 1. Selected differences in APR between humans and mice.

Characteristic	Humans	Mice
Number of identified genes involved in acute phase response	61	62
Major acute phase proteins	CRP, SAA	Haptoglobin, SAA, serum amyloid P
Moderate and minor acute phase proteins	Haptoglobin, fibrinogen, α1 acid glycoprotein	CRP, fibrinogen
SAA isoforms	Saa1, Saa2 and Saa4	Saa1, Saa2, Saa3 and Saa4

References: (Cray, 2012; Gabay & Kushner, 1999; NCBI, 2023; Tannock et al., 2018).

Atherosclerosis is a disease influenced by multiple factors including high levels of lipoproteins in blood, elevated blood pressure, smoking, obesity, type 2 diabetes, diet, and physical activity (Herrington, Lacey, Sherliker, Armitage, & Lewington, 2016; Libby et al., 2019; Raitakari, Pahkala, & Magnussen, 2022). Inflammation is also involved in atherosclerosis, providing pathways via which risk factors might cause the development and advancement of atherosclerotic plaques (Libby, 2021a, 2021b). Therefore, although inflammation and acute phase response are not the only causes of atherosclerosis, the early key events (KE1, KE2 and KE3) can be used to evaluate the particle-induced risk of developing atherosclerosis.

For the development of AOP 237, the MIE and KE1 from AOP 173 have been used (AOP 173: Substance interaction with the pulmonary resident cell membrane components leading to pulmonary fibrosis). The information presented in AOP 173 has not been modified for AOP 237.

The development of the present AOP was supported by the EU project NanoPASS (Grant number: 101092741) and the Focused Research Effort on Chemicals in the Working Environment (FFIKA) form the Danish Government.

Summary of the AOP

Events

Molecular Initiating Events (MIE), Key Events (KE), Adverse Outcomes (AO)

Key Event Relationships

Stressors

Overall Assessment of the AOP

Domain of Applicability

Life Stage Applicability Taxonomic Applicability Sex Applicability

This AOP is applicable to adult humans of both sexes. Although atherosclerosis is a condition that begins during childhood and progresses through life, its clinical manifestation is mostly observed in older individuals (Raitakari et al., 2022).

The AOP is applicable to all stressors that can be inhaled and, therefore, interact with the pulmonary cells and induce pulmonary inflammation.

Essentiality of the Key Events

For the development of AOP 237, the molecular initiating event (MIE) and key event (KE) 1 from AOP 173 have been reused (AOP 173: Substance interaction with the pulmonary resident cell membrane components leading to pulmonary fibrosis). The information presented in AOP 173 has not been modified for AOP 237.

Support for essentiality of KEs	Defining question	High	Moderate	Low
	What is the impact on downstream KEs and/or the AO if an upstream KE is modified or prevented?	Direct evidence from specifically designed experimental studies illustrating prevention or impact on downstream KEs and/or the AO if upstream KEs are blocked or modified	Indirect evidence that modification of one or more upstream KEs is associated with a corresponding (increase or decrease) in the magnitude or frequency of downstream KEs	No or contradictory experimental evidence of the essentiality of any of the KEs.
MIE: Substance interaction with the lung resident cell membrane components (Event 1495)	Moderate. Stressors have a dose-response relationship with transcription of genes encoding acute phase proteins (KE2) and systemic acute phase response (KE3) (Bengtson et al., 2017; Di Ianni et al., 2020; Monse et al., 2018; Poulsen et al., 2017; Saber et al., 2013). Knockout of toll-like receptor 4 (Tlr4) prevents the lipopolysaccharide-induced increase of cytokine/chemokines mRNA levels in lung and liver tissues (KE1) and prevents lipopolysaccharide-induced systemic acute phase response (KE3) in mice (Danielsen et al., 2021). Knockout of toll-like receptor 2 (Tlr2) prevents the multiwalled carbon nanotubes-induced increase of Saa1 mRNA levels in liver tissue (KE2) and serum amyloid A (SAA)1 levels in plasma (KE3) in mice (Danielsen et al., 2021).
KE1: Increased, secretion of proinflammatory mediators (Event 1496)	High. Interleukin (IL) 6 gene disruption (IL-6-/-) reduces the liver mRNA levels (KE2) and serum levels (KE3) of the acute phase proteins haptoglobin, α1-acid glycoprotein and SAA in mice (Kopf et al., 1994). Blockage of IL-6 receptors reduced SAA1 mRNA, while blockage of IL-1β and tumor necrosis factor α receptors partially reduces the expression of SAA1 mRNA (KE2), in hepatic cell lines (Hagihara et al., 2004). Administration of monoclonal antibodies for IL-1β reduces blood levels of C-reactive protein (CRP) (KE2 and KE3), and decreased the incidence rates of recurrent cardiovascular events (AO), in patients with a history of myocardial infarction (Ridker et al., 2017)
KE2: Transcription of genes encoding acute phase proteins, Increased (Event 1438)	High. Gene transcription is necessary for the synthesis of proteins (KE3) (Alberts, 2017). Suppression of SAA3 and double knockout of SAA1/SAA2 reduces atherosclerotic plaque area (AO), in ApoE-/- mice (Thompson et al., 2018).
KE3: Systemic acute phase response (Event 1439)	High. Elevated levels of SAA induce plaque progression (AO) (Christophersen et al., 2021; Dong et al., 2011; Thompson et al., 2018). CRP and SAA levels are predictive of risk of cardiovascular disease (Pai et al., 2004; Ridker, Hennekens, Buring, & Rifai, 2000).
AO: Atherosclerosis (Event 1443)	N/A. This is the AO and it is essential for the AOP.

Uncertainties or Inconsistencies

• Physicochemical characteristics of nanomaterials such as size, surface area, surface functionalization, shape, composition, among others, affect the magnitude and duration of acute phase response in mice (Bengtson et al., 2017; Gutierrez et al., 2023; Poulsen et al., 2017). In animal models, both inflammatory and acute phase response are predicted by the total surface area of the retained, insoluble particles (Cosnier et al., 2021; Gutierrez et al., 2023).
• C-reactive protein (CRP) and serum amyloid A (SAA) are risk factors for cardiovascular disease (Ridker et al., 2000). However, Mendelian randomization studies have shown that CRP genotypes are not associated with risk of coronary heart disease and that genetically elevated levels of CRP are not associated with coronary heart disease risk (Collaboration et al., 2011; Elliott et al., 2009).
• In mice studies, it is possible to measure both Saa gene expression and SAA protein levels, however the dynamic range for Saa gene expression is larger. In humans, measuring gene expression of acute phase proteins is not very common, as a tissue sample is needed, while measuring acute phase protein in blood is more common.
• It is suggested that acute phase proteins are mainly produced in the liver (Gabay & Kushner, 1999), however in mice the liver has little upregulation of Saa genes after exposure to ultrafine carbon particles or diesel exhaust particle. On the other hand, the lung shows a marked expression of Saa3 mRNA (Saber et al., 2009; Saber et al., 2013).
• A level of inconsistency between the results from human studies exists. It has been observed that in most controlled human studies, an increase in CRP and/or SAA was observed after exposure to particulate matter (Baumann et al., 2018; Haase et al., 2022; Monse et al., 2018; Monse et al., 2021; Walker et al., 2022; Wyatt, Devlin, Rappold, Case, & Diaz-Sanchez, 2020). However, in other  studies the exposure did not induce acute phase response (Andersen, Saber, Clausen, et al., 2018; Andersen, Saber, Pedersen, et al., 2018), maybe due to low levels of exposure (Andersen et al., 2019) or limited statistical power.

Weight of Evidence Summary

Biological plausibility of each KER

Please also refer to AOP173: Substance interaction with the pulmonary resident cell membrane components leading to pulmonary fibrosis, which shares MIE and KE1 with the present AOP.

Support for Biological Plasuibility of KERs	Defining question	High	Moderate	Low
	Is there a mechanistic (i.e., structural or functional) relationship between KEup and KEdown consistent with established biological knowledge?	Extensive understanding based on extensive previous documentation and broad acceptance -Established mechanistic basis	The KER is plausible based on analogy to accepted biological relationships but scientific understanding is not completely established.	There is empirical support for a statistical association between KEs (See 3.), but the structural or functional relationship between them is not understood.
MIE => KE1: Interaction with the lung cell membrane leads to Increased proinflammatory mediators (Relationship 1702)	Biological Plausibility of the MIE => KE1 is High. Rationale: There is extensive evidence showing that interaction of stressors with the respiratory system induces the release of proinflammatory markers (Behzadi et al., 2017; Denholm & Phan, 1990; Dostert et al., 2008; Mossman & Churg, 1998).
KE1 => KE2: Increased proinflammatory mediators leads to Increased transcription of genes encoding acute phase proteins (Relationship 2053)	Biological Plausibility of the KE1 => KE2 is High. Rationale: Acute phase proteins are induced by pro-inflammatory cytokines. These cytokines are produced at sites of inflammation mainly by monocytes and macrophages (Gabay & Kushner, 1999; Mantovani & Garlanda, 2023; Uhlar & Whitehead, 1999; Venteclef, Jakobsson, Steffensen, & Treuter, 2011).
KE2 => KE3: Increased transcription of genes encoding acute phase proteins leads to Systemic acute phase response (Relationship 1589)	Biological Plausibility of the KE2 => KE3 is High. Rationale: After gene expression of acute phase proteins in tissues mRNA is translated and folded into proteins (Alberts, 2017). These proteins are then release to the systemic circulation (Van Eeden, Leipsic, Paul Man, & Sin, 2012).
KE3 => AO:  Systemic acute phase response leads to Atherosclerosis (Relationship 2860)	Biological Plausibility of the KE3 => KE2 is High. Rationale: During acute phase response, serum amyloid A (SAA), one of the major acute phase proteins, replaces apolipoprotein A-1 from high density lipoprotein (HDL). This replacement obstructs the reverse transport of cholesterol to the liver, allowing the accumulation of cholesterol in cells (Lindhorst, Young, Bagshaw, Hyland, & Kisilevsky, 1997; McGillicuddy et al., 2009; Meek, Urieli-Shoval, & Benditt, 1994).
Non-adjacent MIE => KE2: Interaction with the lung cell membrane leads to Increased transcription of genes encoding acute phase proteins (Relationship 2958)	Biological Plausibility of the MIE => KE2 is High. Rationale: After cells sense pathogens, tissue damage or dysmetabolism, production of acute phase proteins is triggered by cellular pattern-recognition molecules, through a cytokine cascade (Mantovani & Garlanda, 2023). There is extensive evidence that nanomaterials induce the expression of acute phase response genes in mice (Bengtson et al., 2017; Di Ianni et al., 2020; Erdely, Liston, et al., 2011; Gutierrez et al., 2023; Hadrup et al., 2019; Halappanavar et al., 2015; Poulsen, Saber, Mortensen, et al., 2015; Saber et al., 2013).
Non-adjacent MIE => KE3: Interaction with the lung cell membrane leads to Systemic acute phase response (Relationship 2959)	Biological Plausibility of the MIE => KE3 is High. Rationale: Pulmonary inflammation occurs when stressors interact with the airways (Moldoveanu et al., 2009) and acute phase response is induced during inflammatory conditions (Gabay & Kushner, 1999). There is plenty of evidence showing that inhalation or instillation of stressors induces systemic acute phase response in humans and mice mice (Baumann et al., 2016; Bendtsen et al., 2019; Bengtson et al., 2017; Bourdon et al., 2012; Erdely, Liston, et al., 2011; Kim, Chen, Boyce, & Christiani, 2005; Monse et al., 2018; Monse et al., 2021; Poulsen et al., 2017; Poulsen, Saber, Williams, et al., 2015; Westberg et al., 2016).
Non-adjacent KE1 => KE3: Increased proinflammatory mediators leads to Systemic APR (Relantionship 3052)	Biological Plausibility of the KE1 => KE3 is High. Rationale: Pro-inflammatory cytokines induce the release of acute phase proteins. These proteins are released from inflammatory sites to the systemic circulation (Gabay & Kushner, 1999; Mantovani & Garlanda, 2023).
Non-adjacent MIE => AO: Interaction with the lung cell membrane leads to Atherosclerosis (Relantionship 2960)	Biological Plausibility of the MIE => AO is Moderate. Rationale: There is evidence that the interaction of the lungs with stressor induces atherosclerotic plaque progression; however, the mechanistic relationship has not been clarified (Christophersen et al., 2021; Erdely, Hulderman, et al., 2011; M. R. Miller et al., 2013; M. R. Miller & Newby, 2020; Van Eeden et al., 2012).

Empirical support for each KER

Please also refer to AOP173: Substance interaction with the pulmonary resident cell membrane components leading to pulmonary fibrosis, which shares MIE and KE1 with the present AOP.

Empirical Support	Defining question	High	Moderate	Low
	Does KEup occur at lower doses and earlier time points than KE down and at the same dose of prototypical stressor, is the incidence of KEup > than that for KEdown? Are there inconsistencies in empirical support across taxa, species and prototypical stressor that don’t align with expected pattern for hypothesised AOP?	Multiple studies showing dependent change in both events following exposure to a wide range of specific prototypical stressors. (Extensive evidence for temporal, dose- response and incidence concordance) and no or few critical data gaps or conflicting data	Demonstrated dependent change in both events following exposure to a small number of specific prototypical stressors and some evidence inconsistent with expected pattern that can be explained by factors such as experimental design, technical considerations, differences among laboratories, etc.	Limited or no studies reporting dependent change in both events following exposure to a specific prototypical stressor (i.e., endpoints never measured in the same study or not at all); and/or significant inconsistencies in empirical support across taxa and species that don’t align with expected pattern for hypothesised AOP
MIE => KE1: Interaction with the lung cell membrane leads to Increased proinflammatory mediators (Relationship 1702)	Empirical Support of the MIE => KE1 is Moderate.  Rationale: There are limited in vitro studies which show a temporal and dose-dependent relationship between these two events (Chan et al., 2018; Denholm & Phan, 1990; Roy, Singh, Das, Tripathi, & Dwivedi, 2014).
KE1 => KE2: Increased proinflammatory mediators leads to Increased transcription of genes encoding acute phase proteins (Relationship 2053)	Empirical Support of the KE1 => KE2 is High. Rationale: There are several studies showing a dose concordance and temporal concordance between KEs (Bendtsen et al., 2019; Di Ianni et al., 2020; Kyjovska et al., 2015; Saber et al., 2012; Saber et al., 2013; Wallin et al., 2017).
KE2 => KE3: Increased transcription of genes encoding acute phase proteins leads to Systemic acute phase response (Relationship 1589)	Empirical Support of the KE2 => KE3 is High. Rationale: There are studies showing a dose concordance and temporal concordance between KE (Bengtson et al., 2017; Gutierrez et al., 2023; Poulsen et al., 2017). However, there are inconsistencies between gene expression and translation of acute phase proteins.
KE3 => AO:  Systemic acute phase response leads to Atherosclerosis (Relationship 2860)	Empirical Support of the KE3 => AO is Moderate. Rationale: There is a limited number of animal studies showing the relationship between the KEs, in addition of epidemiological studies showing association between the KEs (Christophersen et al., 2021; Dong et al., 2011; Pai et al., 2004; Rivera et al., 2013; Thompson et al., 2015; Thompson et al., 2018).
Non-adjacent MIE => KE2: Interaction with the lung cell membrane leads to Increased transcription of genes encoding acute phase proteins (Relationship 2958)	Empirical Support of the MIE => KE2 is Moderate. Rationale: There are several studies showing a dose concordance and temporal concordance in animal studies. However, in the case of nanomaterials it has been shown that physicochemical characteristics affect the magnitude and duration of the expression of acute phase proteins in mice (Bengtson et al., 2017; Bourdon et al., 2012; Gutierrez et al., 2023; Kyjovska et al., 2015; Poulsen et al., 2017; Saber et al., 2013; Wallin et al., 2017).
Non-adjacent MIE => KE3: Interaction with the lung cell membrane leads to Systemic acute phase response (Relationship 2959)	Empirical Support of the MIE => KE3 is Moderate. Rationale: There are plenty of studies showing a dose concordance and temporal concordance in animal and controlled human studies (Brand et al., 2014; Erdely, Liston, et al., 2011; Kim et al., 2005; Monse et al., 2018; Monse et al., 2021; Poulsen et al., 2017; Walker et al., 2022; Wyatt et al., 2020). However, it has been observed that systemic acute phase response is not always observed after exposure.
Non-adjacent KE1 => KE3: Increased proinflammatory mediators leads to Systemic APR (Relantionship 3052)	Empirical Support of the KE1 => KE3 is Moderate. Rationale: There are several studies showing a dose concordance and temporal concordance. However, there are inconsistencies between changes in blood levels of pro-inflammatory mediators and systemic APR (Baumann et al., 2016; Kim et al., 2005; Monse et al., 2018; Monse et al., 2021; Poulsen et al., 2017).
Non-adjacent MIE => AO: Interaction with the lung cell membrane leads to Atherosclerosis (Relantionship 2960)	Empirical Support of the MIE => AO is Moderate. Rationale: There are several studies showing the relationship between the key events (Christophersen et al., 2021; Li et al., 2007; Mikkelsen et al., 2011; M. R. Miller et al., 2013).

Quantitative Consideration

The table below presents the quantitative understanding of every KER.

It is important to clarify that when assessing stressors in mice studies, it is possible to measure the gene expression of acute phase proteins (KE2) in different tissues, whereas in humans this is not likely as a tissue sample would be required. On the other hand, in humans it is much more common and easier to measure systemic acute phase response (KE3) through a blood sample. In mice, it has been shown that Saa3 mRNA in lung tissue and blood levels of serum amyloid A (SAA)3 are correlated (Gutierrez et al., 2023). In addition, SAA levels in mice and humans seem to be in level in magnitude after exposure to zinc oxide nanoparticles (Gutierrez et al., 2023). This suggest that systemic acute phase response in humans can be estimated from mice studies.

Saa3 mRNA in lung tissue is also correlated to pulmonary inflammation measured as neutrophil numbers in broncheoalveolar lavage fluid (i.e. indirect marker of the release of pro-inflammatory factors because the release of inflammatory mediators) in mice after pulmonary exposure to nanomaterials. Both of these endpoints can be estimated by calculating the dosed surface area (specific surface area multiplied by dose level) (Gutierrez et al., 2023).

Finally, the relative risk of people developing a cardiovascular disease can be calculated from blood levels of acute phase proteins in epidemiological studies.

KER	Quantitative understanding
MIE => KE1: Interaction with the lung cell membrane leads to Increased proinflammatory mediators (Relationship 1702)	The quantitative understanding of MIE => KE1 is Low. Rationale: The quantitative prediction of the release of proinflammatory factors can be made from the interaction of the stressors with the pulmonary system. In the case of some stressors (nanomaterials) it is possible to make a prediction using the dosed surface area of the materials and neutrophil numbers in broncheoalveolar lavage (BALF) as an indirect marker of the release of pro-inflammatory factors (Gutierrez et al., 2023; Oberdorster, Ferin, Gelein, Soderholm, & Finkelstein, 1992; Oberdorster, Ferin, & Lehnert, 1994; Schmid & Stoeger, 2016; Stoeger et al., 2006).
KE1 => KE2: Increased proinflammatory mediators leads to Increased transcription of genes encoding acute phase proteins (Relationship 2053)	The quantitative understanding is of KE1 => KE2 is Moderate. Rationale: In mice, the gene expression of Saa after exposure to metal oxide nanomaterials can be estimated using an indirect marker of the release of pro-inflammatory factors (neutrophil numbers in BALF)  (Gutierrez et al., 2023).
KE2 => KE3: Increased transcription of genes encoding acute phase proteins leads to Systemic acute phase response (Relationship 1589)	The quantitative understanding of KE2 => KE3 is Moderate. Rationale: In mice, the systemic levels of SAA after exposure to metal oxide nanomaterials can be estimated from the gene expression in lung tissue (Gutierrez et al., 2023).
KE3 => AO:  Systemic acute phase response leads to Atherosclerosis (Relationship 2860)	The quantitative understanding is of KE3 => AO is High. Rationale: The risk of developing a cardiovascular disease at population level can be calculated from blood levels of acute phase proteins (KER 2860).
Non-adjacent MIE => KE2: Interaction with the lung cell membrane leads to Increased transcription of genes encoding acute phase proteins (Relationship 2958)	The quantitative understanding of MIE => KE2 is Moderate. Rationale: In mice, the gene expression of Saa after exposure to metal oxide nanomaterials can be estimated from the dosed surface area (Gutierrez et al., 2023).
Non-adjacent MIE => KE3: Interaction with the lung cell membrane leads to Systemic acute phase response (Relationship 2959)	The quantitative understanding of MIE => KE3 is Moderate. Rationale: In mice, the blood levels of SAA after exposure to metal oxide nanomaterials can be estimated from the dosed surface area (Gutierrez et al., 2023).
Non-adjacent KE1 => KE3: Increased proinflammatory mediators leads to Systemic APR (Relantionship 3052)	The quantitative understanding of KE1 => KE3 is Moderate. Rationale: In mice, the blood levels of SAA after exposure to metal oxide nanomaterials and multiwalled carbon nanotubes can be estimated from neutrophil numbers in BALF (Gutierrez et al., 2023; Poulsen et al., 2017).
Non-adjacent MIE => AO: Interaction with the lung cell membrane leads to Atherosclerosis (Relantionship 2960)	The quantitative understanding of MIE => AO is Moderate. Rationale: Epidemiological studies have shown the risk ratios of having a cardiovascular event per increase or decrease of exposure to particulate matter (Beelen et al., 2014; Cesaroni et al., 2014; Clancy et al., 2002; K. A. Miller et al., 2007)

Considerations for Potential Applications of the AOP (optional)

Particle-induced acute phase response can be regarded as a critical effect linking particle-exposure to cardiovascular disease. Dose-response relationships can be used to establish no-observed-adverse-effect levels (NOAEL) for regulatory purposes and occupational exposure limits for inhalable materials can be determined through health-based risk assessments. This approach was taken by the Danish National Research Centre for the Working Environment at request of the Danish Working Environment Authority and an occupational exposure limit for zinc oxide was proposed based on the induction of acute phase response as the critical effect (the report can be found in: Dokumentation for helbredsbaserede grænseværdier for kemiske stoffer i arbejdsmiljøet (nfa.dk)).

As mentioned previously, not all KE can easily be measured in humans, therefore animal studies can be used to measure early KEs and perform a risk assessment of different stressors. Additionally, physicochemical properties, such as specific surface area and dissolution, are important predictors of particle-induced acute phase response that can be used for hazard assessment (Gutierrez et al., 2023).

References

Alberts, B. (2017). Molecular biology of the cell (Sixth edition. ed.). Boca Raton, FL: CRC Press, an imprint of Garland Science.

Andersen, M. H. G., Frederiksen, M., Saber, A. T., Wils, R. S., Fonseca, A. S., Koponen, I. K., . . . Vogel, U. (2019). Health effects of exposure to diesel exhaust in diesel-powered trains. Part Fibre Toxicol, 16(1), 21. doi:10.1186/s12989-019-0306-4

Andersen, M. H. G., Saber, A. T., Clausen, P. A., Pedersen, J. E., Lohr, M., Kermanizadeh, A., . . . Vogel, U. (2018). Association between polycyclic aromatic hydrocarbon exposure and peripheral blood mononuclear cell DNA damage in human volunteers during fire extinction exercises. Mutagenesis, 33(1), 105-115. doi:10.1093/mutage/gex021

Andersen, M. H. G., Saber, A. T., Pedersen, J. E., Pedersen, P. B., Clausen, P. A., Lohr, M., . . . Moller, P. (2018). Assessment of polycyclic aromatic hydrocarbon exposure, lung function, systemic inflammation, and genotoxicity in peripheral blood mononuclear cells from firefighters before and after a work shift. Environ Mol Mutagen, 59(6), 539-548. doi:10.1002/em.22193

Baumann, R., Gube, M., Markert, A., Davatgarbenam, S., Kossack, V., Gerhards, B., . . . Brand, P. (2018). Systemic serum amyloid A as a biomarker for exposure to zinc and/or copper-containing metal fumes. J Expo Sci Environ Epidemiol, 28(1), 84-91. doi:10.1038/jes.2016.86

Baumann, R., Joraslafsky, S., Markert, A., Rack, I., Davatgarbenam, S., Kossack, V., . . . Gube, M. (2016). IL-6, a central acute-phase mediator, as an early biomarker for exposure to zinc-based metal fumes. Toxicology, 373, 63-73. doi:10.1016/j.tox.2016.11.001

Beelen, R., Stafoggia, M., Raaschou-Nielsen, O., Andersen, Z. J., Xun, W. W., Katsouyanni, K., . . . Hoek, G. (2014). Long-term exposure to air pollution and cardiovascular mortality: an analysis of 22 European cohorts. Epidemiology, 25(3), 368-378. doi:10.1097/EDE.0000000000000076

Behzadi, S., Serpooshan, V., Tao, W., Hamaly, M. A., Alkawareek, M. Y., Dreaden, E. C., . . . Mahmoudi, M. (2017). Cellular uptake of nanoparticles: journey inside the cell. Chem Soc Rev, 46(14), 4218-4244. doi:10.1039/c6cs00636a

Bendtsen, K. M., Brostrom, A., Koivisto, A. J., Koponen, I., Berthing, T., Bertram, N., . . . Vogel, U. (2019). Airport emission particles: exposure characterization and toxicity following intratracheal instillation in mice. Part Fibre Toxicol, 16(1), 23. doi:10.1186/s12989-019-0305-5

Bengtson, S., Knudsen, K. B., Kyjovska, Z. O., Berthing, T., Skaug, V., Levin, M., . . . Vogel, U. (2017). Differences in inflammation and acute phase response but similar genotoxicity in mice following pulmonary exposure to graphene oxide and reduced graphene oxide. PLoS One, 12(6), e0178355. doi:10.1371/journal.pone.0178355

Bourdon, J. A., Halappanavar, S., Saber, A. T., Jacobsen, N. R., Williams, A., Wallin, H., . . . Yauk, C. L. (2012). Hepatic and pulmonary toxicogenomic profiles in mice intratracheally instilled with carbon black nanoparticles reveal pulmonary inflammation, acute phase response, and alterations in lipid homeostasis. Toxicol Sci, 127(2), 474-484. doi:10.1093/toxsci/kfs119

Brand, P., Bauer, M., Gube, M., Lenz, K., Reisgen, U., Spiegel-Ciobanu, V. E., & Kraus, T. (2014). Relationship between welding fume concentration and systemic inflammation after controlled exposure of human subjects with welding fumes from metal inert gas brazing of zinc-coated materials. J Occup Environ Med, 56(1), 1-5. doi:10.1097/JOM.0000000000000061

Cao, Y., Jacobsen, N. R., Danielsen, P. H., Lenz, A. G., Stoeger, T., Loft, S., . . . Moller, P. (2014). Vascular effects of multiwalled carbon nanotubes in dyslipidemic ApoE-/- mice and cultured endothelial cells. Toxicol Sci, 138(1), 104-116. doi:10.1093/toxsci/kft328

Cesaroni, G., Forastiere, F., Stafoggia, M., Andersen, Z. J., Badaloni, C., Beelen, R., . . . Peters, A. (2014). Long term exposure to ambient air pollution and incidence of acute coronary events: prospective cohort study and meta-analysis in 11 European cohorts from the ESCAPE Project. BMJ, 348, f7412. doi:10.1136/bmj.f7412

Chan, J. Y. W., Tsui, J. C. C., Law, P. T. W., So, W. K. W., Leung, D. Y. P., Sham, M. M. K., . . . Chan, C. W. H. (2018). Regulation of TLR4 in silica-induced inflammation: An underlying mechanism of silicosis. Int J Med Sci, 15(10), 986-991. doi:10.7150/ijms.24715

Christophersen, D. V., Moller, P., Thomsen, M. B., Lykkesfeldt, J., Loft, S., Wallin, H., . . . Jacobsen, N. R. (2021). Accelerated atherosclerosis caused by serum amyloid A response in lungs of ApoE(-/-) mice. FASEB J, 35(3), e21307. doi:10.1096/fj.202002017R

Clancy, L., Goodman, P., Sinclair, H., & Dockery, D. W. (2002). Effect of air-pollution control on death rates in Dublin, Ireland: an intervention study. Lancet, 360(9341), 1210-1214. doi:10.1016/S0140-6736(02)11281-5

Collaboration, C. R. P. C. H. D. G., Wensley, F., Gao, P., Burgess, S., Kaptoge, S., Di Angelantonio, E., . . . Danesh, J. (2011). Association between C reactive protein and coronary heart disease: mendelian randomisation analysis based on individual participant data. BMJ, 342, d548. doi:10.1136/bmj.d548

Cosnier, F., Seidel, C., Valentino, S., Schmid, O., Bau, S., Vogel, U., . . . Gate, L. (2021). Retained particle surface area dose drives inflammation in rat lungs following acute, subacute, and subchronic inhalation of nanomaterials. Part Fibre Toxicol, 18(1), 29. doi:10.1186/s12989-021-00419-w

Cray, C. (2012). Acute phase proteins in animals. Prog Mol Biol Transl Sci, 105, 113-150. doi:10.1016/B978-0-12-394596-9.00005-6

Cybulsky, M. I., Iiyama, K., Li, H., Zhu, S., Chen, M., Iiyama, M., . . . Milstone, D. S. (2001). A major role for VCAM-1, but not ICAM-1, in early atherosclerosis. J Clin Invest, 107(10), 1255-1262. doi:10.1172/JCI11871

Danielsen, P. H., Bendtsen, K. M., Knudsen, K. B., Poulsen, S. S., Stoeger, T., & Vogel, U. (2021). Nanomaterial- and shape-dependency of TLR2 and TLR4 mediated signaling following pulmonary exposure to carbonaceous nanomaterials in mice. Part Fibre Toxicol, 18(1), 40. doi:10.1186/s12989-021-00432-z

Denholm, E. M., & Phan, S. H. (1990). Bleomycin binding sites on alveolar macrophages. J Leukoc Biol, 48(6), 519-523. doi:10.1002/jlb.48.6.519

Di Ianni, E., Moller, P., Mortensen, A., Szarek, J., Clausen, P. A., Saber, A. T., . . . Jacobsen, N. R. (2020). Organomodified nanoclays induce less inflammation, acute phase response, and genotoxicity than pristine nanoclays in mice lungs. Nanotoxicology, 14(7), 869-892. doi:10.1080/17435390.2020.1771786

Dockery, D. W., Pope, C. A., 3rd, Xu, X., Spengler, J. D., Ware, J. H., Fay, M. E., . . . Speizer, F. E. (1993). An association between air pollution and mortality in six U.S. cities. N Engl J Med, 329(24), 1753-1759. doi:10.1056/NEJM199312093292401

Dong, Z., Wu, T., Qin, W., An, C., Wang, Z., Zhang, M., . . . An, F. (2011). Serum amyloid A directly accelerates the progression of atherosclerosis in apolipoprotein E-deficient mice. Mol Med, 17(11-12), 1357-1364. doi:10.2119/molmed.2011.00186

Dostert, C., Petrilli, V., Van Bruggen, R., Steele, C., Mossman, B. T., & Tschopp, J. (2008). Innate immune activation through Nalp3 inflammasome sensing of asbestos and silica. Science, 320(5876), 674-677. doi:10.1126/science.1156995

Elliott, P., Chambers, J. C., Zhang, W., Clarke, R., Hopewell, J. C., Peden, J. F., . . . Kooner, J. S. (2009). Genetic Loci associated with C-reactive protein levels and risk of coronary heart disease. JAMA, 302(1), 37-48. doi:10.1001/jama.2009.954

Erdely, A., Hulderman, T., Salmen-Muniz, R., Liston, A., Zeidler-Erdely, P. C., Chen, B. T., . . . Simeonova, P. P. (2011). Inhalation exposure of gas-metal arc stainless steel welding fume increased atherosclerotic lesions in apolipoprotein E knockout mice. Toxicol Lett, 204(1), 12-16. doi:10.1016/j.toxlet.2011.03.030

Erdely, A., Liston, A., Salmen-Muniz, R., Hulderman, T., Young, S. H., Zeidler-Erdely, P. C., . . . Simeonova, P. P. (2011). Identification of systemic markers from a pulmonary carbon nanotube exposure. J Occup Environ Med, 53(6 Suppl), S80-86. doi:10.1097/JOM.0b013e31821ad724

Gabay, C., & Kushner, I. (1999). Acute-phase proteins and other systemic responses to inflammation. N Engl J Med, 340(6), 448-454. doi:10.1056/NEJM199902113400607

Gutierrez, C. T., Loizides, C., Hafez, I., Brostrom, A., Wolff, H., Szarek, J., . . . Vogel, U. (2023). Acute phase response following pulmonary exposure to soluble and insoluble metal oxide nanomaterials in mice. Part Fibre Toxicol, 20(1), 4. doi:10.1186/s12989-023-00514-0

Haase, L. M., Birk, T., Poland, C. A., Holz, O., Muller, M., Bachand, A. M., & Mundt, K. A. (2022). Cross-sectional Study of Workers Employed at a Copper Smelter-Effects of Long-term Exposures to Copper on Lung Function and Chronic Inflammation. J Occup Environ Med, 64(9), e550-e558. doi:10.1097/JOM.0000000000002610

Hadrup, N., Rahmani, F., Jacobsen, N. R., Saber, A. T., Jackson, P., Bengtson, S., . . . Vogel, U. (2019). Acute phase response and inflammation following pulmonary exposure to low doses of zinc oxide nanoparticles in mice. Nanotoxicology, 13(9), 1275-1292. doi:10.1080/17435390.2019.1654004

Hagihara, K., Nishikawa, T., Isobe, T., Song, J., Sugamata, Y., & Yoshizaki, K. (2004). IL-6 plays a critical role in the synergistic induction of human serum amyloid A (SAA) gene when stimulated with proinflammatory cytokines as analyzed with an SAA isoform real-time quantitative RT-PCR assay system. Biochem Biophys Res Commun, 314(2), 363-369. doi:10.1016/j.bbrc.2003.12.096

Halappanavar, S., Saber, A. T., Decan, N., Jensen, K. A., Wu, D., Jacobsen, N. R., . . . Vogel, U. (2015). Transcriptional profiling identifies physicochemical properties of nanomaterials that are determinants of the in vivo pulmonary response. Environ Mol Mutagen, 56(2), 245-264. doi:10.1002/em.21936

Hansson, G. K., & Libby, P. (2006). The immune response in atherosclerosis: a double-edged sword. Nat Rev Immunol, 6(7), 508-519. doi:10.1038/nri1882

Herrington, W., Lacey, B., Sherliker, P., Armitage, J., & Lewington, S. (2016). Epidemiology of Atherosclerosis and the Potential to Reduce the Global Burden of Atherothrombotic Disease. Circ Res, 118(4), 535-546. doi:10.1161/CIRCRESAHA.115.307611

Kim, J. Y., Chen, J. C., Boyce, P. D., & Christiani, D. C. (2005). Exposure to welding fumes is associated with acute systemic inflammatory responses. Occup Environ Med, 62(3), 157-163. doi:10.1136/oem.2004.014795

Kopf, M., Baumann, H., Freer, G., Freudenberg, M., Lamers, M., Kishimoto, T., . . . Kohler, G. (1994). Impaired immune and acute-phase responses in interleukin-6-deficient mice. Nature, 368(6469), 339-342. doi:10.1038/368339a0

Kyjovska, Z. O., Jacobsen, N. R., Saber, A. T., Bengtson, S., Jackson, P., Wallin, H., & Vogel, U. (2015). DNA strand breaks, acute phase response and inflammation following pulmonary exposure by instillation to the diesel exhaust particle NIST1650b in mice. Mutagenesis, 30(4), 499-507. doi:10.1093/mutage/gev009

Li, Z., Hulderman, T., Salmen, R., Chapman, R., Leonard, S. S., Young, S. H., . . . Simeonova, P. P. (2007). Cardiovascular effects of pulmonary exposure to single-wall carbon nanotubes. Environ Health Perspect, 115(3), 377-382. doi:10.1289/ehp.9688

Libby, P. (2012). Inflammation in atherosclerosis. Arterioscler Thromb Vasc Biol, 32(9), 2045-2051. doi:10.1161/ATVBAHA.108.179705

Libby, P. (2021a). The changing landscape of atherosclerosis. Nature, 592(7855), 524-533. doi:10.1038/s41586-021-03392-8

Libby, P. (2021b). Inflammation during the life cycle of the atherosclerotic plaque. Cardiovasc Res, 117(13), 2525-2536. doi:10.1093/cvr/cvab303

Libby, P., Buring, J. E., Badimon, L., Hansson, G. K., Deanfield, J., Bittencourt, M. S., . . . Lewis, E. F. (2019). Atherosclerosis. Nat Rev Dis Primers, 5(1), 56. doi:10.1038/s41572-019-0106-z

Lindhorst, E., Young, D., Bagshaw, W., Hyland, M., & Kisilevsky, R. (1997). Acute inflammation, acute phase serum amyloid A and cholesterol metabolism in the mouse. Biochim Biophys Acta, 1339(1), 143-154. doi:10.1016/s0167-4838(96)00227-0

Mantovani, A., & Garlanda, C. (2023). Humoral Innate Immunity and Acute-Phase Proteins. N Engl J Med, 388(5), 439-452. doi:10.1056/NEJMra2206346

McGillicuddy, F. C., de la Llera Moya, M., Hinkle, C. C., Joshi, M. R., Chiquoine, E. H., Billheimer, J. T., . . . Reilly, M. P. (2009). Inflammation impairs reverse cholesterol transport in vivo. Circulation, 119(8), 1135-1145. doi:10.1161/CIRCULATIONAHA.108.810721

Meek, R. L., Urieli-Shoval, S., & Benditt, E. P. (1994). Expression of apolipoprotein serum amyloid A mRNA in human atherosclerotic lesions and cultured vascular cells: implications for serum amyloid A function. Proc Natl Acad Sci U S A, 91(8), 3186-3190. doi:10.1073/pnas.91.8.3186

Mikkelsen, L., Sheykhzade, M., Jensen, K. A., Saber, A. T., Jacobsen, N. R., Vogel, U., . . . Moller, P. (2011). Modest effect on plaque progression and vasodilatory function in atherosclerosis-prone mice exposed to nanosized TiO(2). Part Fibre Toxicol, 8, 32. doi:10.1186/1743-8977-8-32

Miller, K. A., Siscovick, D. S., Sheppard, L., Shepherd, K., Sullivan, J. H., Anderson, G. L., & Kaufman, J. D. (2007). Long-term exposure to air pollution and incidence of cardiovascular events in women. N Engl J Med, 356(5), 447-458. doi:10.1056/NEJMoa054409

Miller, M. R., McLean, S. G., Duffin, R., Lawal, A. O., Araujo, J. A., Shaw, C. A., . . . Hadoke, P. W. (2013). Diesel exhaust particulate increases the size and complexity of lesions in atherosclerotic mice. Part Fibre Toxicol, 10, 61. doi:10.1186/1743-8977-10-61

Miller, M. R., & Newby, D. E. (2020). Air pollution and cardiovascular disease: car sick. Cardiovasc Res, 116(2), 279-294. doi:10.1093/cvr/cvz228

Moldoveanu, B., Otmishi, P., Jani, P., Walker, J., Sarmiento, X., Guardiola, J., . . . Yu, J. (2009). Inflammatory mechanisms in the lung. J Inflamm Res, 2, 1-11.

Moller, P., Christophersen, D. V., Jacobsen, N. R., Skovmand, A., Gouveia, A. C., Andersen, M. H., . . . Loft, S. (2016). Atherosclerosis and vasomotor dysfunction in arteries of animals after exposure to combustion-derived particulate matter or nanomaterials. Crit Rev Toxicol, 46(5), 437-476. doi:10.3109/10408444.2016.1149451

Monse, C., Hagemeyer, O., Raulf, M., Jettkant, B., van Kampen, V., Kendzia, B., . . . Merget, R. (2018). Concentration-dependent systemic response after inhalation of nano-sized zinc oxide particles in human volunteers. Part Fibre Toxicol, 15(1), 8. doi:10.1186/s12989-018-0246-4

Monse, C., Raulf, M., Jettkant, B., van Kampen, V., Kendzia, B., Schurmeyer, L., . . . Bunger, J. (2021). Health effects after inhalation of micro- and nano-sized zinc oxide particles in human volunteers. Arch Toxicol, 95(1), 53-65. doi:10.1007/s00204-020-02923-y

Mossman, B. T., & Churg, A. (1998). Mechanisms in the pathogenesis of asbestosis and silicosis. Am J Respir Crit Care Med, 157(5 Pt 1), 1666-1680. doi:10.1164/ajrccm.157.5.9707141

NCBI. (2023). Retrieved from https://www.ncbi.nlm.nih.gov/gene

Oberdorster, G., Ferin, J., Gelein, R., Soderholm, S. C., & Finkelstein, J. (1992). Role of the alveolar macrophage in lung injury: studies with ultrafine particles. Environ Health Perspect, 97, 193-199. doi:10.1289/ehp.97-1519541

Oberdorster, G., Ferin, J., & Lehnert, B. E. (1994). Correlation between particle size, in vivo particle persistence, and lung injury. Environ Health Perspect, 102 Suppl 5(Suppl 5), 173-179. doi:10.1289/ehp.102-1567252

Pai, J. K., Pischon, T., Ma, J., Manson, J. E., Hankinson, S. E., Joshipura, K., . . . Rimm, E. B. (2004). Inflammatory markers and the risk of coronary heart disease in men and women. N Engl J Med, 351(25), 2599-2610. doi:10.1056/NEJMoa040967

Pope, C. A., 3rd, Burnett, R. T., Thurston, G. D., Thun, M. J., Calle, E. E., Krewski, D., & Godleski, J. J. (2004). Cardiovascular mortality and long-term exposure to particulate air pollution: epidemiological evidence of general pathophysiological pathways of disease. Circulation, 109(1), 71-77. doi:10.1161/01.CIR.0000108927.80044.7F

Pope, C. A., 3rd, Thun, M. J., Namboodiri, M. M., Dockery, D. W., Evans, J. S., Speizer, F. E., & Heath, C. W., Jr. (1995). Particulate air pollution as a predictor of mortality in a prospective study of U.S. adults. Am J Respir Crit Care Med, 151(3 Pt 1), 669-674. doi:10.1164/ajrccm/151.3_Pt_1.669

Poulsen, S. S., Knudsen, K. B., Jackson, P., Weydahl, I. E., Saber, A. T., Wallin, H., & Vogel, U. (2017). Multi-walled carbon nanotube-physicochemical properties predict the systemic acute phase response following pulmonary exposure in mice. PLoS One, 12(4), e0174167. doi:10.1371/journal.pone.0174167

Poulsen, S. S., Saber, A. T., Mortensen, A., Szarek, J., Wu, D., Williams, A., . . . Vogel, U. (2015). Changes in cholesterol homeostasis and acute phase response link pulmonary exposure to multi-walled carbon nanotubes to risk of cardiovascular disease. Toxicol Appl Pharmacol, 283(3), 210-222. doi:10.1016/j.taap.2015.01.011

Poulsen, S. S., Saber, A. T., Williams, A., Andersen, O., Kobler, C., Atluri, R., . . . Vogel, U. (2015). MWCNTs of different physicochemical properties cause similar inflammatory responses, but differences in transcriptional and histological markers of fibrosis in mouse lungs. Toxicol Appl Pharmacol, 284(1), 16-32. doi:10.1016/j.taap.2014.12.011

Raitakari, O., Pahkala, K., & Magnussen, C. G. (2022). Prevention of atherosclerosis from childhood. Nat Rev Cardiol, 19(8), 543-554. doi:10.1038/s41569-021-00647-9

Ridker, P. M., Everett, B. M., Thuren, T., MacFadyen, J. G., Chang, W. H., Ballantyne, C., . . . Group, C. T. (2017). Antiinflammatory Therapy with Canakinumab for Atherosclerotic Disease. N Engl J Med, 377(12), 1119-1131. doi:10.1056/NEJMoa1707914

Ridker, P. M., Hennekens, C. H., Buring, J. E., & Rifai, N. (2000). C-reactive protein and other markers of inflammation in the prediction of cardiovascular disease in women. N Engl J Med, 342(12), 836-843. doi:10.1056/NEJM200003233421202

Rivera, M. F., Lee, J. Y., Aneja, M., Goswami, V., Liu, L., Velsko, I. M., . . . Kesavalu, L. N. (2013). Polymicrobial infection with major periodontal pathogens induced periodontal disease and aortic atherosclerosis in hyperlipidemic ApoE(null) mice. PLoS One, 8(2), e57178. doi:10.1371/journal.pone.0057178

Roy, R., Singh, S. K., Das, M., Tripathi, A., & Dwivedi, P. D. (2014). Toll-like receptor 6 mediated inflammatory and functional responses of zinc oxide nanoparticles primed macrophages. Immunology, 142(3), 453-464. doi:10.1111/imm.12276

Saber, A. T., Halappanavar, S., Folkmann, J. K., Bornholdt, J., Boisen, A. M., Moller, P., . . . Wallin, H. (2009). Lack of acute phase response in the livers of mice exposed to diesel exhaust particles or carbon black by inhalation. Part Fibre Toxicol, 6, 12. doi:10.1186/1743-8977-6-12

Saber, A. T., Jacobsen, N. R., Mortensen, A., Szarek, J., Jackson, P., Madsen, A. M., . . . Wallin, H. (2012). Nanotitanium dioxide toxicity in mouse lung is reduced in sanding dust from paint. Part Fibre Toxicol, 9, 4. doi:10.1186/1743-8977-9-4

Saber, A. T., Lamson, J. S., Jacobsen, N. R., Ravn-Haren, G., Hougaard, K. S., Nyendi, A. N., . . . Vogel, U. (2013). Particle-induced pulmonary acute phase response correlates with neutrophil influx linking inhaled particles and cardiovascular risk. PLoS One, 8(7), e69020. doi:10.1371/journal.pone.0069020

Schmid, O., & Stoeger, T. (2016). Surface area is the biologically most effective dose metric for acute nanoparticle toxicity in the lung. Journal of Aerosol Science, 99, 133-143.

Stoeger, T., Reinhard, C., Takenaka, S., Schroeppel, A., Karg, E., Ritter, B., . . . Schulz, H. (2006). Instillation of six different ultrafine carbon particles indicates a surface area threshold dose for acute lung inflammation in mice. Environ Health Perspect, 114(3), 328-333. doi:10.1289/ehp.8266

Tannock, L. R., De Beer, M. C., Ji, A., Shridas, P., Noffsinger, V. P., den Hartigh, L., . . . Webb, N. R. (2018). Serum amyloid A3 is a high density lipoprotein-associated acute-phase protein. J Lipid Res, 59(2), 339-347. doi:10.1194/jlr.M080887

Thompson, J. C., Jayne, C., Thompson, J., Wilson, P. G., Yoder, M. H., Webb, N., & Tannock, L. R. (2015). A brief elevation of serum amyloid A is sufficient to increase atherosclerosis. J Lipid Res, 56(2), 286-293. doi:10.1194/jlr.M054015

Thompson, J. C., Wilson, P. G., Shridas, P., Ji, A., de Beer, M., de Beer, F. C., . . . Tannock, L. R. (2018). Serum amyloid A3 is pro-atherogenic. Atherosclerosis, 268, 32-35. doi:10.1016/j.atherosclerosis.2017.11.011

Uhlar, C. M., & Whitehead, A. S. (1999). Serum amyloid A, the major vertebrate acute-phase reactant. Eur J Biochem, 265(2), 501-523. doi:10.1046/j.1432-1327.1999.00657.x

Van Eeden, S., Leipsic, J., Paul Man, S. F., & Sin, D. D. (2012). The relationship between lung inflammation and cardiovascular disease. Am J Respir Crit Care Med, 186(1), 11-16. doi:10.1164/rccm.201203-0455PP

Venteclef, N., Jakobsson, T., Steffensen, K. R., & Treuter, E. (2011). Metabolic nuclear receptor signaling and the inflammatory acute phase response. Trends Endocrinol Metab, 22(8), 333-343. doi:10.1016/j.tem.2011.04.004

Virmani, R., Kolodgie, F. D., Burke, A. P., Finn, A. V., Gold, H. K., Tulenko, T. N., . . . Narula, J. (2005). Atherosclerotic plaque progression and vulnerability to rupture: angiogenesis as a source of intraplaque hemorrhage. Arterioscler Thromb Vasc Biol, 25(10), 2054-2061. doi:10.1161/01.ATV.0000178991.71605.18

Walker, E. S., Fedak, K. M., Good, N., Balmes, J., Brook, R. D., Clark, M. L., . . . Peel, J. L. (2022). Acute differences in blood lipids and inflammatory biomarkers following controlled exposures to cookstove air pollution in the STOVES study. Int J Environ Health Res, 32(3), 565-578. doi:10.1080/09603123.2020.1785402

Wallin, H., Kyjovska, Z. O., Poulsen, S. S., Jacobsen, N. R., Saber, A. T., Bengtson, S., . . . Vogel, U. (2017). Surface modification does not influence the genotoxic and inflammatory effects of TiO2 nanoparticles after pulmonary exposure by instillation in mice. Mutagenesis, 32(1), 47-57. doi:10.1093/mutage/gew046

Westberg, H., Elihn, K., Andersson, E., Persson, B., Andersson, L., Bryngelsson, I. L., . . . Sjogren, B. (2016). Inflammatory markers and exposure to airborne particles among workers in a Swedish pulp and paper mill. Int Arch Occup Environ Health, 89(5), 813-822. doi:10.1007/s00420-016-1119-5

Wyatt, L. H., Devlin, R. B., Rappold, A. G., Case, M. W., & Diaz-Sanchez, D. (2020). Low levels of fine particulate matter increase vascular damage and reduce pulmonary function in young healthy adults. Part Fibre Toxicol, 17(1), 58. doi:10.1186/s12989-020-00389-5

Appendix 1

List of MIEs in this AOP

Event: 1495: Substance interaction with the lung resident cell membrane components

Short Name: Interaction with the lung cell membrane

Key Event Component

AOPs Including This Key Event

Biological Context

Cell term

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

Human, mouse, rat.

Although the expression of DAMPs following exposure to pro-fibrotic substances is not assessed across species, it is known that alarmins are released after trauma or injury, and their release is important for initiating the inflammatory response in all species including humans. The immediate acute inflammatory response involving DAMP signalling is also observed in human idiopathic pulmonary fibrosis (IPF); however, anti-inflammatory drugs have proven ineffective for treating IPF. Danger signalling axis including uric acid, adenosine triphosphate and IL-33/ST2 has been proven to promote lung fibrosis in animals.

Key Event Description

The human lung consists of approximately 40 different resident cell types that play different roles during homeostasis, injury, repair and disease states (Franks et al., 2008; Luettich et al., 2021). Of these, resident airway epithelial cells, alveolar/interstitial macrophages and dendritic cells are well characterised for their ability to sense the danger upon interaction with harmful substances and relay the message to mount the necessary immune/inflammatory response. The resident macrophages are present in all tissues, and in a steady state, macrophages contribute to epithelial integrity, survey the tissue for invading pathogens or chemicals and maintain an immunosuppressive environment. Their main function is to clear the incoming irritants and microbes. They are named differently based on the tissue type and their specific functions (Kierdorf et al., 2015).

Substance interactions:

The chemicals or pathogens interact with cellular membrane to gain access to the organisms’ interior. A predominant interaction mechanism involves the recognition of innate immune response agonists by pattern recognition receptors (PRRs) present on resident cells such as epithelial and alveolar macrophages. PRRs are also present on other immune and parenchymal cells. PRRs can be activated by two classes of ligands. Pathogen associated molecular patterns (PAMPs) are microbial molecules derived from invading pathogens. PAMPs will not be discussed further as pathogens are not the focus for the AOP presented here. The other class of ligands are called danger associated molecular patterns (DAMPs) that include cellular fragments, nucleic acids, small molecules, proteins and even cytokines released from injured or dying cells (Bianchi, 2007). Most fibrogenic stressors discussed in this AOP act via DAMPs-driven PRR activation. High aspect ratio (HAR) materials such as asbestos or carbon nanotubes (CNTs) pierce the cellular membrane of epithelial cells or resident macrophages resulting in cell injury or non-programmed cellular death. Alveolar macrophages trying to engulf HAR fibres that are long and stiff undergo frustrated phagocytosis because of their inability to engulf the piercing fibres and subsequently lead to cell injury (Boyles et al., 2015; Brown et al., 2007; Donaldson K et al., 2010; Dörger et al., 2001; Mossman and Churg, 1998). The cellular debris from injured or dying cell then serves as ligands for PRRs (Nakayama, 2018), leading to cell activation. In case of pro-fibrotic insoluble particles such as silica, coal dust and nanomaterials (NMs), the particle adsorbed opsonins such as immunoglobulins, complement proteins, or serum proteins act as ligands to the receptors on the macrophage cell surface (Behzadi et al., 2017). The tissue response to these materials resembles that observed following foreign body invasion in lungs.

Toll-like receptors (TLRs) are highly conserved PRRs that are associated with fibrogenic stressors (Desai et al., 2018). Inhibition of TLR-4 is protective against bleomycin-induced fibrosis (Li et al., 2015). However, the exact role and mechanisms by which TLRs mediate lung fibrosis are yet to be uncovered and some studies have shown TLRs to be protective against lung fibrosis (Desai et al., 2018). Asbestos and silica crystals are suggested to engage scavenger receptors present on the macrophages. Mice deficient in class A scavenger macrophage receptor with collagenous structure (MARCO) are shown to induce reduced fibrogenic response following chrysotile asbestos exposure; although, the direct binding of MARCO by asbestos is not investigated in the study (Murthy et al., 2015). In case of soluble substances such as bleomycin, paraquat (Dinis-Oliveira et al., 2008) (N,N'-dimethyl-4, 4′-bipyridinium dichloride) and other soluble fibrogenic chemicals, direct damage of lung epithelial cells and resulting cellular debris or secreted cytokines (DAMPs) serve as triggers for downstream cascading pro-inflammatory events, tissue injury and fibrosis. Engagement of PRRs and consequent cell activation is observed in various organisms including flies and mammals (Denholm and Phan, 1990; Matzinger, 2002).

How it is Measured or Detected

Detection of DAMPs or homeostasis-altering molecular processes:

Cellular interaction with substances or particles can be measured by assessing the release of DAMPs from stressed, injured or dying cells - indicative of binding of PRRs on the cell surface. Release of DAMPs is reflective of substance interaction with resident cells and their activation, a key step in the process of inflammation.

The release of DAMPs can be measured by the techniques listed in the published literature (Nikota et al., 2017; Rabolli et al., 2014; Suwara et al., 2014).

Targeted enzyme-linked immunosorbent assays (ELISA) (routinely used and recommended):

ELISA  – permits quantitative measurement of antigens in biological samples. For example, in a cytokine ELISA (sandwich ELISA), an antibody (capture antibody) specific to a cytokine is immobilised on microtitre wells (96-well, 386-well, etc.). Experimental samples or samples containing a known amount of the specific recombinant cytokine are then reacted with the immobilised antibody. Following removal of unbound antibody by thorough washing, plates are reacted with the secondary antibody (detection antibody) that is conjugated to an enzyme such as horseradish peroxidase, which when bound, will form a sandwich with the capture antibody and the cytokine (Amsen and De Visser, 2009). The secondary antibody can be conjugated to biotin, which is then detected by addition of streptavidin linked to horseradish peroxidase. A chromogenic substrate can also be added, which is the most commonly used method. Chromogenic substrate is chemically converted by the enzyme coupled to the detection antibody, resulting in colour change. The amount of colour detected is directly proportional to the amount of cytokine in the sample that is bound to the capture antibody. The results are read using a spectrophotometer and compared to the levels of cytokine in control samples where cytokine is not expected to be secreted or to the samples containing known recombinant cytokine levels.

Interleukin (IL)-1α and -1β is activated or secreted into the cytosol following stimulus (Di Paolo and Shayakhmetov, 2016). Targeted ELISA can be used to quantify IL-1α  or IL-1β that is released in the culture supernatant of the cells exposed to toxicants, in bronchoalveolar lavage fluid and serum of exposed animals. The assay is also applicable to human serum, cerebrospinal fluid, and peritoneal fluids.

Similarly, other alarmins can also be quantified by ELISA. Western blot is another method that can be used to quantify the release of various alarmins using specific antibodies. ELISA or real-time reverse transcription-polymerase chain reaction (qRT-PCR) assays can also be used to quantify the expression of genes or proteins that are regulated by the receptor binding – e.g. downstream of TLR binding.

Frustrated phagocytosis and cellular uptake of NMs:

In vitro, interaction of NMs with the cellular membrane is investigated by assessing their uptake by lysosomes (Chen et al., 2013; Nel et al., 2009; Varela et al., 2012). Immunohistochemistry methods targeting lysosome specific proteins are regularly employed for this purpose. In co-localisation experiments, lysosomal marker Lysosomal-associated membrane protein 1 (LAMP1) antibody is used to detect particle co-localisation with lysosomes. A combination of Cytoviva hyperspectral microscope and immunolocalisation (Decan et al., 2016) or confocal microscopy to visualise co-localisation of fluorescence labelled nanoparticles with lysosomal markers have been used.

Frustrated phagocytosis is assessed using microscopic techniques such as time-lapse microscopy, backscatter electron microscopy and others (Donaldson et al., 2010; Murphy et al., 2012; Padmore et al., 2017; Pascolo et al., 2013; Schinwald et al., 2012). In addition, MIE 1668 of AOP303 notes other indirect methods for measuring frustrated phagocytosis.  

Cellular co-culture models of the pulmonary epithelium:

Complex co-culture systems, such as those containing epithelial cells and immune cells, better model the environment of the lung epithelium and can be used to study the interaction of potentially pro-fibrotic fibres and particles with resident lung cells. This type of model has been used, alongside electron microscopy, to study lung cell interactions with CNTs following 24 h in vitro exposure (Clift et al., 2014). More recently, the EpiAlveolar model, which contains primary human alveolar epithelial cells, endothelial cells, as well as fibroblasts was assessed for its ability to predict fibrosis induced by CNTs (Barasova et al., 2020). Using laser scanning, fluorescence, and enhanced darkfield microscopy, CNT interaction with the resident cells of the model was shown, and this interaction induced the formation of holes in the epithelial model (Barasova et al., 2020). While new co-culture models are a better recapitulation of the native lung environment as compared to traditional mono-cultures, the increased complexity necessitates enhanced expertise in tissue culture techniques, and can make them less practical as compared to submerged mono culture methods. 

Ex vivo model of the lung – Precision cut lung slices (PCLS):

Even closer to the in vivo condition than co-culture models, PCLS techniques capture the native lung architecture, cell-cell communication and cellularity of the lung. Advancement in culturing and cryopreservation techniques has increased accessibility and use of PCLS for longer term studies (Bai et al., 2016, Neuhaus et al., 2017). These slices can be cultured ex vivo for up to a week with minimal reduction in viability, and the technique has recently been assessed for its applicability to assess nanomaterial induced fibrosis ex vivo (Rahman et al., 2020). Using multi-walled carbon nanotubes (MWCNTs) and darkfield microscopy, interaction between the nanofibers and the lung epithelium could be determined. The main downside of this technique is the animal requirement, which precludes their use in a first-pass screening context for the MIE.

References

1. Amsen D, de Visser KE, Town T. Approaches to determine expression of inflammatory cytokines. Methods Mol Biol. 2009;511:107-42. doi: 10.1007/978-1-59745-447-6_5.

2. Bai Y, Krishnamoorthy N, Patel KR, Rosas I, Sanderson MJ, Ai X. Cryopreserved Human Precision-Cut Lung Slices as a Bioassay for Live Tissue Banking. A Viability Study of Bronchodilation with Bitter-Taste Receptor Agonists. Am J Respir Cell Mol Biol. 2016 May;54(5):656-63. doi: 10.1165/rcmb.2015-0290MA. 

3. Barosova H, Maione AG, Septiadi D, Sharma M, Haeni L, Balog S, O'Connell O, Jackson GR, Brown D, Clippinger AJ, Hayden P, Petri-Fink A, Stone V, Rothen-Rutishauser B. Use of EpiAlveolar Lung Model to Predict Fibrotic Potential of Multiwalled Carbon Nanotubes. ACS Nano. 2020 Apr 28;14(4):3941-3956. doi: 10.1021/acsnano.9b06860. 

4. Behzadi S, Serpooshan V, Tao W, Hamaly MA, Alkawareek MY, Dreaden EC, Brown D, Alkilany AM, Farokhzad OC, Mahmoudi M. Cellular uptake of nanoparticles: journey inside the cell. Chem Soc Rev. 2017 Jul 17;46(14):4218-4244. doi: 10.1039/c6cs00636a. 

5. Bianchi ME. DAMPs, PAMPs and alarmins: all we need to know about danger. J Leukoc Biol. 2007 Jan;81(1):1-5. doi: 10.1189/jlb.0306164. 

6. Boyles MS, Young L, Brown DM, MacCalman L, Cowie H, Moisala A, Smail F, Smith PJ, Proudfoot L, Windle AH, Stone V. Multi-walled carbon nanotube induced frustrated phagocytosis, cytotoxicity and pro-inflammatory conditions in macrophages are length dependent and greater than that of asbestos. Toxicol In Vitro. 2015 Oct;29(7):1513-28. doi: 10.1016/j.tiv.2015.06.012.

7. Brown DM, Kinloch IA, Bangert U, Windle AH, Walter DM, Walker GS, et al. An in vitro study of the potential of carbon nanotubes and nanofibres to induce inflammatory mediators and frustrated phagocytosis. Carbon. 2007;45(9):1743-56. doi: https://doi.org/10.1016/j.carbon.2007.05.011.

8. Centers for Disease Control and Prevention, National Institute for Occupational Safety and Health. (2011). Current Intelligence Bulletin 62: Asbestos Fibers and Other Elongate Mineral Particles: State of the Science and Roadmap for Research. Retrieved from https://www.cdc.gov/niosh/docs/2011-159/.

9. Cheng LC, Jiang X, Wang J, Chen C, Liu RS. Nano-bio effects: interaction of nanomaterials with cells. Nanoscale. 2013 May 7;5(9):3547-69. doi: 10.1039/c3nr34276j. 

10. Clift MJ, Endes C, Vanhecke D, Wick P, Gehr P, Schins RP, Petri-Fink A, Rothen-Rutishauser B. A comparative study of different in vitro lung cell culture systems to assess the most beneficial tool for screening the potential adverse effects of carbon nanotubes. Toxicol Sci. 2014 Jan;137(1):55-64. doi: 10.1093/toxsci/kft216. 

11. Decan N, Wu D, Williams A, Bernatchez S, Johnston M, Hill M, Halappanavar S. Characterization of in vitro genotoxic, cytotoxic and transcriptomic responses following exposures to amorphous silica of different sizes. Mutat Res Genet Toxicol Environ Mutagen. 2016 Jan 15;796:8-22. doi: 10.1016/j.mrgentox.2015.11.011.

12. Denholm EM, Phan SH. Bleomycin binding sites on alveolar macrophages. J Leukoc Biol. 1990 Dec;48(6):519-23. doi: 10.1002/jlb.48.6.519.

13. Desai O, Winkler J, Minasyan M, Herzog EL. The Role of Immune and Inflammatory Cells in Idiopathic Pulmonary Fibrosis. Front Med (Lausanne). 2018 Mar 20;5:43. doi: 10.3389/fmed.2018.00043. 

14. Di Paolo NC, Shayakhmetov DM. Interleukin 1α and the inflammatory process. Nat Immunol. 2016 Jul 19;17(8):906-13. doi: 10.1038/ni.3503.

15. Dinis-Oliveira RJ, Duarte JA, Sánchez-Navarro A, Remião F, Bastos ML, Carvalho F. Paraquat poisonings: mechanisms of lung toxicity, clinical features, and treatment. Crit Rev Toxicol. 2008;38(1):13-71. doi: 10.1080/10408440701669959.

16. Donaldson K, Murphy FA, Duffin R, Poland CA. Asbestos, carbon nanotubes and the pleural mesothelium: a review of the hypothesis regarding the role of long fibre retention in the parietal pleura, inflammation and mesothelioma. Part Fibre Toxicol. 2010 Mar 22;7:5. doi: 10.1186/1743-8977-7-5.

17. Dörger M, Münzing S, Allmeling AM, Messmer K, Krombach F. Differential responses of rat alveolar and peritoneal macrophages to man-made vitreous fibers in vitro. Environ Res. 2001 Mar;85(3):207-14. doi: 10.1006/enrs.2001.4234.

18. Franks TJ, Colby TV, Travis WD, Tuder RM, Reynolds HY, Brody AR, Cardoso WV, Crystal RG, Drake CJ, Engelhardt J, Frid M, Herzog E, Mason R, Phan SH, Randell SH, Rose MC, Stevens T, Serge J, Sunday ME, Voynow JA, Weinstein BM, Whitsett J, Williams MC. Resident cellular components of the human lung: current knowledge and goals for research on cell phenotyping and function. Proc Am Thorac Soc. 2008 Sep 15;5(7):763-6. doi: 10.1513/pats.200803-025HR.

19. Kierdorf K, Prinz M, Geissmann F, Gomez Perdiguero E. Development and function of tissue resident macrophages in mice. Semin Immunol. 2015 Dec;27(6):369-78. doi: 10.1016/j.smim.2016.03.017.

20. Kim JE, Lim HT, Minai-Tehrani A, Kwon JT, Shin JY, Woo CG, Choi M, Baek J, Jeong DH, Ha YC, Chae CH, Song KS, Ahn KH, Lee JH, Sung HJ, Yu IJ, Beck GR Jr, Cho MH. Toxicity and clearance of intratracheally administered multiwalled carbon nanotubes from murine lung. J Toxicol Environ Health A. 2010;73(21-22):1530-43. doi: 10.1080/15287394.2010.511578. 

21. Luettich K, Sharma M, Yepiskoposyan H, Breheny D, Lowe FJ. An Adverse Outcome Pathway for Decreased Lung Function Focusing on Mechanisms of Impaired Mucociliary Clearance Following Inhalation Exposure. Front Toxicol. 2021 Dec 14;3:750254. doi: 10.3389/ftox.2021.750254.

22. Li XX, Jiang DY, Huang XX, Guo SL, Yuan W, Dai HP. Toll-like receptor 4 promotes fibrosis in bleomycin-induced lung injury in mice. Genet Mol Res. 2015 Dec 21;14(4):17391-8. doi: 10.4238/2015.

23. Matzinger P. The danger model: a renewed sense of self. Science. 2002 Apr 12;296(5566):301-5. doi: 10.1126/science.1071059.

24. Mossman BT, Churg A. Mechanisms in the pathogenesis of asbestosis and silicosis. Am J Respir Crit Care Med. 1998 May;157(5 Pt 1):1666-80. doi: 10.1164/ajrccm.157.5.9707141.

25. Murphy FA, Schinwald A, Poland CA, Donaldson K. The mechanism of pleural inflammation by long carbon nanotubes: interaction of long fibres with macrophages stimulates them to amplify pro-inflammatory responses in mesothelial cells. Part Fibre Toxicol. 2012 Apr 3;9:8. doi: 10.1186/1743-8977-9-8. 

26. Murthy S, Larson-Casey JL, Ryan AJ, He C, Kobzik L, Carter AB. Alternative activation of macrophages and pulmonary fibrosis are modulated by scavenger receptor, macrophage receptor with collagenous structure. FASEB J. 2015 Aug;29(8):3527-36. doi: 10.1096/fj.15-271304.

27. Nakayama M. Macrophage Recognition of Crystals and Nanoparticles. Front Immunol. 2018 Jan 29;9:103. doi: 10.3389/fimmu.2018.00103. 

28. Nel AE, Mädler L, Velegol D, Xia T, Hoek EM, Somasundaran P, Klaessig F, Castranova V, Thompson M. Understanding biophysicochemical interactions at the nano-bio interface. Nat Mater. 2009 Jul;8(7):543-57. doi: 10.1038/nmat2442. 

29. Neuhaus V, Schaudien D, Golovina T, Temann UA, Thompson C, Lippmann T, Bersch C, Pfennig O, Jonigk D, Braubach P, Fieguth HG, Warnecke G, Yusibov V, Sewald K, Braun A. Assessment of long-term cultivated human precision-cut lung slices as an ex vivo system for evaluation of chronic cytotoxicity and functionality. J Occup Med Toxicol. 2017 May 26;12:13. doi: 10.1186/s12995-017-0158-5.

30. Nikota J, Banville A, Goodwin LR, Wu D, Williams A, Yauk CL, Wallin H, Vogel U, Halappanavar S. Stat-6 signaling pathway and not Interleukin-1 mediates multi-walled carbon nanotube-induced lung fibrosis in mice: insights from an adverse outcome pathway framework. Part Fibre Toxicol. 2017 Sep 13;14(1):37. doi: 10.1186/s12989-017-0218-0.

31. Padmore T, Stark C, Turkevich LA, Champion JA. Quantitative analysis of the role of fiber length on phagocytosis and inflammatory response by alveolar macrophages. Biochim Biophys Acta Gen Subj. 2017 Feb;1861(2):58-67. doi: 10.1016/j.bbagen.2016.09.031.

32. Pascolo L, Gianoncelli A, Schneider G, Salomé M, Schneider M, Calligaro C, Kiskinova M, Melato M, Rizzardi C. The interaction of asbestos and iron in lung tissue revealed by synchrotron-based scanning X-ray microscopy. Sci Rep. 2013;3:1123. doi: 10.1038/srep01123. 

33. Poland CA, Duffin R, Kinloch I, Maynard A, Wallace WA, Seaton A, Stone V, Brown S, Macnee W, Donaldson K. Carbon nanotubes introduced into the abdominal cavity of mice show asbestos-like pathogenicity in a pilot study. Nat Nanotechnol. 2008 Jul;3(7):423-8. doi: 10.1038/nnano.2008.111.

34. Rabolli V, Badissi AA, Devosse R, Uwambayinema F, Yakoub Y, Palmai-Pallag M, Lebrun A, De Gussem V, Couillin I, Ryffel B, Marbaix E, Lison D, Huaux F. The alarmin IL-1α is a master cytokine in acute lung inflammation induced by silica micro- and nanoparticles. Part Fibre Toxicol. 2014 Dec 13;11:69. doi: 10.1186/s12989-014-0069-x.

35. Rahman L, Williams A, Gelda K, Nikota J, Wu D, Vogel U, Halappanavar S. 21st Century Tools for Nanotoxicology: Transcriptomic Biomarker Panel and Precision-Cut Lung Slice Organ Mimic System for the Assessment of Nanomaterial-Induced Lung Fibrosis. Small. 2020 Sep;16(36):e2000272. doi: 10.1002/smll.202000272.

36. Schinwald A, Donaldson K. Use of back-scatter electron signals to visualise cell/nanowires interactions in vitro and in vivo; frustrated phagocytosis of long fibres in macrophages and compartmentalisation in mesothelial cells in vivo. Part Fibre Toxicol. 2012 Aug 28;9:34. doi: 10.1186/1743-8977-9-34. 

37. Suwara MI, Green NJ, Borthwick LA, Mann J, Mayer-Barber KD, Barron L, Corris PA, Farrow SN, Wynn TA, Fisher AJ, Mann DA. IL-1α released from damaged epithelial cells is sufficient and essential to trigger inflammatory responses in human lung fibroblasts. Mucosal Immunol. 2014 May;7(3):684-93. doi: 10.1038/mi.2013.87.

38. Varela JA, Bexiga MG, Åberg C, Simpson JC, Dawson KA. Quantifying size-dependent interactions between fluorescently labeled polystyrene nanoparticles and mammalian cells. J Nanobiotechnology. 2012 Sep 24;10:39. doi: 10.1186/1477-3155-10-39. 

List of Key Events in the AOP

Event: 1496: Increased, secretion of proinflammatory mediators

Short Name: Increased proinflammatory mediators

Key Event Component

AOPs Including This Key Event

Biological Context

Cell term

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

Human, mouse, rat

Cytokines are the common pro-inflammatory mediators secreted following inflammogenic stimuli. Cytokines can be defined as a diverse group of signaling protein molecules. They are secreted by different cell types in different tissues and in all mammalian species, irrespective of gender, age or sex. A lot of literature is available to support cross species, gender and developmental stage application for this KE. The challenge is the specificity; most cytokines exhibit redundant functions and many are pleotropic.

Key Event Description

Pro-inflammatory mediators are the chemical and biological molecules that initiate and regulate inflammatory reactions. Pro-inflammatory mediators are secreted following exposure to an inflammogen in a gender/sex or developmental stage independent manner. They are secreted during inflammation in all species. Different types of pro-inflammatory mediators are secreted during innate or adaptive immune responses across various species (Mestas and Hughes, 2004). Cell-derived pro-inflammatory mediators include cytokines, chemokines, and growth factors. Blood derived pro-inflammatory mediators include vasoactive amines, complement activation products and others. These modulators can be grouped based on the cell type that secrete them, their cellular localisation and also based on the type of immune response they trigger. For example, members of the interleukin (IL) family including IL-2, IL-4, IL-7, IL-9, IL-15, IL-21, IL-3, IL-5 and Granulocyte-macrophage colony stimulating factor (GM-CSF) are involved in the adaptive immune responses. The pro-inflammatory cytokines include IL-1 family (IL-1α , IL-1β, IL-1rα, IL-18, IL-36α, IL-36β, IL-36γ, IL-36Rα, IL-37), IL-6 family, Tumor necrosis factor (TNF) family, IL-17, and Interferon gamma (IFN-γ) (Turner et al., 2014). While IL-4 and IL-5 are considered T helper (Th) cell type 2 response, IFN-γ is suggested to be Th1 type response.

Different types of pro-inflammatory mediators are secreted during innate or adaptive immune responses across various species (Mestas and Hughes, 2004). However, IL-1 family cytokines, IL-4, IL-5, IL-6, TNF-α, IFN-γ are the commonly measured mediators in experimental animals and in humans. Similar gene expression patterns involving inflammation and matrix remodelling are observed in human patients of pulmonary fibrosis and mouse lungs exposed to bleomycin (Kaminski, 2002).

Literature evidence for its perturbation:

Several studies show increased proinflammatory mediators in rodent lungs and bronchoalveolar lavage fluid, and in cell culture supernatants following exposure to a variety of carbon nanotube (CNT) types and other materials. Poland et al., 2008 showed that long and thin CNTs (>5 µm) can elicit asbestos-like pathogenicity through the continual release of pro-inflammatory cytokines and reactive oxygen species. Exposure to crystalline silica induces release of inflammatory cytokines (TNF-α, IL-1, IL-6), transcription factors (Nuclear factor kappa B [NF-κB], Activator protein-1 [AP-1]) and kinase signalling pathways in mice that contain NF-κB luciferase reporter (Hubbard et al., 2002). Boyles et al., 2015 found that lung responses to long multi-walled carbon nanotubes (MWCNTs) included high expression levels of pro-inflammatory mediators Monocyte chemoattractant protein 1 (MCP-1), Transforming growth factor beta 1 (TGF-β1), and TNF-α (Boyles et al., 2015). Bleomycin administration in rodents induces lung inflammation and increased expression of pro-inflammatory mediators (Park et al., 2019). Inflammation induced by bleomycin, paraquat and CNTs is characterised by the altered expression of pro-inflammatory mediators. A large number of nanomaterials induce expression of cytokines and chemokines in lungs of rodents exposed via inhalation (Halappanavar et al., 2011; Husain et al., 2015a). Similarities are observed in gene programs involving pro-inflammatory event is observed in both humans and experimental mice (Zuo et al., 2002).

How it is Measured or Detected

The selection of pro-inflammatory mediators for investigation varies based on the expertise of the lab, cell types studied and the availability of the specific antibodies.

Real-time reverse transcription-polymerase chain reaction (qRT-PCR) – will measure the abundance of cytokine mRNA in a given sample. The method involves three steps: conversion of RNA into cDNA by reverse transcription method, amplification of cDNA using the PCR, and the real-time detection and quantification of amplified products (amplicons) (Nolan et al., 2006). Amplicons are detected using fluorescence, increase in which is directly proportional to the amplified PCR product. The number of cycles required per sample to reach a certain threshold of fluorescence (set by the user – usually set in the linear phase of the amplification, and the observed difference in samples to cross the set threshold reflects the initial amount available for amplification) is used to quantify the relative amount in the samples. The amplified products are detected by the DNA intercalating minor groove-binding fluorophore SYBR green, which produces a signal when incorporated into double-stranded amplicons. Since the cDNA is single stranded, the dye does not bind enhancing the specificity of the results. There are other methods such as nested fluorescent probes for detection, but SYBR green is widely used. RT-PCR primers specific to several pro-inflammatory mediators in several species including mouse, rat and humans, are readily available commercially.

Enzyme-linked immunosorbent assays (ELISA) – permit quantitative measurement of antigens in biological samples. The method is the same as described for the MIE. Both ELISA and qRT-PCR assays are used in vivo and are readily applicable to in vitro cell culture models, where cell culture supernatants or whole cell homogenates are used for ELISA or mRNA assays. Both assays are straight forward, quantitative and require relatively a small amount of input sample.

Apart from assaying single protein or gene at a time, cytokine bead arrays or cytokine PCR arrays can also be used to detect a whole panel of inflammatory mediators in a multiplex method (Husain et al., 2015b). This method is quantitative and especially advantageous when the sample amount available for testing is scarce. Lastly, immunohistochemistry can also be used to detect specific immune cell types producing the pro-inflammatory mediators and its downstream effectors in any given tissue (Costa et al., 2017). Immunohistochemistry results can be used as weight of evidence; however, the technique is not quantitative and depending on the specific antibodies used, the assay sensitivity may also become an issue (Amsen and De Visser, 2009).

Cell models - of varying complexity have been used to assess the expression of pro-inflammatory mediators. Two dimensional submerged monocultures of the main fibrotic effector cells – lung epithelial cells, macrophages, and fibroblasts – have routinely been used in vitro due to the large literature base, and ease of use, but do not adequately mimic the in vivo condition (Sharma et al., 2016; Sundarakrishnan et al., 2018). Recently, the EpiAlveolar in vitro lung model (containing epithelial cells, endothelial cells, and fibroblasts) was used to predict the fibrotic potential of MWCNTs, and researchers noted increases in the pro-inflammatory molecules TNF-α, IL-1β, and the pro-fibrotic TGF-β using ELISA (Barasova et al., 2020). A similar, but less complicated co-culture model of immortalized human alveolar epithelial cells and idiopathic pulmonary fibrosis patient derived fibroblasts was used to assess pro-fibrotic signalling, and noted enhanced secretion of Platelet derived growth factor (PDGF) and Basic fibroblast growth factor (bFGF), as well as evidence for epithelial to mesenchymal transition of epithelial cells in this system (Prasad et al., 2014). Models such as these better capitulate the in vivo pulmonary alveolar capillary, but have lower reproducibility as compared to traditional submerged mono-culture experiments.

References

1. Amsen D, de Visser KE, Town T. Approaches to determine expression of inflammatory cytokines. Methods Mol Biol. 2009;511:107-42. doi: 10.1007/978-1-59745-447-6_5. 

2. Barosova H, Maione AG, Septiadi D, Sharma M, Haeni L, Balog S, O'Connell O, Jackson GR, Brown D, Clippinger AJ, Hayden P, Petri-Fink A, Stone V, Rothen-Rutishauser B. Use of EpiAlveolar Lung Model to Predict Fibrotic Potential of Multiwalled Carbon Nanotubes. ACS Nano. 2020 Apr 28;14(4):3941-3956. doi: 10.1021/acsnano.9b06860. 

3. Boyles MS, Young L, Brown DM, MacCalman L, Cowie H, Moisala A, Smail F, Smith PJ, Proudfoot L, Windle AH, Stone V. Multi-walled carbon nanotube induced frustrated phagocytosis, cytotoxicity and pro-inflammatory conditions in macrophages are length dependent and greater than that of asbestos. Toxicol In Vitro. 2015 Oct;29(7):1513-28. doi: 10.1016/j.tiv.2015.06.012. 

4. Costa PM, Gosens I, Williams A, Farcal L, Pantano D, Brown DM, Stone V, Cassee FR, Halappanavar S, Fadeel B. Transcriptional profiling reveals gene expression changes associated with inflammation and cell proliferation following short-term inhalation exposure to copper oxide nanoparticles. J Appl Toxicol. 2018 Mar;38(3):385-397. doi: 10.1002/jat.3548.

5. Halappanavar S, Jackson P, Williams A, Jensen KA, Hougaard KS, Vogel U, Yauk CL, Wallin H. Pulmonary response to surface-coated nanotitanium dioxide particles includes induction of acute phase response genes, inflammatory cascades, and changes in microRNAs: a toxicogenomic study. Environ Mol Mutagen. 2011 Jul;52(6):425-39. doi: 10.1002/em.20639. 

6. Hubbard AK, Timblin CR, Shukla A, Rincón M, Mossman BT. Activation of NF-kappaB-dependent gene expression by silica in lungs of luciferase reporter mice. Am J Physiol Lung Cell Mol Physiol. 2002 May;282(5):L968-75. doi: 10.1152/ajplung.00327.2001.

7. Husain M, Kyjovska ZO, Bourdon-Lacombe J, Saber AT, Jensen KA, Jacobsen NR, Williams A, Wallin H, Halappanavar S, Vogel U, Yauk CL. Carbon black nanoparticles induce biphasic gene expression changes associated with inflammatory responses in the lungs of C57BL/6 mice following a single intratracheal instillation. Toxicol Appl Pharmacol. 2015a Dec 15;289(3):573-88. doi: 10.1016/j.taap.2015.11.003.

8. Husain M, Wu D, Saber AT, Decan N, Jacobsen NR, Williams A, Yauk CL, Wallin H, Vogel U, Halappanavar S. Intratracheally instilled titanium dioxide nanoparticles translocate to heart and liver and activate complement cascade in the heart of C57BL/6 mice. Nanotoxicology. 2015b;9(8):1013-22. doi: 10.3109/17435390.2014.996192.

9. Kaminski N. Microarray analysis of idiopathic pulmonary fibrosis. Am J Respir Cell Mol Biol. 2003 Sep;29(3 Suppl):S32-6.

10. Mestas J, Hughes CC. Of mice and not men: differences between mouse and human immunology. J Immunol. 2004 Mar 1;172(5):2731-8. doi: 10.4049/jimmunol.172.5.2731.

11. Nolan T, Hands RE, Bustin SA. Quantification of mRNA using real-time RT-PCR. Nat Protoc. 2006;1(3):1559-82. doi: 10.1038/nprot.2006.236.

12. Park SJ, Im DS. Deficiency of Sphingosine-1-Phosphate Receptor 2 (S1P₂) Attenuates Bleomycin-Induced Pulmonary Fibrosis. Biomol Ther (Seoul). 2019 May 1;27(3):318-326. doi: 10.4062/biomolther.2018.131.

13. Poland CA, Duffin R, Kinloch I, Maynard A, Wallace WA, Seaton A, Stone V, Brown S, Macnee W, Donaldson K. Carbon nanotubes introduced into the abdominal cavity of mice show asbestos-like pathogenicity in a pilot study. Nat Nanotechnol. 2008 Jul;3(7):423-8. doi: 10.1038/nnano.2008.111.

14. Prasad S, Hogaboam CM, Jarai G. Deficient repair response of IPF fibroblasts in a co-culture model of epithelial injury and repair. Fibrogenesis Tissue Repair. 2014 Apr 29;7:7. doi: 10.1186/1755-1536-7-7. 

15. Sharma M, Nikota J, Halappanavar S, Castranova V, Rothen-Rutishauser B, Clippinger AJ. Predicting pulmonary fibrosis in humans after exposure to multi-walled carbon nanotubes (MWCNTs). Arch Toxicol. 2016 Jul;90(7):1605-22. doi: 10.1007/s00204-016-1742-7. 

16. Sundarakrishnan A, Chen Y, Black LD, Aldridge BB, Kaplan DL. Engineered cell and tissue models of pulmonary fibrosis. Adv Drug Deliv Rev. 2018 Apr;129:78-94. doi: 10.1016/j.addr.2017.12.013.

17. Turner MD, Nedjai B, Hurst T, Pennington DJ. Cytokines and chemokines: At the crossroads of cell signalling and inflammatory disease. Biochim Biophys Acta. 2014 Nov;1843(11):2563-2582. doi: 10.1016/j.bbamcr.2014.05.014. 

18. Zuo F, Kaminski N, Eugui E, Allard J, Yakhini Z, Ben-Dor A, Lollini L, Morris D, Kim Y, DeLustro B, Sheppard D, Pardo A, Selman M, Heller RA. Gene expression analysis reveals matrilysin as a key regulator of pulmonary fibrosis in mice and humans. Proc Natl Acad Sci U S A. 2002 Apr 30;99(9):6292-7. doi: 10.1073/pnas.092134099.

Event: 1438: Transcription of genes encoding acute phase proteins, Increased

Short Name: Increased transcription of genes encoding acute phase proteins

Key Event Component

AOPs Including This Key Event

Biological Context

Organ term

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

• Taxonomic applicability: Acute phase response is part of the immune response and is observed in vertebrate species (Cray et al., 2009).
• Life stage applicability: This key event is applicable to all life stages.
• Sex applicability: This key event is applicable to male and female sexes.

Key Event Description

Acute phase proteins (APPs) are proteins that have an increase in plasma concentration of at least 25% during an acute phase response (Gabay & Kushner, 1999; Mantovani & Garlanda, 2023). In humans, the major APPs are C reactive protein (CRP) and serum amyloid A (SAA), while in mice the major APPs are SAA, haptoglobin and serum amyloid P (Cray, Zaias, & Altman, 2009; Gabay & Kushner, 1999).

It is widely accepted than APPs are mainly produced in the liver, while several other tissues have been shown to express APPs. In humans, APP genes have been shown to be expressed in the adrenal gland, adipose tissue, appendix, gall bladder, heart, kidney, liver, lung, placenta, prostate, salivary gland, skin, small intestine, stomach, thymus, thyroid, trachea and uterus (de Dios et al., 2018; NCBI, 2023; Schrödl et al., 2016; Urieli-Shoval, Cohen, Eisenberg, & Matzner, 1998; Venteclef, Jakobsson, Steffensen, & Treuter, 2011). In mice, APPs have been shown to be expressed in the adrenal gland, bladder, central nervous system, colon, duodenum, genital fat pad, heart, kidney, large intestine, limbs, liver, lung, mammary gland, ovary, placenta, small intestine, spleen, subcutaneous fat pad, testis and thymus (Kalmovarin et al., 1991; NCBI, 2023; Saber et al., 2013). 

Table 1 presents a list of acute phase response genes in humans and mice according the National Center for Biotechnology Information (NCBI): Table 1.

It is important to note that humans and mice express four SAA isoforms (Saa1, Saa2, Saa3 and Saa4), while Saa3 is a pseudogene in humans (Shridas & Tannock, 2019). CRP is expressed in humans and mice, although only moderately expressed in mice (Pepys & Hirschfield, 2003).

It has been shown that in mice, the Saa3 isoform is the most differentially expressed APP gene in lung tissue and it is not highly expressed in the liver, while Saa1 gene is the most differentially expressed in liver tissue after exposure to particles (Halappanavar et al., 2011; Poulsen et al., 2017; Saber et al., 2014).

How it is Measured or Detected

Gene expression of acute phase proteins (APPs) can be measured from tissue samples using quantitative Polymerase Chain Reaction (PCR). This technique allows the amplification of selected fragments of DNA or cDNA by using primers (i.e. known end-portions of the selected DNA). By repeated cycles of transcription, DNA is amplified. The use of fluorescent probes to quantify the expression the targeted DNA, as the binding of the probe to this DNA emits a fluorescent signal. Sequences of RNA can be quantify using PCR, by first synthetizing DNA from a RNA sample, resulting in cDNA. This technique is called reverse transcriptase PCR (Nelson, Nelson, Lehninger, & Cox, 2017).

Other techniques for evaluating the expression of several APPs at the same time are microarray analysis and total RNA sequencing (Halappanavar et al., 2011; Nelson et al., 2017).

To evaluate this key event in mice, gene expression of Saa3 can be quantified in lung tissue and Saa1 gene in liver tissue after exposure to a stressor (Halappanavar et al., 2011; Poulsen et al., 2017; Saber et al., 2014).

In humans, it is not common to measure gene expression as a tissue sample is required, however gene expression of crp, saa1, saa2 and saa4 can be measured from samples taken during surgery (Calero et al., 2014).

References

Calero, C., Arellano, E., Lopez-Villalobos, J. L., Sanchez-Lopez, V., Moreno-Mata, N., & Lopez-Campos, J. L. (2014). Differential expression of C-reactive protein and serum amyloid A in different cell types in the lung tissue of chronic obstructive pulmonary disease patients. BMC Pulm Med, 14, 95. doi:10.1186/1471-2466-14-95

Cray, C., Zaias, J., & Altman, N. H. (2009). Acute phase response in animals: a review. Comp Med, 59(6), 517-526.

de Dios, O., Gavela-Perez, T., Aguado-Roncero, P., Perez-Tejerizo, G., Ricote, M., Gonzalez, N., . . . Soriano-Guillen, L. (2018). C-reactive protein expression in adipose tissue of children with acute appendicitis. Pediatr Res, 84(4), 564-567. doi:10.1038/s41390-018-0091-z

Gabay, C., & Kushner, I. (1999). Acute-phase proteins and other systemic responses to inflammation. N Engl J Med, 340(6), 448-454. doi:10.1056/NEJM199902113400607

Halappanavar, S., Jackson, P., Williams, A., Jensen, K. A., Hougaard, K. S., Vogel, U., . . . Wallin, H. (2011). Pulmonary response to surface-coated nanotitanium dioxide particles includes induction of acute phase response genes, inflammatory cascades, and changes in microRNAs: a toxicogenomic study. Environ Mol Mutagen, 52(6), 425-439. doi:10.1002/em.20639

Kalmovarin, N., Friedrichs, W. E., O'Brien, H. V., Linehan, L. A., Bowman, B. H., & Yang, F. (1991). Extrahepatic expression of plasma protein genes during inflammation. Inflammation, 15(5), 369-379. doi:10.1007/BF00917353

Mantovani, A., & Garlanda, C. (2023). Humoral Innate Immunity and Acute-Phase Proteins. N Engl J Med, 388(5), 439-452. doi:10.1056/NEJMra2206346

NCBI. (2023). Retrieved from https://www.ncbi.nlm.nih.gov/gene

Nelson, D. L., Nelson, D. L., Lehninger, A. L., & Cox, M. M. (2017). Lehninger Principles of biochemistry (Seventh edition ed.). Macmillan Higher Education: Basingstoke.

Pepys, M. B., & Hirschfield, G. M. (2003). C-reactive protein: a critical update. J Clin Invest, 111(12), 1805-1812. doi:10.1172/JCI18921

Poulsen, S. S., Knudsen, K. B., Jackson, P., Weydahl, I. E., Saber, A. T., Wallin, H., & Vogel, U. (2017). Multi-walled carbon nanotube-physicochemical properties predict the systemic acute phase response following pulmonary exposure in mice. PLoS One, 12(4), e0174167. doi:10.1371/journal.pone.0174167

Saber, A. T., Jacobsen, N. R., Jackson, P., Poulsen, S. S., Kyjovska, Z. O., Halappanavar, S., . . . Vogel, U. (2014). Particle-induced pulmonary acute phase response may be the causal link between particle inhalation and cardiovascular disease. Wiley Interdiscip Rev Nanomed Nanobiotechnol, 6(6), 517-531. doi:10.1002/wnan.1279

Saber, A. T., Lamson, J. S., Jacobsen, N. R., Ravn-Haren, G., Hougaard, K. S., Nyendi, A. N., . . . Vogel, U. (2013). Particle-induced pulmonary acute phase response correlates with neutrophil influx linking inhaled particles and cardiovascular risk. PLoS One, 8(7), e69020. doi:10.1371/journal.pone.0069020

Schrödl, W., Büchler, R., Wendler, S., Reinhold, P., Muckova, P., Reindl, J., & Rhode, H. (2016). Acute phase proteins as promising biomarkers: Perspectives and limitations for human and veterinary medicine. 10(11), 1077-1092. doi:https://doi.org/10.1002/prca.201600028

Shridas, P., & Tannock, L. R. (2019). Role of serum amyloid A in atherosclerosis. Curr Opin Lipidol, 30(4), 320-325. doi:10.1097/MOL.0000000000000616

Urieli-Shoval, S., Cohen, P., Eisenberg, S., & Matzner, Y. (1998). Widespread expression of serum amyloid A in histologically normal human tissues. Predominant localization to the epithelium. J Histochem Cytochem, 46(12), 1377-1384. doi:10.1177/002215549804601206

Venteclef, N., Jakobsson, T., Steffensen, K. R., & Treuter, E. (2011). Metabolic nuclear receptor signaling and the inflammatory acute phase response. Trends Endocrinol Metab, 22(8), 333-343. doi:10.1016/j.tem.2011.04.004

Event: 1439: Systemic acute phase response

Short Name: Systemic acute phase response

Key Event Component

AOPs Including This Key Event

Biological Context

Organ term

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

• Taxonomic applicability: Acute phase response is part of the immune response and is observed in vertebrates (Cray et al., 2009).
• Life stages applicability: This key event is applicable to all life stages.
• Sex applicability: This key event is applicable to male and females sexes.

Key Event Description

Acute phase response (APR) is the systemic response to acute and chronic inflammatory states, for example bacterial infection, trauma and infarction. These changes include variations in plasma concentration of proteins, along with other physiological changes. Proteins are considered acute phase proteins (APPs) when their plasma concentration changes at least 25% (Gabay & Kushner, 1999).

APPs that increase their concentration during APR are called positive APP, while negative APP are decreased during APR (Gabay & Kushner, 1999). In humans, the two major APPs are C-reactive protein (CRP) and serum amyloid A (SAA), whose concentration can increase more than 1000-fold during an acute phase response (Gabay & Kushner, 1999). In mice, the major APPs (i.e. the APPs whose plasma concentration increases the most during APR) are SAA, haptoglobin and serum amyloid P (Cray, Zaias, & Altman, 2009).

How it is Measured or Detected

Systemic acute phase response is assessed by measuring acute phase proteins (APPs) concentrations in blood plasma or serum.

In mice, serum amyloid A (SAA) isoforms are measured using enzyme-linked immunosorbent assay (ELISA) assays or Western blot.  (Christophersen et al., 2021; Gutierrez et al., 2023; Hadrup et al., 2019; Halappanavar et al., 2011; Poulsen et al., 2017). ELISA assays allows the measurement of proteins in a sample by adsorbing the desired proteins (antigens) to an inert surface. Using antibodies specific to the protein and an enzyme that catalyzes the reaction, a colorimetric signal is produced and measured (Nelson, Nelson, Lehninger, & Cox, 2017). In the case of Western blot, also called immunoblot assay, proteins in a sample are first separated by gel electrophoresis. Following, the proteins are transferred to a membrane and treated with antibodies, obtaining a coloured precipitate along the band of the desired protein (Nelson et al., 2017).

 

In humans, most often C-reactive protein (CRP) and SAA are measured. These proteins are measured by immunoassays detecting single or multiple proteins (Adetona et al., 2017; Andersen et al., 2019; Baumann et al., 2018; Meier et al., 2014; Monse et al., 2018; Walker et al., 2022; Wyatt, Devlin, Rappold, Case, & Diaz-Sanchez, 2020). In addition, CRP is measured by turbidimetric (Barregard et al., 2006; Kim, Chen, Boyce, & Christiani, 2005; Sikkeland et al., 2018) and nephelometric assays (Brand et al., 2014).  

For both techniques, a blood sample (serum or plasma) is mixed with a suspension of latex beads coated with CRP antibodies. When CRP binds to the beads, a complex is formed that produces the scattering of light. The amount of light scattered is proportional to the amount of complexes formed. While in nephelometry the amount of light scaterred is measure, in turbidimetry the amount of light that passes through the suspension is measured. The measurements are later converted to CRP concentration using a calibration curve (Drieghe, Alsaadi, Tugirimana, & Delanghe, 2014; Hamilton, 2014).

CRP is used in the clinical setting as a marker of systemic inflammation.

References

Adetona, A. M., Adetona, O., Gogal, R. M., Jr., Diaz-Sanchez, D., Rathbun, S. L., & Naeher, L. P. (2017). Impact of Work Task-Related Acute Occupational Smoke Exposures on Select Proinflammatory Immune Parameters in Wildland Firefighters. J Occup Environ Med, 59(7), 679-690. doi:10.1097/JOM.0000000000001053

Andersen, M. H. G., Frederiksen, M., Saber, A. T., Wils, R. S., Fonseca, A. S., Koponen, I. K., . . . Vogel, U. (2019). Health effects of exposure to diesel exhaust in diesel-powered trains. Part Fibre Toxicol, 16(1), 21. doi:10.1186/s12989-019-0306-4

Barregard, L., Sallsten, G., Gustafson, P., Andersson, L., Johansson, L., Basu, S., & Stigendal, L. (2006). Experimental exposure to wood-smoke particles in healthy humans: effects on markers of inflammation, coagulation, and lipid peroxidation. Inhal Toxicol, 18(11), 845-853. doi:10.1080/08958370600685798

Baumann, R., Gube, M., Markert, A., Davatgarbenam, S., Kossack, V., Gerhards, B., . . . Brand, P. (2018). Systemic serum amyloid A as a biomarker for exposure to zinc and/or copper-containing metal fumes. J Expo Sci Environ Epidemiol, 28(1), 84-91. doi:10.1038/jes.2016.86

Brand, P., Bauer, M., Gube, M., Lenz, K., Reisgen, U., Spiegel-Ciobanu, V. E., & Kraus, T. (2014). Relationship between welding fume concentration and systemic inflammation after controlled exposure of human subjects with welding fumes from metal inert gas brazing of zinc-coated materials. J Occup Environ Med, 56(1), 1-5. doi:10.1097/JOM.0000000000000061

Christophersen, D. V., Moller, P., Thomsen, M. B., Lykkesfeldt, J., Loft, S., Wallin, H., . . . Jacobsen, N. R. (2021). Accelerated atherosclerosis caused by serum amyloid A response in lungs of ApoE(-/-) mice. FASEB J, 35(3), e21307. doi:10.1096/fj.202002017R

Cray, C., Zaias, J., & Altman, N. H. (2009). Acute phase response in animals: a review. Comp Med, 59(6), 517-526.

Drieghe, S. A., Alsaadi, H., Tugirimana, P. L., & Delanghe, J. R. (2014). A new high-sensitive nephelometric method for assaying serum C-reactive protein based on phosphocholine interaction. Clinical chemistry and laboratory medicine, 52(6), 861-867. doi:10.1515/cclm-2013-0669

Gabay, C., & Kushner, I. (1999). Acute-phase proteins and other systemic responses to inflammation. N Engl J Med, 340(6), 448-454. doi:10.1056/NEJM199902113400607

Gutierrez, C. T., Loizides, C., Hafez, I., Brostrom, A., Wolff, H., Szarek, J., . . . Vogel, U. (2023). Acute phase response following pulmonary exposure to soluble and insoluble metal oxide nanomaterials in mice. Part Fibre Toxicol, 20(1), 4. doi:10.1186/s12989-023-00514-0

Hadrup, N., Rahmani, F., Jacobsen, N. R., Saber, A. T., Jackson, P., Bengtson, S., . . . Vogel, U. (2019). Acute phase response and inflammation following pulmonary exposure to low doses of zinc oxide nanoparticles in mice. Nanotoxicology, 13(9), 1275-1292. doi:10.1080/17435390.2019.1654004

Halappanavar, S., Jackson, P., Williams, A., Jensen, K. A., Hougaard, K. S., Vogel, U., . . . Wallin, H. (2011). Pulmonary response to surface-coated nanotitanium dioxide particles includes induction of acute phase response genes, inflammatory cascades, and changes in microRNAs: a toxicogenomic study. Environ Mol Mutagen, 52(6), 425-439. doi:10.1002/em.20639

Hamilton, R. G. (2014). Methods (In Vitro and In Vivo): Nephelometry and Turbidimetry. In Encyclopedia of Medical Immunology.

Kim, J. Y., Chen, J. C., Boyce, P. D., & Christiani, D. C. (2005). Exposure to welding fumes is associated with acute systemic inflammatory responses. Occup Environ Med, 62(3), 157-163. doi:10.1136/oem.2004.014795

Meier, R., Cascio, W. E., Ghio, A. J., Wild, P., Danuser, B., & Riediker, M. (2014). Associations of short-term particle and noise exposures with markers of cardiovascular and respiratory health among highway maintenance workers. Environ Health Perspect, 122(7), 726-732. doi:10.1289/ehp.1307100

Monse, C., Hagemeyer, O., Raulf, M., Jettkant, B., van Kampen, V., Kendzia, B., . . . Merget, R. (2018). Concentration-dependent systemic response after inhalation of nano-sized zinc oxide particles in human volunteers. Part Fibre Toxicol, 15(1), 8. doi:10.1186/s12989-018-0246-4

Nelson, D. L., Nelson, D. L., Lehninger, A. L., & Cox, M. M. (2017). Lehninger Principles of biochemistry (Seventh edition ed.). Macmillan Higher Education: Basingstoke.

Poulsen, S. S., Knudsen, K. B., Jackson, P., Weydahl, I. E., Saber, A. T., Wallin, H., & Vogel, U. (2017). Multi-walled carbon nanotube-physicochemical properties predict the systemic acute phase response following pulmonary exposure in mice. PLoS One, 12(4), e0174167. doi:10.1371/journal.pone.0174167

Sikkeland, L. I. B., Borander, A. K., Voie, O. A., Aass, H. C. D., Ovstebo, R., Aukrust, P., . . . Ueland, T. (2018). Systemic and Airway Inflammation after Exposure to Fumes from Military Small Arms. Am J Respir Crit Care Med, 197(10), 1349-1353. doi:10.1164/rccm.201709-1857LE

Walker, E. S., Fedak, K. M., Good, N., Balmes, J., Brook, R. D., Clark, M. L., . . . Peel, J. L. (2022). Acute differences in blood lipids and inflammatory biomarkers following controlled exposures to cookstove air pollution in the STOVES study. Int J Environ Health Res, 32(3), 565-578. doi:10.1080/09603123.2020.1785402

Wyatt, L. H., Devlin, R. B., Rappold, A. G., Case, M. W., & Diaz-Sanchez, D. (2020). Low levels of fine particulate matter increase vascular damage and reduce pulmonary function in young healthy adults. Part Fibre Toxicol, 17(1), 58. doi:10.1186/s12989-020-00389-5

List of Adverse Outcomes in this AOP

Event: 1443: Atherosclerosis

Short Name: Atherosclerosis

Key Event Component

AOPs Including This Key Event

Biological Context

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

• Taxonomic applicability: Human.
• Life stages applicability: All life stages. Although atherosclerosis is mostly observed in adult humans, this condition begins early in life, and progresses through adulthood (McGill, McMahan, & Gidding, 2008; McMahan et al., 2005). Children with chronic inflammation diseases have shown to develop atherosclerosis in early childhood. (Tyrrell et al., 2010; Yamamura et al., 2014)
• Sex applicability: Unspecific, atherosclerosis is manifested in males and females (Libby, 2021).

Key Event Description

Atherosclerosis is defined as the thickening of the arterial wall towards the lumen. The thickening, called atherosclerotic plaques, is composed of macrophages, smooth muscle cells, lymphocytes, lipids (including cholesterol), and connective tissue. This thickening reduces the space in the blood vessels which is available for the blood flow (Widmaier, Raff, Strang, & Vander, 2016).

Atherosclerosis is initiated with an endothelial injury that allows the translocation of low density lipoprotein (LDL) molecules to the intima layer of the artery, where they become oxidized (oxLDL). The endothelial cells release chemokines and adhesion molecules that recruits blood monocytes to the injury site, where monocytes cross to the sub-endothelial space. Monocytes then differentiate into macrophages and take up oxLDL, thus becoming laden with lipoproteins (“foam cells”). The lipid accumulation and foam cell formation continues over time, and the migration of smooth muscle cells from the media layer to the intima space helps establishing an atherosclerotic plaque with the release extracellular matrix molecules (Libby et al., 2019).

How it is Measured or Detected

Current in vitro models have been used to evaluate different parts of the atherosclerosis development, for example: expression of adhesion molecules, adhesion of monocytes to endothelial cells, monocytes migration and foam cell formation (Chen et al., 2021).

In animal models, the induction and/or progression of atherosclerosis after exposure to a stressor can be studied. Examples of these are the ApoE^-/- and LdLr^-/- mouse models and Watanabe rabbit model, where the development of atherosclerotic can be assessed (Gistera, Ketelhuth, Malin, & Hansson, 2022).

In humans, atheroscleorosis is diagnosed by clinicians. Techniques that allow direct visualization of atherosclerotic plaques include ultrasonography, computed tomography angiography, magnetic resonance imaging, and optical coherence tomography (Libby et al., 2019). These techniques can measure the intima thickness of arteries, along with detection of calcified components (Poyrazoglu, Vurdem, Arslan, & Uytun, 2016; van der Meer et al., 2004). Techniques that allow the evaluation of atherosclerosis without direct visualization of plaques include angiography, aortic pulse wave velocity and the ankle-arm systolic blood pressure index (Libby et al., 2019; Rodondi et al., 2010; van der Meer et al., 2004). Finally, nonspecific, inflammatory markers are also used to evaluate atherosclerosis. These include blood levels of interleukin 6 (IL-6), C-reactive protein and tumor necrosis factor α (TNF-α) (Rodondi et al., 2010).

Regulatory Significance of the AO

Atherosclerosis is the principal cause of cardiovascular diseases including myocardial infarction, stroke and angina pectoris (Frostegard, 2013; Jebari-Benslaiman et al., 2022; Libby et al., 2019). In turn, cardiovascular diseases are the principal cause of deaths worldwide and measures have been made by many countries to control risk factors and prevent this disease (Vaduganathan, Mensah, Turco, Fuster, & Roth, 2022). It is pertinent to remark that ambient (outdoor) and indoor particulate matter are risk factors for cardiovascular and the World Health Organization (WHO) has estimated that 6.7 million annual premature deaths are associated with these risk factors (Vaduganathan et al., 2022; WHO, 2023).

References

Chen, J., Zhang, X., Millican, R., Lynd, T., Gangasani, M., Malhotra, S., . . . Jun, H. W. (2021). Recent Progress in in vitro Models for Atherosclerosis Studies. Front Cardiovasc Med, 8, 790529. doi:10.3389/fcvm.2021.790529

Frostegard, J. (2013). Immunity, atherosclerosis and cardiovascular disease. BMC Med, 11, 117. doi:10.1186/1741-7015-11-117

Gistera, A., Ketelhuth, D. F. J., Malin, S. G., & Hansson, G. K. (2022). Animal Models of Atherosclerosis-Supportive Notes and Tricks of the Trade. Circ Res, 130(12), 1869-1887. doi:10.1161/CIRCRESAHA.122.320263

Jebari-Benslaiman, S., Galicia-Garcia, U., Larrea-Sebal, A., Olaetxea, J. R., Alloza, I., Vandenbroeck, K., . . . Martin, C. (2022). Pathophysiology of Atherosclerosis. Int J Mol Sci, 23(6). doi:10.3390/ijms23063346

Libby, P. (2021). The changing landscape of atherosclerosis. Nature, 592(7855), 524-533. doi:10.1038/s41586-021-03392-8

Libby, P., Buring, J. E., Badimon, L., Hansson, G. K., Deanfield, J., Bittencourt, M. S., . . . Lewis, E. F. (2019). Atherosclerosis. Nat Rev Dis Primers, 5(1), 56. doi:10.1038/s41572-019-0106-z

McGill, H. C., Jr., McMahan, C. A., & Gidding, S. S. (2008). Preventing heart disease in the 21st century: implications of the Pathobiological Determinants of Atherosclerosis in Youth (PDAY) study. Circulation, 117(9), 1216-1227. doi:10.1161/CIRCULATIONAHA.107.717033

McMahan, C. A., Gidding, S. S., Fayad, Z. A., Zieske, A. W., Malcom, G. T., Tracy, R. E., . . . McGill, H. C., Jr. (2005). Risk scores predict atherosclerotic lesions in young people. Arch Intern Med, 165(8), 883-890. doi:10.1001/archinte.165.8.883

Poyrazoglu, H. G., Vurdem, U. E., Arslan, A., & Uytun, S. (2016). Evaluation of carotid intima-media thickness in children with migraine: a marker of subclinical atherosclerosis. Neurol Sci, 37(10), 1663-1669. doi:10.1007/s10072-016-2648-0

Rodondi, N., Marques-Vidal, P., Butler, J., Sutton-Tyrrell, K., Cornuz, J., Satterfield, S., . . . Body Composition, S. (2010). Markers of atherosclerosis and inflammation for prediction of coronary heart disease in older adults. Am J Epidemiol, 171(5), 540-549. doi:10.1093/aje/kwp428

Tyrrell, P. N., Beyene, J., Feldman, B. M., McCrindle, B. W., Silverman, E. D., & Bradley, T. J. (2010). Rheumatic disease and carotid intima-media thickness: a systematic review and meta-analysis. Arterioscler Thromb Vasc Biol, 30(5), 1014-1026. doi:10.1161/ATVBAHA.109.198424

Vaduganathan, M., Mensah, G. A., Turco, J. V., Fuster, V., & Roth, G. A. (2022). The Global Burden of Cardiovascular Diseases and Risk: A Compass for Future Health. J Am Coll Cardiol, 80(25), 2361-2371. doi:10.1016/j.jacc.2022.11.005

van der Meer, I. M., Bots, M. L., Hofman, A., del Sol, A. I., van der Kuip, D. A., & Witteman, J. C. (2004). Predictive value of noninvasive measures of atherosclerosis for incident myocardial infarction: the Rotterdam Study. Circulation, 109(9), 1089-1094. doi:10.1161/01.CIR.0000120708.59903.1B

WHO. (2023). Ambient air pollution. Retrieved from https://www.who.int/news-room/fact-sheets/detail/ambient-(outdoor)-air-quality-and-health

Widmaier, E. P., Raff, H., Strang, K. T., & Vander, A. J. (2016). Vander's human physiology : the mechanisms of body function (Fourteenth edition. ed.). New York, NY: McGraw-Hill.

Yamamura, K., Takada, H., Uike, K., Nakashima, Y., Hirata, Y., Nagata, H., . . . Hara, T. (2014). Early progression of atherosclerosis in children with chronic infantile neurological cutaneous and articular syndrome. Rheumatology (Oxford), 53(10), 1783-1787. doi:10.1093/rheumatology/keu180

Appendix 2

List of Key Event Relationships in the AOP