Putative adverse outcome pathway for silver nanoparticle toxicity on male reproductive system: A literature review

doi:10.21203/rs.3.rs-2173816/v1

Download PDF

Research Article

Putative adverse outcome pathway for silver nanoparticle toxicity on male reproductive system: A literature review

https://doi.org/10.21203/rs.3.rs-2173816/v1

This work is licensed under a CC BY 4.0 License

Journal Publication

published 05 Jan, 2023

Read the published version in Particle and Fibre Toxicology →

You are reading this latest preprint version

Background: Adverse outcome pathways (AOPs) are conceptual frameworks that organize knowledge about biological interactions and toxicity mechanisms. They present a sequence of events commencing with initial interaction(s) of a stressor, which defines the perturbation in a biological system (molecular initiating event, MIE), and a dependent series of key events (KEs), ending with an adverse outcome (AO). AOPs have recently become the subject of intense studies in a view to better understand the mechanisms of nanomaterial (NM) toxicity. Silver nanoparticles (Ag NPs) are one of the most explored nanostructures and are extensively used in various application. This, in turn, has increased the potential for interactions of Ag NPs with environments, and toxicity to human health. The aim of this study was to construct a putative AOPs (pAOP) related to reproductive toxicity of Ag NPs, in order to lay the groundwork for a better comprehension of mechanism affecting both undesired toxicity (against human cell) and expected toxicity (against microorganisms).

Methods: PubMed and Scopus were systematically searched for peer-reviewed studies examining reproductive toxicity potential of Ag NPs. The quality of selected studies was assessed through ToxRtool. Eventually, forty-eightstudies published between 2005 and 2022 were selected to identify the mechanisms of Ag NPs impact on reproductive function in male. The biological endpoints, measurements, and results were extracted from these studies. Where possible, endpoints were assigned to a potential KE and an AO using expert judgment. Then, KEs were classified at each major level of biological organization.

Results: We identified the impairment of intracellular SH-containing biomolecules, which are major cellular antioxidants, as a putative MIE, with subsequent KEs defined as ROS accumulation, mitochondrial damage, DNA damage and lipid peroxidation, apoptosis, reduced production of reproductive hormones and reduced quality of sperm. These successive KEs may result in impaired male fertility (AO).

Conclusion: This research recapitulates and schematically represents complex literature data gathered from different biological levels and propose a pAOP related to the reproductive toxicity induced by AgNPs. The development of AOPs specific to NMs should be encouraged in order to provide new insights to gain a better understanding of NP toxicity.

Adverse outcome pathways

Silver Nanoparticles

Reproductive toxicity

Molecular initiating event

key events

Adverse outcome

Silver nanoparticles (Ag NPs) are one of the most studied NMs and they have been extensively used in various fields such as in food, cosmetic, textile, and medical industries (1–3). Their high preference in such areas is generally attributed to their unique characteristics differing from bulk materials, including optical, electrical, magnetic and antibacterial properties (4,5). Given that Ag NPs are incorporated into commercially available products primarily for antibacterial and antifungal purposes, their abundance in everyday products leads to concerns related to their health effects and the possible consequences of their dispersal in the environment (3,4).

When taken up into the human body, the reactivity of NMs depends on complex physicochemical properties such as size, agglomeration state, dissolution kinetics, capping agent, surface charge, and specific surface area (6–8). The small size and remarkably high surface area of NPs enhance their interaction with biomolecules, biological membranes, cells or tissues. The high surface area of metal- and metal-oxide based NPs such as copper, zinc oxide, silver, manganese oxide, and cerium oxide increases the potential that metal ions are released from these NPs as they dissolve (9,10). The so-called “Trojan-horse” mechanism, through which Ag NPs act as a vehicle that carries silver across the cell membrane followed by intra-cellular dissolution of Ag NPs to release Ag ions, has been proposed as one mechanism of Ag NP toxicity (11–13). Still, Ag ion release cannot alone account for the observed toxic effects (14). The toxicity mechanism for Ag NP could be related to their small size, the amount of released silver ions, or a combination of both (12,15).

Released ions from Ag NPs bind to ligands, creating a mixture of metal ion-ligand complexes. Silver is a soft acid according to the Pearson (HSAB) acid-base theory, therefore it shows high affinity for soft bases, and among them, it is particularly affine for thiols (9,16,17). Thiol groups of cysteine residues are crucial for many proteins to maintain their integrity and function (18,19). The most abundant thiolated molecules in human cells include some proteins, especially metallothioneins and Zn-finger proteins, and small molecules such as glutathione (GSH). These molecules play an important role in maintaining the oxidative balance (20), metal homeostasis and DNA integrity in cells (21). Ag ions complexation with these thiol groups may induce protein misfolding, scavenge thiolated molecules, thereby hindering their function and impairing cellular antioxidant mechanisms (13,22,23). Due to the complex nature of Ag NP exposure, there remains uncertainty and to some extent controversy, regarding the level to which each constituent —ion, ion-protein complex, particle— contributes to cellular toxicity.

Considering their presence in food additives, food packaging, in textiles such as clothing and bedding and in toothbrushes, hair straighteners, disinfectant sprays as cosmetic and hygiene products, the major routes of exposure of Ag NPs are dermal contact (24), ingestion (25), and inhalation (26). According to European Commission Scientific Committees, the main target organs for Ag NP in the human body are the spleen, liver, and kidney, with less distribution to other organs (14). There are studies that recorded high levels of silver accumulation in the brain and testicles (14,25,27–31), although the significance for toxicity is unknown (14). In 2018, the Scientific Committee on Consumer Safety (SCCS) of the European Commission recommended to collect information on the reproductive toxicity of Ag NPs (32). The SCCS also noted the lack of information on systemic availability via the relevant uptake route(s) that would allow drawing conclusions on reproductive system toxicity (32). Therefore, the potential toxicity mechanism of Ag NPs is a matter of great concern with regards to reproductive toxicity (33).

Exposure to nanoparticles may cause adverse effects on the reproductive function and fertility in adult males, including impact on reproductive cells, spermatogenesis, the seminiferous tubules, and testes (34). In vitro studies show that Ag NPs cause the alteration of germ cells and somatic cells function mainly due to cell membrane peroxidation, oxidative stress, mitochondrial damage, DNA damage, and apoptosis (35–37). Necrotic spermatogonial cells, degenerative alterations in the cellular architecture of testes and epididymis are reported in in vivo models (38,39). In some animal models, the accumulation of NPs in the testes is demonstrated (40,41). It is also reported that Ag NPs affect reproductive hormone levels such as testosterone and androgen hormones (39), sperm quantity and quality (38), which suggest potential consequences for male fecundity.

Likewise, in non-mammalian models, Ag NPs reproductive toxicity has been extensively studied. Ag NP considerably decrease reproductive potential in Caenorhabditis elegans (42). Yan et al prove the maternal transfer of Ag NPs to offsprings in Daphnia magna together with inhibition of the reproduction capability of F₀ and F₁ generations (43).

To support effective risk assessments of chemicals, The Organisation for Economic Co-operation and Development (OECD) has introduced the Adverse Outcome Pathway (AOP) conceptual framework (44), which is designed to organize toxicological information, thereby assisting integrated approaches to testing and assessment strategies (44). Considering the creation of non-animal testing approaches, emphasis has been placed on AOPs as a conceptual support for developing in vitro and in silico testing strategies (44). This framework presents a sequence of events commencing with initial interaction(s) of a stressor, which defines the perturbation in a biological system (i.e., molecular initiating event, MIE), and then a dependent series of intermediate key events (KEs), ending up in an adverse outcome (AO) (44,45). KEs describe a toxicological response and are linked to one another by a Key Event Relationship (KER), which establishes one KE as upstream and one KE as downstream (44,45). The AOP-Wiki database (46) serves as the primary repository of qualitative information for the international AOP development effort.

Within AOP-Wiki, one AOP that describes reproductive failure due to Ag NP exposure is available, which is AOP207 (47). It has been established on the non-mammalian model Caenorhabditis elegans by using a Bayesian network (BN) model (48). The MIE of this AOP is oxidative stress through NADPH oxidase activity, reproduction failure is the outcome. PMK-1 (P38 MAPK) activation, HIF-1 activation, mitochondrial damage, DNA damage, and apoptosis are as the described key events. Another AOP regarding reproductive toxicity of Ag NPs has been reported in zebrafish (49). It describes that Ag NPs exposure causes oxidative stress, induces germ cells apoptosis via mitochondrial-dependent pathway, and ultimately impairs the reproduction in zebrafish (49). Moreover, some reproductive failure-releated AOPs exist for other NPs such as graphene oxide and UV-activated Titanium dioxide NPs (TiO₂ NPs). First, in AOP210 (50) the comprehensive mechanism of stress response to graphene oxide NPs is investigated in C. elegans using transcriptomics, metabolomics and lipidomics. Based on the results, the authors constructed an AOP for oxidative stress leading to reproductive failure in C. elegans. This AOP includes the KEs increased oxidative stress and activation of c-Jun N-terminal kinase (JNK) and activation of transcription factor DAF-16/FOXO, inhibition of WNT signaling, defect embryogenesis, with the AO being reproductive. In addition, in AOP208 (51), JAK/STAT and TGF-beta pathway activation leading the reproductive failure has been used to describe the mechanism by which UV-activated TiO₂ NPs affect the reproductive function (52).

Anticipating Safety Issues at the Design Stage of NAno Product Development (ASINA) EU Horizon 2020 (H2020) project is developing a specific Safe by Design (SbD) Management Methodology, consistent with modern business management systems, to deliver SbD solutions and inform design decisions. Within this project, the investigation of toxicity mechanisms of antimicrobial NMs that born to exert a toxicity effect is pivotal to support their design and match safety requirements. The current literature is reviewed in order to identify major areas of concerns related to the toxicity of the NM categories under investigation, using an AOP-oriented approach. With this perspective, in the present study, the reproductive toxicity of Ag NPs toward male mammalian models has been reviewed and a putative testable AOP is proposed, since previous related work has only focused on non-mammalian models. Based on the existing literature, we evaluated the MIE, KEs and AOs related to the induction of reproductive failure. As a follow-up of the present investigation, we expect to compare the AOPs of different models, including identified toxicity thresholds, so as to propose the most promising design options (concentration, physicochemical features, stabilizing agents) able to guarantee a safe use of Ag NMs.

2.1 Literature search

The searches for peer-reviewed research publications on the toxicity of Ag NPs were conducted in PubMed and Scopus and limited between 2007 and 2022 according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) recommendations (53). As shown in Figure 1, a total of 4633 articles were screened on the toxicity induced by Ag NPs. Among them, the articles not written in English, inaccessible or systematic reviews were excluded and this resulted in 3319 articles. After, articles related to environmental species, or target organs outside of reproductive system were eliminated. Finally, 330 articles were found to be related to reproductive toxicity of Ag NPs. Articles reporting data collected on in vivo female models, marine organisms and non-mammalian models such as drosophila, medaka, nematodes and zebrafish were further excluded. Finally, 52 articles were selected to identify how Ag NPs affect reproductive function in male in vivo and in vitro models.

The articles are grouped into two parts, in vivo and in vitro. While 44 studies were in vivo studies, 6 of them were in vitro studies. Two study comprises both in vivo and in vitro models; therefore, they were evaluated in both groups for the corresponding parts. That is, 46 in vivo data and 8 in vitro data were collected from 52 studies in total.

2.2 Quality assessment

Study quality, study design adequacy for their intended measures and the reliability of outcome were assessed in these 52 studies by using the Toxicological data Reliability Assessment Tool (ToxRTool) (54). These studies were evaluated in terms of the nanoparticle identification (synthesis, characterization, p-chem informations) (group I), in vivo or in vitro test system characterization (animal source, age, body weight information, housing-feeding conditions, cell types, cultivation conditions) (group II), study design description (administration route, dose, frequency/duration of exposure etc.) (group III), study results documentation (endpoints, methods judgment) (group IV), and plausibility of study design and results (group V).

The studies were scored between 0-21 points or 0-18 points for in vivo and in vitro, respectively (reliable without restrictions, in vivo: 18–21 points, in vitro: 15-18 points; reliable with restrictions, in vivo: 13–17 points, in vitro: 11-14 points; not reliable, in vivo: <13 points, in vitro <11 points). Studies with a score of ≥13 (in vivo) and ≥11 (in vitro) points were classified as high quality.

2.3 Database analysis to identify potential KEs

The selected high quality studies were analyzed and reported in detail in our database as recommended by AOP-expert groups (45,55,56). The physicochemical properties of the nanoparticles used in these articles (particle size, shape, surface area, surface charge, agglomeration status, surface coating, purity), characterization of the test organism used (animal sex, age, weight, cell type), route of administration, exposure dose and duration, methods, biological endpoints, and key results were reported separately for each article. The aim of this evaluation was to understand connection between p-chem properties and relevant key events and adverse outcomes, which would feed the ASINA SbD strategy.

Biological endpoints, measurements, methods were identified to define the standard strategy for nanoparticle reproductive toxicity and listed in Table 1. The terminology used in the title of KEs were chosen according to the OECD guideline on AOP development (44). All KEs were evaluated for three main criteria: credibility, measurability, and regulatory compliance. After that, KEs were classified at each major level of biological organization (molecular, cellular, tissue, organ, individual) and selected with respect to their potency to be measured in a relatively routine manner, in opposition to those that require highly specialized expertise or equipment. To establish the AOP frame, KEs that were reported as either transient or reversible were avoided.

3.1. Quality assessment of the identified studies

The quality of 52 selected studies were assessed using ToxRTool. Four records were analysed as poor studies because of missing material characterization and/or usage of ultra-high concentrations or doses and/or no controls for biological endpoints have been included. Seven records were acceptable studies, with most quality criteria fulfilled but not strictly all. Finally, forty-one records were considered good studies, with all quality criteria fulfilled.

Overall, the 48 selected studies were classified as good and acceptable quality and these were later used in subsequent data evaluations.

3.2. Dataset evaluation

Datasets were created through our database containing a summary of the selected in vitro and in vivo studies. The in vitro datasets comprised studies performed on germ cells, Leydig, Sertoli cells or human semen. In in vivo studies, 17 studies used the oral route of exposure, i.e. Ag NPs were administered via oral gavage or through food/water, 13 used intraperitoneal injection, 9 used intravenous injection, 1 used subdermal, 1 intratracheal, and 1 intratesticular administration routes, which were mostly conducted in rats (25 studies), mice (15 studies) and rabbits (2 studies).

The dose ranges used in these studies were quite wide, for example in the 17 oral administration studies, 0.015 to 500 mg/kg/bw doses were applied. The dose was generally linked with those selected in previous studies. Some of the studies pointed that the investigated doses were in the range between the lowest observable adverse effect level (LOAEL) and a no observable adverse effect level (NOAEL) (38,57), i.e., 125 mg/kg and 30 mg/kg, respectively, as suggested by a 90-day oral toxicity study, based on signs of liver toxicity (58).

In each study, the physicochemical properties of the used Ag NP was strictly reviewed, so that we could further identify the influence of these properties on the adverse outcome on the reproductive system. Ag NPs size range varied between 8.92 nm to 200 nm (Fig. 2).

Nine studies reported that the used Ag NPs were coated with polyvinyl pyrrolidone (PVP) or citrate. Moreover, some studies used Ag NPs produced via green method such as biogenic production in Bacillus funiculus, baker’s yeast (Saccharomyces cerevisiae) or V. opulus L. fruit extract. The studied Ag NPs were mostly spherically-shaped.

3.3. Potential molecular initiating events

Two mechanisms involved in the toxicity of Ag NPs have been proposed. First, silver ions are formed and released, mediated by the oxidation of the metallic silver core, inducing the formation of reactive oxygen species (ROS) (2). Silver ions interaction with enzymes and proteins containing thiol groups, such as metallothioneins or zinc-finger proteins, affecting cellular processes such as cellular respiration and antioxidant defense system, possibly resulting in cell death (12,13,22,59,60). Silver ions, indeed, are soft acid according to the HSAB principle, consequently they show high affinity towards soft bases and among them silver is highly affine for biomolecules containing thiols. However, it is important to go one-step back and explore the link between the oxidative stress and Ag NP dissolution to understand via which mechanism Ag NPs induce oxidative stress, in order to identify molecular initiating event(s).

Ag NP dissolution mechanisms are well described (9,61–63). The release of ions from Ag NPs has been shown to be an oxidation involving dissolved oxygen and protons. The reaction stoichiometry is as shown in the equation [1] (64),

$${2Ag}_{\left(s\right) }+ \frac{1}{2} {O}_{2\left(aq\right)}+2{H}_{\left(aq\right)}^{+}\leftrightarrow {2Ag}^{+}+ {H}_{2}O \left[1\right]$$

However, studies have shown that the oxidation of Ag NPs to Ag ions occurs through simple redox reactions that produce peroxide intermediates rather than a four-electron transfer process that directly reduces O₂ to water (64,65). This is illustrated by the equation [2].

$${O}_{2}+{H}^{+} \underrightarrow{{Ag}^{0}} {Ag}^{+}+peroxide intermediates \underrightarrow{{Ag}^{0}} {Ag}^{+}+ {H}_{2}O \left[2\right]$$

Dissolution of Ag NPs to form Ag ions may accompany the decomposition of hydrogen peroxide under acidic conditions leading to the formation of hydroxyl radicals (63). The formation of hydroxyl radicals can go through a process similar to the Fenton reaction, in which Ag NPs act as a Fenton-like reagent (63). Under neutral and alkaline conditions, the reaction of Ag NPs with H₂O₂ generates oxygen instead of °OH radical (64). The pH of the environment plays a role in the Ag NPs dissolution, as illustrated by equation [3] (62).

$$Ag+ {H}_{2}{O}_{2}+ {H}^{+ }\to {Ag}^{+}+ \bullet OH+{H}_{2}O \left[3\right]$$

The ability of generating radicals has been reported across a variety of metal and metal oxide NPs, such as copper nanoparticles, zinc oxide nanoparticles, Ag NPs (66–68). Previous reports using electron spin resonance (ESR) coupled with spin trapping and spin labeling prove that free radicals are derived from the surface of Ag NPs (69,70).

In cellular system, Ag NPs are internalized within cells by endocytosis and are stored in endosomes, which then mature to lysosomes. Ag NPs undergo intracellular dissolution in the favor of low lysosomal pH, leading to Ag(I) species (11,16). This behavior is related to the so-called “Trojan horse” mechanism and leads to high Ag(I) concentrations in cellular compartments that Ag ions would otherwise not reach. Intracellular Ag(I) is a chemically reactive form of silver and shows remarkable affinity to zinc-finger domains of proteins, thiol-containing enzymes and molecules, mainly GSH and metallothionein. Binding to these ligands leads to the subsequent formation of intracellular Ag(I)-thiolate complexes (22,71,72). Such interaction affects the native domain structure of these proteins, which plays a role in maintaining the cellular homeostasis and antioxidant systems. As a consequence, it will influence their biological functions (13,60,71–73).

Due to the abovementioned mechanisms, since a MIE describes an initial point of interaction between stressors and the biomolecule, we propose that the impairment of intracellular SH-containing biomolecules can be defined as a MIE of the putative AOP described here. Indeed, the release of Ag(I) in solution, the consequent production of ROS together with the thiol-Ag ion complexation would activate such MIE and lead to a chain of intracellular consequences ultimately leading to reproductive toxicity.

In our MIE evaluations, we reviewed AOP207 (47), examining the reproductive toxicity study of Ag NPs in worms. This AOP focuses on identifying potential MIEs on Ag NPs induced reproductive toxicity in C. elegans (Table 2) and is still under development. The authors examine the question of how Ag NPs cause ROS production in C. elegans. They state that ROS can be formed on the surface of nanomaterials or that following the NP internalization endosomes are formed and ROS are produced by NADPH oxidase. They examine whether ROS arise directly from Ag NPs or indirectly through the action of NADPH oxidase. Finally, they identify NADPH oxidase as MIE, and reproduction failure as the outcome in C. elegans. However, any general correlation between the findings from C. elegans and in vitro and in vivo mammalian studies on the toxicity of Ag NPs is lacking (74).

3.4. Identification and selection of key events

AOP-Wiki was screened to identify already-existing KEs that could describe the biological events reported in the 48 selected studies. The result of this analysis and screening is reported in Additional file 1, an extract of which, reduced to the analysis of 4 articles, is presented in Table 1.

Disruption of SH-containing molecules (MIE), such as glutathione, can cause oxidative stress through disruption of the antioxidant system, as described in the section above. In our database, the most reported of the biological events is Ag NPs exposure triggering oxidative stress, which is described in 22 out of the 48 articles at both cellular and tissue level. It has been shown that accumulation of Ag NPs depleted cells from the molecular antioxidant GSH (39,75–77), and decreased super oxide dismutase (SOD) and catalase (CAT) activities (29,39,77–79), altered enzymatic oxidative defense system in male reproductive system (29,38,39,77,80,81) and lead to increased ROS levels in human sperm (82), in mouse Sertoli cells (15P-1) (83), in somatic Leydig (TM3) and Sertoli (TM4) cells (36,84) which eventually cause oxidative stress. In addition, mitochondrial damage due to the impairment of metallothionein (MIE) would result in oxidative stress by inhibition of electron transfer chain enzymes and perturbation of antioxidant system. Thus, it would increase mitochondrial ROS production, which may lead to mitochondrial damage including damage to respiratory chain and its membrane permeability. It has been shown that Ag NPs within the intracellular space has the potential to cause mitochondrial dysfunction by the depolarization of the mitochondrial membrane (84,85). Wang et al reported damaged mitochondria in the testis upon Ag NP exposure to Balb/c mice (86). These findings support the hypothesis in the biological plausibility perspective that Ag NPs interact with the thiol groups of the biomolecules, causing disruptions in the antioxidant system and thus triggering oxidative stress.

In addition to outlining the evidence supported by biological plausibility, there is also empirical evidence supporting this association in AOP-Wiki. AOP17 (87) has proposed a MIE similar to ours as the binding of electrophilic chemicals to SH(thiol)-group of proteins and /or to seleno-proteins, generating neurotoxicity. It is stated in AOP17 that soft metals like mercury binding to thiol/sulfhydryl/SH/SeH- groups results in structural modifications affecting the catalytic capacity of enzymes, and thereby reducing their capacity to neutralize ROS (88). The relationship of this MIE and oxidative stress is classified as moderate in quantitative manner. The same could occur with Ag ions. Therefore, it can be assumed that impairment of SH-containing molecules like glutathione and metallothionein can lead both mitochondrial damage (KE1a) and ROS production (KE1b), eventually result in oxidative stress (KE2) as shown in Fig. 3.

Table 1

Database summary of analysis 4 studies out of 48 selected studies
Ag NP p-chem		Exposure dose, sampling time		Actual measurements and methods	Result	Potential key event(s) (KE nr. from AOP-Wiki) ^a	Potential adverse outcomes (AOs) associated with the key event (AO nr. from AOP-Wiki^a	Ref
Size: 50 nm Shape: Spherical Hydrodynamic size :113.4 ± 12.1 nm Zeta potential: −12.30 ± 0.4 mV	Adult male SD rats Oral gavage	50 mg/kg bw, 3 months	Sperm Evaluation	Sperm Motility, Concentration, and Viability by eosin staining	Increased sperm morphological abnormalities Decreased sperm concentrations motility and viability	Decreased sperm quantity or quality in the adult, Decreased fertility (ID505, ID520)	Impaired fertility (ID330 , ID406)	(38)
			Oxidative Status	GSH level, CAT activity and lipid peroxidation MDA content in testicular tissue by commercial kits	Decreased CAT activity, Increased MDA content, Non-significant change in GSH level	Decreased protection against oxidative stress, Occurance oxidative stress (ID210, ID1112, ID1249, ID1538, ID1869) Lipid Peroxidation (ID1445 or ID1511)	Oxidative Damage (ID356)
			Hormonal Assessment	Serum testosterone, LH and FSH by ELISA kit	Decreased testosterone, LH and FSH levels	Reduction, testosterone level (ID1613, ID1689, ID1612) Reduced, Gonadotropins (ID1986)	Decreased sperm quantity or quality in the adult, Decreased fertility (ID505, ID520)
			DNA damage	DNA strand breaks in testicular tissue by COMET assay	Increased DNA damage	Increased DNA damage (ID1194)	DNA Damage (ID1194)
			Histopathological Examination	Testis and Seminiferous tubules by Hematoxylin and Eosin (HE) staining	Testis and Seminiferous tubules Histological alterations Necrotic spermatogonial cells	Testicular atrophy (ID1506)	Male reproductive tract malformations (ID348) Reduced, Reproductive Success (ID675)
Size:45 nm PVP (< 1%) Zeta potential:−20 mV	New Zealand White male rabbits Intravenous	5 mM AgNP solution (0.6 mg/kg bw) 2 months (weekly)	Sperm Evaluation	Sperm Volume, Concentration, and Viability	Decreased sperm motility, concentration and volume	Decreased sperm quantity or quality in the adult, Decreased fertility (ID505, ID520)	Impaired fertility (ID330 , ID406)	(75)
Size:45 nm PVP (< 1%) Zeta potential:−20 mV	New Zealand White male rabbits Intravenous	5 mM AgNP solution (0.6 mg/kg bw) 2 months (weekly)	Oxidative Status	MDA content, NO concentration, CAT and GPX Activity in sperm and blood samples by commercial kits	Increased NO, and MDA content Decreased CAT activity, and GSH level	Decreased protection against oxidative stress, Occurance oxidative stress (ID210, ID1112, ID1249, ID1538, ID1869) Lipid Peroxidation (ID1445 or ID1511)	Oxidative Damage (ID356)	(75)
Size:40 nm Shape: Spherical	Adult male NMRI mice Oral gavage	500 mg/kg bw with a time interval of 24 hr for 35 days	Oxidative Status	Total antioxidant capacity and Lipid peroxidation parameters	Decrease in the total antioxidant capacity, Increased MDA concent	Decreased protection against oxidative stress, Occurance oxidative stress (ID210, ID1112, ID1249, ID1538, ID1869) Lipid Peroxidation (ID1445 or ID1511)	Oxidative Damage (ID356)	(89)
			Hormonal Assessment	Serum testosterone level by comercial kits	Decreased testosterone hormone	Reduction, testosterone level (ID1613, ID1689, ID1612)	Decreased sperm quantity or quality in the adult, Decreased fertility (ID505, ID520)
			Histological parametres	Testis, the volume of interstitial tissue and seminiferous tubules	Decreased mean volume of testicular tissue and the volume of seminiferous tubules Decreased sperm density, mean number of spermatocytes, mean number of Sertoli cells	Testicular atrophy (ID1506)	Male reproductive tract malformations (ID348) Reduced, Reproductive Success (ID675)
Size:100 nm Shape: Spherical SSA : 7.5329m²/g Zeta potential:− 18.9 mV	Male Rats Sub dermal	10 and 50 mg/kg bw, 7 and 28 days	Sperm Evaluation	Sperm motility, velocity by HE staining	Decreased sperm motility and velocity	Decreased sperm quantity or quality in the adult, Decreased fertility (ID505, ID520)	Impaired fertility	(39)
			Oxidative Status	MDA, GSH, CAT	Increased Lipid peroxidization Decreased SOD, CAT, GSH and total thiols	Decreased protection against oxidative stress, Occurance oxidative stress (ID210, ID1112, ID1249, ID1538, ID1869)	Oxidative Damage (ID356)
			Hormonal Assessment	Testosterone, LH and FSH	Decreased testosteron, LH and FSH levels (dose dependent	Reduction, testosterone level (ID1613, ID1689, ID1612) Reduced, Gonadotropins (ID1986)	Decreased sperm quantity or quality in the adult, Decreased fertility (ID505, ID520)
			Histological parametres	Cellular achitecture of testes and epididymis	Degenerative alterations in the cellular architecture of testes and epididymis	Testicular atrophy (ID1506)	Male reproductive tract malformations (ID348) Reduced, Reproductive Success (ID675)
^aAs several KEs and AOs are related to some of these cellular mechanisms, we indicate the title of only some of them, but the IDs of all of them are cited.

Lipid peroxidation following Ag NP exposure has also been identified in our database. Lipid peroxidation byproducts including malondialdehyde (MDA), and Thiobarbituric acid reactive substances (TBARS) have been shown to be significantly increased in serum, testicular tissue or in reproductive cells exposed to Ag NPs (38,83,89–91). ROS-mediated lipid peroxidation is shown in at least two studies out of the 48 studies (29,75). Collodel et al (75) confirmed the correlation between excessive radical generation, lipid peroxidation, and damage to the sperm membrane. Evidence supporting the KER between oxidative stress and lipid peroxidation was also provided by AOP-Wiki and the relation (KER ID:1727) is classified as high by weight of evidence and quantitative understanding. Therefore, it can be postulated that oxidative stress leads to lipid peroxidation, as reported in Figure 3.

Ag NPs induced DNA damage is reported in some of the evaluated studies in in vitro germ cells, somatic cells (82,92) and in in vivo sperm samples and testicular tissues (38,76,79,80,93–96). According to AshaRani et al and Carlson et al, ROS formation/oxidative stress was suggested to be a key event in DNA damage induction (85,97).

Apoptosis is a widely observed response to Ag NP exposure, which is frequently reported through measurements of apoptosis-related proteins at the cellular level, or the tissue level with histopathological observations. At the cellular level, Ag NPs induce apoptosis in the mouse germ cell line C18-4 (98), in the mouse male-derived Sertoli cell line TM4 (84). It is also suggested that accumulated ROS lead to apoptosis as downstream event in somatic Leydig and Sertoli cells (36). Ag NPs induce expression of autophagy-related genes and activate signaling molecules involved in apoptosis (36). Ntera2 cells (NT2, human testicular embryonic carcinoma cell line) are affected by Ag NPs which cause DNA strand breaks, reduce the cell proliferation and trigger apoptosis and necrosis (94).

Moreover, several in vivo studies demonstrate alterations in apoptosis-related gene expressions, increased ratio of Bax/Bcl-2 expressions (77,80,86,99,100) and mitochondria-dependent intrinsic apoptotic pathway in testes (80,99). An extensive gene expression analysis conducted on 383 genes by microarray shows great changes in apoptosis-related genes and proteins (caspase3 and Myc). This analysis shows apoptosis-related changes of testis morphology and sperm production, with the evidence of apoptotic nuclei in spermatogonia and spermatocytes in the testis (80,86). The histopathology assessment of tubular cross-sections of seminiferous tubules provides evidence of increased number of apoptotic germ and somatic cells (30,101). Testicular sections in rats treated with Ag NPs show decrease and disturbance in the spermatogenic cells arrangements, atrophied seminiferous tubules with degenerative Sertoli cell, and depletion in Leydig cells (76). In addition, other studies using different target systems such as liver (23), colon (102), and endothelial cells (103) show that Ag NPs cause apoptosis in a p53-dependent process involving ROS and the c-Jun N-terminal kinase cascade, or via the IKK/NF-κB pathway. These results suggest the appropriateness of the KE ‘apoptosis’. Moreover, it is widely recognized that if cells fail to handle oxidative stress, then apoptosis will be triggered through downstream signaling pathways (104–107). Therefore, we chose to define apoptosis as a downstream event of mitochondrial damage, DNA damage and lipid peroxidation in the putative AOP that we propose (KE4).

In AOP-Wiki, apoptosis, DNA damage, and sperm count relation was evaluated in AOP 322 (108) (Fig. 4). In this network, DNA alkylation (MIE) cause subsequent key events as inadequate DNA repair, increased DNA strand breaks, increased apoptosis, and reduced sperm counts (Table 2). This AOP 322 is still under development, however, it provides a key sub-network that is possibly relevant to our proposed AOPs (Fig. 4). The AOP examining the reproductive toxicity study of Ag NPs in worms (47) classified PMK-1(P38 MAPK) activation, HIF-1 activation, mitochondrial damage, DNA damage, and apoptosis as key events (Fig. 4). They performed correlation analysis between each key events on their AOPs and it has been proved that there is significant positive correlations between the exposure concentration of Ag NPs, ROS formation, the expression of bli-3 (NADPH oxidase), and mitochondrial damage. The most significant negative correlations were observed between the concentration of Ag NPs, reproduction and DNA repair gene expressions in C. elegans (48). Our in vivo and in vitro data analysis is in good agreement with this AOP framework developed by Jeong et al which has similar key events as in our proposed putative AOP on mammalian models.

Significant alterations of serum and intratesticular testosterone levels was observed upon Ag NPs exposure, as reported in a number of studies from our database (38,39,78,79,100,109). According to Attia et al the significant decrease in the level of serum testosterone could be related with the adverse effects of Ag NPs in Leydig cells (78). Circulating testosterone levels depend on the steroidogenic capacity of individual Leydig cells and the total number of Leydig cells per testis (110). Leydig cell apoptosis causes the decrease in their number in the testis, which in turn affects testosterone level as shown in some studies (30,111,112) and further impact the spermatogenesis (113). Therefore, the relationship between apoptosis of Leydig cells and alterations of serum and intratesticular testosterone levels is consistent with established biological knowledge.

On the other hand, there are studies showing that low testosterone levels are associated with impaired cholesterol transport in damaged mitochondria of Leydig cells (114,115). Mitochondrial steroidogenic acute regulatory protein (StAR) or translocator protein (TSPO) are responsible for cholesterol transport from the outer to the inner mitochondrial membrane (116). In the inner membrane, cholesterol is converted into the Pregnenolone by CYP11a1 (116). Afterwards, 3β-Hydroxysteroid dehydorgenase (Hsd3b), 17β-Hydroxysteroid dehydorgenase (Hsd17b), CYP17A1 transform pregnenolone to testosterone (111,116). In our database, steroidogenesis perturbation by Ag NPs were shown in some reproductive toxicology studies. Garcia et al (117) reported no change in the expression level of StAR after Ag NP administration although they detected increased Cyp11a1 and Hsd3b1 expression levels, which were also verified through increased testosterone level in their study. However, Dziendzikowska et al (118) reported decreased expression level of Star, Cyp11a1, Hsd3b1 and Hsd17b3 in Wistar rats treated with Ag NPs. Zhang et al showed inhibited StAR, Hsd3b1, and Hsd17b transcription, which can negatively affect testosterone production in TM3 cells (36). As suggested by Dziendzikowska et al (119), impaired steroidogenesis is probably resulting from the interactions of Ag ions with the thiol groups present in the inner mitochondrial membrane (120,121). This concurs well with the MIE in the putative AOP that we propose.

In AOP-Wiki, impairment of steroidogenesis was investigating regarding its role in reproductive toxicity, and we identified AOP18 (122) that describes the AO impaired fertility following a MIE that is PPARα activation in Leydig cells (123). The development of this AOP relies on evidence collected from rodent models and incorporates human mechanistic and epidemiological data. The pathway comprise the activation of PPARα, followed by the disruption cholesterol transport in mitochondria, impairment of hormonal balance which leads to malformation of the reproductive tract in males. In their evidence assessment, the authors found a moderate relation between cholesterol transport in mitochondria and testosterone synthesis. It is stated that decreasing the amount of cholesterol inside the mitochondria (e. g by decreasing the expression of enzymes like StAR or TSOP) will result in a diminished amount of substrate for hormone (testosterone) synthesis. These results offer compelling evidence for the alteration of testosterone level in our pAOP. Therefore, we can assume that apoptosis in Leydig cells or its mitochondrial damage may lead to the endpoint of testosterone level reduction that we define as KE5 in our putative AOP.

In our database, after intravenous injection or oral route administration Ag NPs are shown to accumulate in the testes and are found in spermatids and ejaculated sperms, which suggest the likelihood that Ag NPs could pass through the blood-testis barrier (BTB) and eventually could impair the endocrine and reproductive functions (25,28,40,41,58,124–126). Arisha et al showed increased Ag NPs levels in testes, reduced expression of tight junction proteins (occludin, claudin-11, and tight junction protein 1) resulting in BTB permeability increase (99). They correlated these results with significantly reduced mRNA expression of hypothalamic GnRH1, testicular AR, and serum levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH) and testosterone concentrations. The authors conclude that decreased testosterone level, mainly due to unbalanced sexual hormones signaling and testicular damage, affects spermatogenesis. In a very recent study investigating steroidogenesis in rat hippocampus upon exposure to Ag NPs, the alterations in expression of Star, Hsd3b3, and Hsd17b1 genes, involved in steroid metabolism, is shown (119). Any interference with the normal functioning of the hypothalamic-pituitary-testicular axis can lead to reduced fertility, and if this interference persists, infertility could develop. Although these events do not occur in the reproductive organ itself but rather in the associated endocrine system, they contribute to Ag-NP reproductive toxicity and are complementary to all the other tested endpoints. Still, we chose not to include them in the current putative AO, which is focused exclusively on the male reproductive organ.

Sperm characteristics have a great importance in the prediction of fertility. Twenty-four out of the 48 identified studies analyzed sperm parameters after exposure to Ag-NPs, including sperm morphology, viability, motility, and DNA damage. Upon Ag NP exposure, abnormal sperm morphology, decreased sperm viability and motility, increased sperm DNA damage were reported in number of studies (38,75,77,79,86,90,109,127). The production of sperm requires a complex interaction between Sertoli, Leydig and germ cells. Any defect of these cells may prevent normal sperm production. Moreover, studies mostly emphasize that inadequate hormonal level may provoke low sperm quality and quantity (128). For example, Cavallin et al (95) showed that reduced sperm storage and reduced sperm transit time after Ag NPs exposure to rats may be due to increased testosterone concentration in the serum. In the AOP-Wiki database, the reduction of testosterone level is defined as an upstream event of decreased sperm quality. Therefore, we assigned decreased sperm quality and quantity (KE6) as a downstream event of reduction, testosterone level (KE5).

At the organ level, in vivo histopathological analysis of testis was conducted in 27 studies out of 48. Testis index and histological structure of testicular tissues, morphology, seminiferous tubule area, circumference, diameter and tubular degeneration/atrophy were the most studied parameters. A prominent atrophy of seminiferous tubules, thinning of the tubule wall, disorganization and vacuolization of germinal epithelium, and loss of spermatogenic cells in testis tissue of rats and mice exposed to Ag NPs are reported (39,77,80,84). In the interstitial tissue, Leydig cells are highly affected, presenting disrupted plasma membrane on extensive areas, with loss of cell organelles (80). Shehata et al showed significant reduction in the area, circumference and mean diameter of seminiferous tubules in Ag NP exposed rats (38). These results suggest that Ag NPs may affect the testicular structure and decrease reproductive success.

As a summary, impairment of intracellular SH-containing biomolecules may lead to mitochondrial damage and ROS accumulation (KE1) and lead to oxidative stress (KE2) which further provoke DNA damage and Lipid peroxidation (KE3). Intracellular perturbations may lead to apoptosis in Leydig cells. At the organ/organ system level, these perturbations result in altered testosterone level (KE5) and decreased sperm quality (KE6). All these biological events, which emerged from our literature analysis, lead to the putative AOP framework shown in Fig. 3.

3.5. Network of AOPs

Then, the pAOPs presented in Fig. 3 was used to tentatively build a network of AOPs for reproductive toxicity. MIEs and KEs involved in male reproduction impairment-related AOPs were extracted from AOP-Wiki and are listed in the Table 2.

Table 2

Male reproductive system AOPs on AOP-Wiki
AOP ID:	Title	KE	AO	Taxonomic Applicability	Ref
18	PPARα activation in utero leading to impaired fertility in males	MIE : Activation, PPARα KE1 : Decrease, Steroidogenic acute regulatory protein (STAR) KE2 : Reduction, Cholesterol transport in mitochondria KE3 : Reduction, Testosterone synthesis in Leydig cells KE4 : Reduction, Testosterone level KE5 : Decrease, Translocator protein (TSPO)	Impaired, Fertility Malformation, Male reproductive tract	Rattus norvegicus Homo sapiens Mus musculus	(122)
64	Glucocorticoid Receptor (GR) Mediated Adult Leydig Cell Dysfunction Leading to Decreased Male Fertility	MIE : Glucocorticoid Receptor Agonist, Activation KE1 : Repressed expression of steroidogenic enzymes KE2 : Increased apoptosis, decreased number of adult Leydig Cells KE3 : Reduction, Testosterone synthesis in Leydig cells KE4 : Reduction, testosterone level KE5 : Decreased sperm quantity or quality in the adult, Decreased fertility	Impaired, Fertility	Rattus norvegicus	(129)
207	NADPH oxidase and P38 MAPK activation leading to reproductive failure in Caenorhabditis elegans	MIE : Activation, NADPH Oxidase KE1 : ROS formation KE2 : Increase, Oxidative Stress / Activation, PMK-1 P38 MAPK KE3 : Activation, HIF-1 KE4 : Increased, DNA Damage-Repair KE5 : Damaging, Mitochondria KE6 : Apoptosis	Reproductive failure	Caenorhabditis elegans	(47)
208	Janus kinase (JAK)/Signal transducer and activator of transcription (STAT) and Transforming growth factor (TGF)-beta pathways activation leading to reproductive failure	KE1 : Activation, JAK/STAT pathway KE2 : Activation, TGF-beta pathway	Reproductive failure	Caenorhabditis elegans	(51)
322	Alkylation of DNA leading to reduced sperm count	MIE : Alkylation, DNA KE1 : Inadequate DNA repair KE2 : Increase, DNA strand breaks KE3 : Increase, Apoptosis	Reduce, Sperm count	No information	(108)
323	PPAR alpha Agonism Impairs Fish Reproduction	MIE : Activation, PPARα KE1 : Decreased, cholesterol KE2 : Decreased, 11KT KE3 : Impaired, Spermatogenesis KE4 : impaired, Fertility	No information	Teleost fish	(130)
444	Ionizing radiation leads to reduced reproduction in Eisenia fetida via reduced spermatogenesis and cocoon hatchability	MIE : Deposition of Energy MIE : Increase in reactive oxygen and nitrogen species (RONS) MIE : Increase, DNA damage KE1 : Increased, Oxidative Stress KE2 : Increase, Apoptosis KE3 : Decreased spermatogenesis KE4 : Decrease, Fecundity KE5 : Decrease, Reproduction	Decrease, Population growth rate	No information	(131)

Some AOPs listed in this table are already discussed in the previous section. We observed that these AOPs share at least one common KE with our pAOPs. For example, oxidative stress, apoptosis, reduction of testosterone levels, decreased sperm quality events are shared by AOP 64, 207, 444. In Fig. 4, we interconnected these events with our pAOPs. While individual AOPs are likely to be activated by a limited number of reprotoxic compounds, interconnected AOPs that are linked by common key events of single AOPs are likely to represent more realistic descriptions of the complexity of disease pathophysiology (132).

The AOP network reported in Fig. 4 also shows the potential knowledge gaps in internal associations between KEs. Complete/pathway driven studies investigating the effects of impairment of SH-containing biomolecules and their role in male reproductive development are lacking. For establishing a solid quantitative linkage, mode of action framework analysis for reproductive toxicity is needed. This figure also could serve as a candidate list of MIE that could provide clues for experimental verifications for future studies.

3.6. Experimental methods for assessing the KEs

As suggested by Halappanavar et al (55), AOPs can be used as a tool in the design of testing strategies to support the safety assessment of nanomaterials. In this regard, our database identified the various in vitro endpoints, methods and assays used to measure the KEs in this pAOP (Table 3).

In studies examining oxidative stress, the intracellular level of ROS has often been evaluated using a fluorescent probe such as H2-DCF-DA (82,84,92). MDA content in ELISA methods can be used to detect lipid peroxidation, which is one of the main indicators of oxidative stress (77,81,89). With commercial kits, GSH levels and total antioxidant capacities can be measured, as well as analyzed at the level of antioxidant biomolecules and enzymes such as CAT, SOD (83,91,133). GSH and GSSG levels can also be determined biochemically in high performance liquid chromatography HPLC, capillary electrophoresis or microplates. Gene expressions of the antioxidant defense system can be measured by RT-PCR. Mitochondrial dysfunction can be measured by colorimetric assays such as 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) through assessment of mitochondrial membrane potential (MMP), mitochondrial ATP production, cytochrome c release, or mtDNA damage (84).

Although in the articles from our database DNA damage is evaluated using the comet assay, micronucleus assay and DNA fragmentation, a roadmap for testing DNA damage caused by nanomaterials was recently proposed by Elespuru et al (134). It includes the use of an in vitro gene mutation assay (OECD TG476 (135), HPRT or TG490 (136), mouse lymphoma TK ± assay) and a chromosomal damage assay (OECD TG487 (137) in vitro micronucleus assay or TG473 (138) chromosomal aberration assay). Eventually, optional assays are proposed, both in vitro (comet assay) and in vivo (comet assay, OECD TG489 (139); transgenic rodent gene mutation assay TG488 (140); erythrocyte micronucleus assay, TG474 (141); bone marrow chromosomal aberration test, TG 475 (142)). Other non-guideline test methods to measure the DNA damage also exist although they are not discussed in the roadmap by Elespuru et al (134). For instance the detection of DNA repair proteins such as H2AX, 53BP1 or XRCC2 can be used, or high performance liquid chromatography (HPLC) coupled to tandem mass spectrometry (MS/MS) that quantifies very low levels of oxidative lesions to DNA (143).

In articles in our database, apoptosis was evaluated by western blot, qPCR, TUNEL Assay or Flow Cytometry (80,84,98,144). Other methods can also be used, including annexin V-FITC probes, with analysis of the relative percentage of Annexin V-FITC-positive/PI-negative cells by flow cytometry. The alteration of procaspases 7 and 3, Caspase-3 and caspase-9 activity, as well as the cleavage of PARP can be determined by western blotting or RT-PCR.

The OECD TG 456 (145) is a validated test guideline for in vitro screening of the effect of chemicals on steroidogenesis, specifically the production of 17ß-estradiol (E2) and testosterone. In vitro testosterone synthesis in Leydig cells can be measured by P450scc, StAR, Hsd3b, and Cyp17a1 gene expression or indirectly by testosterone radioimmunoassay or analytical methods such as LC-MS or by isotope-dilution gas chromatography-mass spectrometry in serum (146,147). Sperm assessment includes the evaluation of sperm count and concentration (haemocytometer, automated image-based system), morphology and motility (microscope, automated image-based system) and viability (for example propidium iodide staining of necrotic cells, TUNEL assay staining apoptotic cells).

Models that can be used as alternatives to animal experimentation for assessing this putative AOP on male reproductive function, i.e., in vitro models of Leydig cells, Sertoli cells, Sertoli-germ cell cocultures, as well as methods to prepare testicular organ and tissue culture systems can be found in the Database Service on Alternative Methods to animal experimentation (DB-ALM) (148). Data generated by alternative methods or in vivo testing can be integrated in quantitative AOPs and can be validated for future studies.

Another purpose of our research in developing this pAOP was actually to observe how physicochemical properties of NPs influence the key events and their relationships. From the selected articles that were analysed, we identified Ag NP size, agglomeration state, surface coating and tendency to dissolve as key physico-chemical parameters that could influence their toxicity (Additional file 1). However, it is difficult to reach a definite conclusion, as the physicochemical characterization of Ag NPs in the considered articles is too diverse and sometimes lacks precision. It seems clear from these studies that controlling ion release could diminish the hazard potential of Ag NPs with respect to SbD approaches (16). Therefore, it is highly recommended to analyze Ag ion release systematically in the published articles. Since ion release is higher when the nanoparticle is smaller and when the nanoparticle surface is uncoated or coated with a ligand that tends to desorb, we consider that both the size and surface coating are important parameters that influence Ag NP hazard potential, as previously suggested in studies related to other organs (13,149,150).

Table 3

Summary information of the biological endpoints measured in the reproductive system toxicology
KE	Biological events/measurement	Methods	Ref
Cell Viability		MTT, MTS, LDH, CCK-8 assay	(83,84,92,94,98)
Oxidative stress	ROS Production	H2DCFDA	(82,84,92)
	Lipid peroxidation, MDA content In testicular tissue and cells	ELISA, Western Blot, qRT-PCR	(76,77,81,83,89,96)
	Enzymatic/non-enzymatic antioxidants GPX, CAT, SOD, TBARS, TAOC, GSH in Sperm, Seminal Plasma, and Blood, serum, testicular tissue homogenate and in vitro cells	ELISA, Western Blot, qRT-PCR	(38,75,76,83,91,133)
DNA damage	DNA Strand breaks, In cells and the testis tissue	Comet Assay, DNA microarray analysis, DNA fragmentation	(38,75,76,83,94,96)
Mitochondrial damage	Cell metabolic activity,	MTT assay,	(98)
Mitochondrial damage	Mitochondrial membrane potential	Quantitative method uses a tetraphenylphosphonium ion (TPP+)-sensitive electrode, or by fluorimetric methods	(84)
Apoptosis, in germ Sertoli and Leydig cell	P53, BAX, bcl-2 gene expressions or cell apoptosis	Western Blot, qPCR, TUNEL Assay Flow Cytometry	(77,80,84,98,99,144)
Reproductive hormone levels	Hormonal Assessment Serum Testosterone, LH and FSH, P450scc, StAR, Hsd3b, and Cyp17a1 gene expression	Radioimmunoassay (145), ELISA, qRT-PCR, chemiluminescent protein immunoassay	(38,76,78,86,89,118,151)
Sperm evaluations	Morphology, Motility, Concentration, Count, Viability, Plasma membrane intergrity	Eosin/nigrosine staining, hemocytometer, Microscope	(38,79,82,90,126,144,152)
	Acrosome status	Fluorescence assessment (eg.chlortetracycline fluorescence assay)	(57,126,152)
	Sperm DNA integrity	Toluidine blue staining, Aniline blue staining, Acridine orange staining Eosin–nigrosine-staining	(29,77,90,109,153)
	Sperm DNA damage	Comet assay	(96,153)
	The mitochondrial activity	Activity of cytochrome c oxidase	(57,95)
	Spermatogenesis	The transcript expression of Gnrh1, Ar, Cyp11a1, Hsd3b1, Hsd17b3, Srd5a1, Cyp19a1, Star by qPCR Intratesticular steroid metabolism enzyme protein level aromatase (Aro) and 5α-reductase type 1 (Srd5a1) measurements.	(95,118)
Determination of silver concentration in organs		ICPMS, UV/vis proton spectrophotometery	(29,100,154)
Histopathological Examination	The seminiferous tubules area, circumference, and diameter, Testis index and histological structure of testicular tissues	Hematoxylin and Eosin light microscope Transmission electron microscopy (TEM)	(38,86,90,109)

This review discusses the effects of Ag NPs on male reproductive system in the concept of AOPs. By reviewing the existing literature, a putative AOP framework was constructed, where the MIE is identified as the impairment of intracellular SH-containing biomolecules, with subsequent key events that are mitochondrial damage, ROS accumulation, DNA damage and lipid peroxidation, apoptosis, reduction of reproductive hormones production and sperm quality. These successive key events may result in impaired male fertility (AO). This AOP study summarizes complex data from different biological levels in the literature. It could serve to predict male fertility impairment caused by some NMs using the proposed methods of KE evaluation. Moreover, since the use of the AOP approach is emerging in the nanotoxicology community, proposing some putative AOPs like this one and linking AOPs as networks would help increasing the improvement of mechanistic understanding of pathways interactions involved in various reproductive disorders. Finally, it should be considered that among the factors contributing to the global population decline we are foreseeing, the fertility outcomes related to the decrease in testosterone level and semen quality and quantity, also induced by environmental pollutants, are of pivotal importance. Thus, more efforts should be devoted in the future to better characterize the risk of using new potential endocrine disrupting compounds, as well as to guarantee the better strategies to develop safer and more environmentally sustainable Ag-based materials.

Ag NPs: Silver nanoparticles;

AO: Adverse Outcome;

AOP: Adverse outcome pathway;

ASINA: Anticipating Safety Issues at the Design Stage of NAno Product Development;

BTB: Blood-testis barrier;

CAT: Catalase;

ESR : Electron spin resonance;

FSH: Follicle-stimulating hormone;

GSH: Glutathione;

HPLC: High performance liquid chromatography;

Hsd17b: 17β-Hydroxysteroid dehydorgenase;

Hsd3b: 3β-Hydroxysteroid dehydorgenase;

KE: Key event;

KER: Key Event Relationship;

LH: Luteinizing hormone;

MDA: Malondialdehyde;

MIE: Molecular initiating event;

MMP: Mitochondrial membrane potential;

MTT: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide;

OECD: The Organisation for Economic Co-operation and Development;

pAOP: Putative Adverse Outcome Pathway;

PVP: Polyvinyl pyrrolidone;

ROS: Reactive Oxygen Species;

SbD: Safe by Design;

SOD: Super oxide dismutase;

StAR: Steroidogenic acute regulatory protein;

TBARS: Thiobarbituric acid reactive substances;

TM3: Leydig Cells;

TM4: Sertoli Cells;

ToxRTool: Toxicological data Reliability Assessment Tool;

TSPO: Translocator protein

Ethics approval and consent to participate: Not applicable

Consent for publication: Not applicable

Availability of data and materials: The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests: The authors declare that they have no competing interests

Funding: This work has received funding from the European Union’s Horizon 2020 research and innovation programme under the grant agreement n. 862444, ASINA (Anticipating Safety Issues at the Design Stage of NAno Product Development).

Authors' contributions: Conceptualization: MC, PM, AC. Funding acquisition: AC, PM, MC. Data curation, Formal analysis: OK. Writing – original draft: OK, MC. Writing – review & editing: OK, PM, AC, MC. All authors read and approved the final manuscript.

Prabhu S, Poulose EK. Silver nanoparticles: mechanism of antimicrobial action, synthesis, medical applications, and toxicity effects. Int Nano Lett 2012 21. 2012;2(1):1–10. doi:10.1186/2228-5326-2-32.
Yoshida Y, Churei H, Takeuch Y, Wada T, Uo M, Izumi Y, et al. Novel antibacterial mouthguard material manufactured using silver-nanoparticle-embedded ethylene-vinyl acetate copolymer masterbatch. Dent Mater J. 2018;37(3):437–44. doi:10.4012/DMJ.2017-226.
ANSES. Exposure to silver nanoparticles | Anses - Agence nationale de sécurité sanitaire de l’alimentation, de l’environnement et du travail. https://www.anses.fr/en/content/exposure-silver-nanoparticles. 2018.
Marambio-Jones C, Hoek EM V. A review of the antibacterial effects of silver nanomaterials and potential implications for human health and the environment. J Nanoparticle Res 2010 125. 2010;12(5):1531–51. doi:10.1007/S11051-010-9900-Y.
Nakamura S, Sato M, Sato Y, Ando N, Takayama T, Fujita M, et al. Synthesis and application of silver nanoparticles (Ag NPs) for the prevention of infection in healthcare workers. Vol. 20, International Journal of Molecular Sciences. 2019.
Silva RM, Anderson DS, Franzi LM, Peake JL, Edwards PC, Van Winkle LS, et al. Pulmonary Effects of Silver Nanoparticle Size, Coating, and Dose over Time upon Intratracheal Instillation. Toxicol Sci. 2015;144(1):151–62. doi:10.1093/toxsci/kfu265.
Anderson DS, Silva RM, Lee D, Edwards PC, Sharmah A, Guo T, et al. Persistence of silver nanoparticles in the rat lung: Influence of dose, size, and chemical composition. Nanotoxicology. 2015;9(5):591–602. doi:10.3109/17435390.2014.958116.
Batista D, Pascoal C, Cássio F. How do physicochemical properties influence the toxicity of silver nanoparticles on freshwater decomposers of plant litter in streams? Ecotoxicol Environ Saf. 2017;140:148–55.
Liu J, Sonshine DA, Shervani S, Hurt RH. Controlled Release of Biologically Active Silver from Nanosilver Surfaces. ACS Nano. 2010;4(11):6903–13. doi:10.1021/nn102272n.
Peretyazhko TS, Zhang Q, Colvin VL. Size-Controlled Dissolution of Silver Nanoparticles at Neutral and Acidic pH Conditions: Kinetics and Size Changes. Environ Sci Technol. 2014;48(20):11954–61. doi:10.1021/ES5023202.
Hsiao IL, Hsieh YK, Wang CF, Chen IC, Huang YJ. Trojan-Horse Mechanism in the Cellular Uptake of Silver Nanoparticles Verified by Direct Intra- and Extracellular Silver Speciation Analysis. Environ Sci Technol. 2015;49(6):3813–21. doi:10.1021/ES504705P.
Beer C, Foldbjerg R, Hayashi Y, Sutherland DS, Autrup H. Toxicity of silver nanoparticles—Nanoparticle or silver ion? Toxicol Lett. 2012;208(3):286–92.
Bobyk L, Tarantini A, Beal D, Veronesi G, Kieffer I, Motellier S, et al. Toxicity and chemical transformation of silver nanoparticles in A549 lung cells: dose-rate-dependent genotoxic impact. Environ Sci Nano. 2021;8(3):806–21. doi:10.1039/D0EN00533A.
European Commission. Nanosilver: Safety, health and environmental effects and role in antimicrobial resistance, Final Opinion Health Effects of Exposure to nanosilver. http://ec.europa.eu/health/scientific_committees/emerging/members_wg/index_en.htm. 2014. doi:10.2772/76851.
Rohde MM, Snyder CM, Sloop J, Solst SR, Donati GL, Spitz DR, et al. The mechanism of cell death induced by silver nanoparticles is distinct from silver cations. Part Fibre Toxicol. 2021;18(1):37. doi:10.1186/s12989-021-00430-1.
Marchioni M, Jouneau PH, Chevallet M, Michaud-Soret I, Deniaud A. Silver nanoparticle fate in mammals: Bridging in vitro and in vivo studies. Coord Chem Rev. 2018;364:118–36.
Barngrover BM, Aikens CM. Incremental Binding Energies of Gold(I) and Silver(I) Thiolate Clusters. J Phys Chem A. 2011;115(42):11818–23. doi:10.1021/JP2061893.
Hogg PJ. Disulfide bonds as switches for protein function. Trends Biochem Sci. 2003;28(4):210–4.
Go YM, Chandler JD, Jones DP. The cysteine proteome. Free Radic Biol Med. 2015;84:227–45. doi:10.1016/j.freeradbiomed.2015.03.022.
Ulrich K, Jakob U. The role of thiols in antioxidant systems. Free Radic Biol Med. 2019;140:14–27. doi:10.1016/j.freeradbiomed.2019.05.035.
Xiong Y, Uys JD, Tew KD, Townsend DM. S-Glutathionylation: From Molecular Mechanisms to Health Outcomes. Antioxid Redox Signal. 2011;15(1):233. doi:10.1089/ARS.2010.3540.
Jiang HS, Zhang Y, Lu ZW, Lebrun R, Gontero B, Li W. Interaction between Silver Nanoparticles and Two Dehydrogenases: Role of Thiol Groups. Small. 2019;15(27):1900860. doi:10.1002/smll.201900860.
Piao MJ, Kang KA, Lee IK, Kim HS, Kim S, Choi JY, et al. Silver nanoparticles induce oxidative cell damage in human liver cells through inhibition of reduced glutathione and induction of mitochondria-involved apoptosis. Toxicol Lett. 2011;201(1):92–100.
George R, Merten S, Wang TT, Kennedy P, Maitz P. In vivo analysis of dermal and systemic absorption of silver nanoparticles through healthy human skin. Australas J Dermatol. 2014;55(3):185–90. doi:10.1111/ajd.12101.
van der Zande M, Vandebriel RJ, Van Doren E, Kramer E, Herrera Rivera Z, Serrano-Rojero CS, et al. Distribution, Elimination, and Toxicity of Silver Nanoparticles and Silver Ions in Rats after 28-Day Oral Exposure. ACS Nano. 2012;6(8):7427–42. doi:10.1021/nn302649p.
Sung JH, Ji JH, Park JD, Yoon JU, Kim DS, Jeon KS, et al. Subchronic Inhalation Toxicity of Silver Nanoparticles. Toxicol Sci. 2009;108(2):452–61. doi:10.1093/toxsci/kfn246.
Antsiferova AA, Kopaeva MY, Kochkin VN, Kashkarov PK. Kinetics of Silver Accumulation in Tissues of Laboratory Mice after Long-Term Oral Administration of Silver Nanoparticles. Nanomaterials. 2021;11(12):3204. doi:10.3390/nano11123204.
Arslan NP, Keles ON, Gonul-Baltaci N. Effect of Titanium Dioxide and Silver Nanoparticles on Mitochondrial Dynamics in Mouse Testis Tissue. Biol Trace Elem Res. 2022;200(4):1650–8. doi:10.1007/s12011-021-02763-6.
Rezazadeh-Reyhani Z, Razi M, Malekinejad H, Sadrkhanlou R. Cytotoxic effect of nanosilver particles on testicular tissue: Evidence for biochemical stress and Hsp70-2 protein expression. Environ Toxicol Pharmacol. 2015;40(2):626–38. doi:10.1016/j.etap.2015.08.024.
Garcia TX, Costa GMJ, França LR, Hofmann MC. Sub-acute intravenous administration of silver nanoparticles in male mice alters Leydig cell function and testosterone levels. Reprod Toxicol. 2014;45:59–70. doi:10.1016/j.reprotox.2014.01.006.
Lankveld DPK, Oomen AG, Krystek P, Neigh A, Troost – de Jong A, Noorlander CW, et al. The kinetics of the tissue distribution of silver nanoparticles of different sizes. Biomaterials. 2010;31(32):8350–61. doi:10.1016/j.biomaterials.2010.07.045.
European Commission. Scientific Committee on Consumer Safety SCCS Opinion on Colloidal Silver (nano). http://ec.europa.eu/health/scientific_committees/experts/declarations/sccs_en.htm. 2018.
Alahmar A. Role of oxidative stress in male infertility: An updated review. J Hum Reprod Sci. 2019;12(1):4. doi:10.4103/jhrs.JHRS_150_18.
Brohi RD, Wang L, Talpur HS, Wu D, Khan FA, Bhattarai D, et al. Toxicity of Nanoparticles on the Reproductive System in Animal Models: A Review. Front Pharmacol. 2017;8:606. doi:10.3389/fphar.2017.00606.
Habas K, Brinkworth MH, Anderson D. Silver nanoparticle-mediated cellular responses in isolated primary Sertoli cells in vitro. Food Chem Toxicol. 2018;116:182–8.
Gurunathan S, Zhang XF, Choi YJ, Han JW, Kim E, Park JH, et al. Differential nanoreprotoxicity of silver nanoparticles in male somatic cells and spermatogonial stem cells. Int J Nanomedicine. 2015;10:1335. doi:10.2147/IJN.S76062.
Braydich-Stolle LK, Lucas B, Schrand A, Murdock RC, Lee T, Schlager JJ, et al. Silver Nanoparticles Disrupt GDNF/Fyn kinase Signaling in Spermatogonial Stem Cells. Toxicol Sci. 2010;116(2):577–89. doi:10.1093/toxsci/kfq148.
Shehata AM, Salem FMS, El-Saied EM, Abd El-Rahman SS, Mahmoud MY, Noshy PA. Zinc Nanoparticles Ameliorate the Reproductive Toxicity Induced by Silver Nanoparticles in Male Rats. Int J Nanomedicine. 2021;Volume 16:2555–68. doi:10.2147/IJN.S307189.
Olugbodi JO, David O, Oketa EN, Lawal B, Okoli BJ, Mtunzi F. Silver Nanoparticles Stimulates Spermatogenesis Impairments and Hematological Alterations in Testis and Epididymis of Male Rats. Molecules. 2020;25(5):1063. doi:10.3390/molecules25051063.
Wang Z, Qu G, Su L, Wang L, Yang Z, Jiang J, et al. Evaluation of the Biological Fate and the Transport Through Biological Barriers of Nanosilver in Mice. Curr Pharm Des. 2013;19(37):6691–7. doi:10.2174/1381612811319370012.
Kim YS, Kim JS, Cho HS, Rha DS, Kim JM, Park JD, et al. Twenty-Eight-Day Oral Toxicity, Genotoxicity, and Gender-Related Tissue Distribution of Silver Nanoparticles in Sprague-Dawley Rats. Inhal Toxicol. 2008;20(6):575–83. doi:10.1080/08958370701874663.
Roh JY, Sang JS, Yi J, Park K, Kyu HC, Ryu DY, et al. Ecotoxicity of silver nanoparticles on the soil nematode Caenorhabditis elegans using functional ecotoxicogenomics. Environ Sci Technol. 2009;43(10):3933–40. doi:10.1021/es803477u.
Yan N, Tsim SMJ, He X, Tang BZ, Wang WX. Direct Visualization and Quantification of Maternal Transfer of Silver Nanoparticles in Zooplankton. Environ Sci Technol. 2020;54(17):10763–71. doi:10.1021/acs.est.0c03228.
OECD. Users’ Handbook supplement to the Guidance Document for developing and assessing Adverse Outcome Pathways | OECD Series on Adverse Outcome Pathways | OECD iLibrary. https://www.oecd-ilibrary.org/environment/users-handbook-supplement-to-the-guidance-document-for-developing-and-assessing-adverse-outcome-pathways_5jlv1m9d1g32-en. 2014.
Halappanavar S, Ede JD, Shatkin JA, Krug HF. A systematic process for identifying key events for advancing the development of nanomaterial relevant adverse outcome pathways. NanoImpact. 2019;15(February):100178. doi:10.1016/j.impact.2019.100178.
AOP-Wiki. Adverse Outcome Pathway Wiki. https://aopwiki.org/.
AOP-Wiki. AOP207: NADPH oxidase and P38 MAPK activation leading to reproductive failure in Caenorhabditis elegans. https://aopwiki.org/aops/207.
Jeong J, Song T, Chatterjee N, Choi I, Cha YK, Choi J. Developing adverse outcome pathways on silver nanoparticle-induced reproductive toxicity via oxidative stress in the nematode Caenorhabditis elegans using a Bayesian network model. Nanotoxicology. 2018;12(10):1182–97. doi:10.1080/17435390.2018.1529835.
Ma YB, Lu CJ, Junaid M, Jia PP, Yang L, Zhang JH, et al. Potential adverse outcome pathway (AOP) of silver nanoparticles mediated reproductive toxicity in zebrafish. Chemosphere. 2018;207:320–8. doi:10.1016/j.chemosphere.2018.05.019.
AOP-Wiki. AOP210: Activation of c-Jun N-terminal kinase (JNK) and Forkhead box O (FOXO) and reduction of WNT pathways leading to reproductive failure: Integrated multi-OMICS approach for AOP building. https://aopwiki.org/aops/210.
AOP-Wiki. AOP208:Janus kinase (JAK)/Signal transducer and activator of transcription (STAT) and Transforming growth factor (TGF)-beta pathways activation leading to reproductive failure. https://aopwiki.org/aops/208.
Kim H, Jeong J, Chatterjee N, Roca CP, Yoon D, Kim S, et al. JAK/STAT and TGF-ß activation as potential adverse outcome pathway of TiO2NPs phototoxicity in Caenorhabditis elegans. Sci Reports 2017 71. 2017;7(1):1–12. doi:10.1038/s41598-017-17495-8.
Moher D, Liberati A, Tetzlaff J, Altman DG. Preferred Reporting Items for Systematic Reviews and Meta-Analyses: The PRISMA Statement. PLoS Med. 2009;6(7):e1000097. doi:10.1371/journal.pmed.1000097.
Schneider K, Schwarz M, Burkholder I, Kopp-Schneider A, Edler L, Kinsner-Ovaskainen A, et al. “ToxRTool”, a new tool to assess the reliability of toxicological data. Toxicol Lett. 2009;189(2):138–44. doi:10.1016/j.toxlet.2009.05.013.
Halappanavar S, Van Den Brule S, Nymark P, Gaté L, Seidel C, Valentino S, et al. Adverse outcome pathways as a tool for the design of testing strategies to support the safety assessment of emerging advanced materials at the nanoscale. Part Fibre Toxicol. 2020;17(1):1–24.
Murugadoss S. A strategy towards the generation of testable adverse outcome pathways for nanomaterials. ALTEX. 2021; doi:10.14573/altex.2102191.
Mathias FT, Romano RM, Kizys MML, Kasamatsu T, Giannocco G, Chiamolera MI, et al. Daily exposure to silver nanoparticles during prepubertal development decreases adult sperm and reproductive parameters. Nanotoxicology. 2015;9(1):64–70. doi:10.3109/17435390.2014.889237.
Kim YS, Song MY, Park JD, Song KS, Ryu HR, Chung YH, et al. Subchronic oral toxicity of silver nanoparticles. Part Fibre Toxicol. 2010;7(1):1–11. doi:10.1186/1743-8977-7-20.
Ratte HT. Bioaccumulation and toxicity of silver compounds: A review. Environ Toxicol Chem. 1999;18(1):89–108. doi:10.1002/etc.5620180112.
Gordon O, Vig Slenters T, Brunetto PS, Villaruz AE, Sturdevant DE, Otto M, et al. Silver Coordination Polymers for Prevention of Implant Infection: Thiol Interaction, Impact on Respiratory Chain Enzymes, and Hydroxyl Radical Induction. Antimicrob Agents Chemother. 2010;54(10):4208–18. doi:10.1128/AAC.01830-09.
Loza K, Diendorf J, Sengstock C, Ruiz-Gonzalez L, Gonzalez-Calbet JM, Vallet-Regi M, et al. The dissolution and biological effects of silver nanoparticles in biological media. J Mater Chem B. 2014;2(12):1634. doi:10.1039/c3tb21569e.
Pallavicini P, Preti L, Vita L De, Dacarro G, Diaz Fernandez YA, Merli D, et al. Fast dissolution of silver nanoparticles at physiological pH. J Colloid Interface Sci. 2020;563:177–88. doi:10.1016/j.jcis.2019.12.081.
He W, Zhou YT, Wamer WG, Boudreau MD, Yin JJ. Mechanisms of the pH dependent generation of hydroxyl radicals and oxygen induced by Ag nanoparticles. Biomaterials. 2012;33(30):7547–55. doi:10.1016/j.biomaterials.2012.06.076.
Liu J, Hurt RH. Ion Release Kinetics and Particle Persistence in Aqueous Nano-Silver Colloids. Environ Sci Technol. 2010;44(6):2169–75. doi:10.1021/es9035557.
Ho CM, Wong CK, Yau SKW, Lok CN, Che CM. Oxidative Dissolution of Silver Nanoparticles by Dioxygen: A Kinetic and Mechanistic Study. Chem - An Asian J. 2011;6(9):2506–11. doi:10.1002/asia.201100034.
Shi M, Kwon HS, Peng Z, Elder A, Yang H. Effects of Surface Chemistry on the Generation of Reactive Oxygen Species by Copper Nanoparticles. ACS Nano. 2012;6(3):2157–64. doi:10.1021/NN300445D.
Jose GP, Santra S, Mandal SK, Sengupta TK. Singlet oxygen mediated DNA degradation by copper nanoparticles: potential towards cytotoxic effect on cancer cells. J Nanobiotechnology 2011 91. 2011;9(1):1–8. doi:10.1186/1477-3155-9-9.
Xia T, Kovochich M, Liong M, Mädler L, Gilbert B, Shi H, et al. Comparison of the Mechanism of Toxicity of Zinc Oxide and Cerium Oxide Nanoparticles Based on Dissolution and Oxidative Stress Properties. ACS Nano. 2008;2(10):2121–34. doi:10.1021/nn800511k.
Kim JS, Kuk E, Yu KN, Kim JH, Park SJ, Lee HJ, et al. Antimicrobial effects of silver nanoparticles. Nanomedicine Nanotechnology, Biol Med. 2007;3(1):95–101. doi:10.1016/j.nano.2006.12.001.
Park MVDZ, Neigh AM, Vermeulen JP, de la Fonteyne LJJ, Verharen HW, Briedé JJ, et al. The effect of particle size on the cytotoxicity, inflammation, developmental toxicity and genotoxicity of silver nanoparticles. Biomaterials. 2011;32(36):9810–7. doi:10.1016/j.biomaterials.2011.08.085.
Kluska K, Peris-Díaz MD, Płonka D, Moysa A, Dadlez M, Deniaud A, et al. Formation of highly stable multinuclear AgnSn clusters in zinc fingers disrupts their structure and function. Chem Commun. 2020;56(9):1329–32. doi:10.1039/C9CC09418K.
Wei Liu, Isabelle Worms, I. Slaveykova V. Interaction of silver nanoparticles with antioxidant enzymes. Environ Sci Nano. 2020;7(5):1507–17. doi:10.1039/C9EN01284B.
Käkinen A, Ding F, Chen P, Mortimer M, Kahru A, Ke PC. Interaction of firefly luciferase and silver nanoparticles and its impact on enzyme activity. Nanotechnology. 2013;24(34):345101. doi:10.1088/0957-4484/24/34/345101.
Lim D, Roh JY, Eom HJ, Choi JY, Hyun J, Choi J. Oxidative stress-related PMK-1 P38 MAPK activation as a mechanism for toxicity of silver nanoparticles to reproduction in the nematode Caenorhabditis elegans. Environ Toxicol Chem. 2012;31(3):585–92. doi:10.1002/etc.1706.
Giulia C, Simona M, Elena M, Daniela C, Lucia M, Anna Ida F, et al. Oxidative and/or Inflammatory Thrust Induced by Silver Nanoparticles in Rabbits: Effect of Vitamin E or NSAID Administration on Semen Parameters. Capasso R, editor. Mediators Inflamm. 2020;2020:1–15. doi:10.1155/2020/6664062.
Altwaijry N, El-Masry TA, Alotaibi B, Tousson E, Saleh A. Therapeutic effects of rocket seeds (Eruca sativa L.) against testicular toxicity and oxidative stress caused by silver nanoparticles injection in rats. Environ Toxicol. 2020;35(9):952–60.
Abu-Taweel GM, Albetran HM, Al-Mutary MG, Ahmad M, Low IM. Alleviation of silver nanoparticle-induced sexual behavior and testicular parameters dysfunction in male mice by yttrium oxide nanoparticles. Toxicol Reports. 2021;8:1121–30. doi:10.1016/j.toxrep.2021.05.014.
Attia AA. Evaluation of the testicular alterations induced by silver nanoparticles in male mice: biochemical, histological and ultrastructural studies. Res J Pharm Biol Chem Sci. 2014;5(4):1558–89.
Moradi-Sardareh H, Basir HRG, Hassan ZM, Davoudi M, Amidi F, Paknejad M. Toxicity of silver nanoparticles on different tissues of Balb/C mice. Life Sci. 2018;211:81–90. doi:10.1016/j.lfs.2018.09.001.
Bidian C, Filip GA, David L, Florea A, Moldovan B, Robu DP, et al. The impact of silver nanoparticles phytosynthesized with Viburnum opulus L. extract on the ultrastrastructure and cell death in the testis of offspring rats. Food Chem Toxicol. 2021;150:112053. doi:10.1016/j.fct.2021.112053.
Lopes IMD, de Oliveira IM, Bargi-Souza P, Cavallin MD, Kolc CSM, Khalil NM, et al. Effects of Silver Nanoparticle Exposure to the Testicular Antioxidant System during the Prepubertal Rat Stage. Chem Res Toxicol. 2019;32(6):986–94. doi:10.1021/acs.chemrestox.8b00281.
Wang E, Huang Y, Du Q, Sun Y. Silver nanoparticle induced toxicity to human sperm by increasing ROS(reactive oxygen species) production and DNA damage. Environ Toxicol Pharmacol. 2017;52:193–9. doi:10.1016/j.etap.2017.04.010.
Zapór L. Effects of silver nanoparticles of different sizes on cytotoxicity and oxygen metabolism disorders in both reproductive and respiratory system cells. Arch Environ Prot. 2016;42(4):32–47. doi:10.1515/aep-2016-0038.
Han JW, Jeong JK, Gurunathan S, Choi YJ, Das J, Kwon DN, et al. Male- and female-derived somatic and germ cell-specific toxicity of silver nanoparticles in mouse. Nanotoxicology. 2016;10(3):361–73. doi:10.3109/17435390.2015.1073396.
Carlson C, Hussain SM, Schrand AM, Braydich-Stolle LK, Hess KL, Jones RL, et al. Unique Cellular Interaction of Silver Nanoparticles: Size-Dependent Generation of Reactive Oxygen Species. J Phys Chem B. 2008;112(43):13608–19. doi:10.1021/JP712087M.
Wang E, Huang Y, Du Q, Sun Y. Alterations in reproductive parameters and gene expression in Balb/c mice testes after exposure to silver nanoparticles. Andrologia. 2021;53(1):1–9. doi:10.1111/and.13841.
AOP-Wiki. AOP17:Binding of electrophilic chemicals to SH(thiol)-group of proteins and /or to seleno-proteins involved in protection against oxidative stress during brain development leads to impairment of learning and memory. https://aopwiki.org/aops/17.
Spiller HA. Rethinking mercury: the role of selenium in the pathophysiology of mercury toxicity. https://doi.org/101080/1556365020171400555. 2017;56(5):313–26. doi:10.1080/15563650.2017.1400555.
Shariatzadeh SMA, Miri SA, Cheraghi E. The protective effect of Kombucha against silver nanoparticles‐induced toxicity on testicular tissue in NMRI mice. Andrologia. 2021;53(3):1–10. doi:10.1111/and.13982.
Elsharkawy EE, Abd El-Nasser M, Kamaly HF. Silver nanoparticles testicular toxicity in rat. Environ Toxicol Pharmacol. 2019;70. doi:10.1016/j.etap.2019.103194.
Hassanpour H, Mirshokraei P, Khalili Sadrabad E, Esmailian Dehkordi A, Layeghi S, Afzali A, et al. In vitro effect of nanosilver on gene expression of superoxide dismutases and nitric oxide synthases in chicken sertoli cells. Animal. 2014;9(2):295–300. doi:10.1017/S1751731114002262.
Zhang XF, Choi YJ, Han JW, Kim E, Park JH, Gurunathan S, et al. Differential nanoreprotoxicity of silver nanoparticles in male somatic cells and spermatogonial stem cells. Int J Nanomedicine. 2015;10:1335–57. doi:10.2147/IJN.S76062.
Asare N, Duale N, Slagsvold HH, Lindeman B, Olsen AK, Gromadzka-Ostrowska J, et al. Genotoxicity and gene expression modulation of silver and titanium dioxide nanoparticles in mice. Nanotoxicology. 2016;10(3):312–21. doi:10.3109/17435390.2015.1071443.
Asare N, Instanes C, Sandberg W, Refsnes M, Schwarze P, Kruszewski M, et al. Cytotoxic and genotoxic effects of silver nanoparticles in testicular cells. Toxicology. 2012;291(1–3):65–72. doi:10.1016/J.TOX.2011.10.022.
Cavallin MD, Wilk R, Oliveira IM, Cardoso NCS, Khalil NM, Oliveira CA, et al. The hypothalamic-pituitary-testicular axis and the testicular function are modulated after silver nanoparticle exposure. Toxicol Res (Camb). 2018;7(1):102–16. doi:10.1039/c7tx00236j.
Mohamed HRH. Studies on the Genotoxicity Behavior of Silver Nanoparticles in the Presence of Heavy Metal Cadmium Chloride in Mice. J Nanomater. 2016;1–12. doi:10.1155/2016/5283162.
AshaRani P V., Low Kah Mun G, Hande MP, Valiyaveettil S. Cytotoxicity and Genotoxicity of Silver Nanoparticles in Human Cells. ACS Nano. 2009;3(2):279–90. doi:10.1021/nn800596w.
Braydich-Stolle L, Hussain S, Schlager J, Hofmann MC. In vitro cytotoxicity of nanoparticles in mammalian germline stem cells. Toxicol Sci. 2005;88(2):412–9. doi:10.1093/TOXSCI/KFI256.
Arisha AH, Ahmed MM, Kamel MA, Attia YA, Hussein MMA. Morin ameliorates the testicular apoptosis, oxidative stress, and impact on blood–testis barrier induced by photo-extracellularly synthesized silver nanoparticles. Environ Sci Pollut Res. 2019;26(28):28749–62. doi:10.1007/s11356-019-06066-1.
de Brito JLM, de Lima VN, Ansa DO, Moya SE, Morais PC, de Azevedo RB, et al. Acute reproductive toxicology after intratesticular injection of silver nanoparticles (AgNPs) in Wistar rats. Nanotoxicology. 2020;14(7):893–907. doi:10.1080/17435390.2020.1774812.
Thakur M, Gupta H, Singh D, Mohanty IR, Maheswari U, Vanage G, et al. Histopathological and ultra structural effects of nanoparticles on rat testis following 90 days (Chronic study) of repeated oral administration. J Nanobiotechnology. 2014;12(1):42. doi:10.1186/s12951-014-0042-8.
Satapathy SR, Mohapatra P, Preet R, Das D, Sarkar B, Choudhuri T, et al. Silver-based nanoparticles induce apoptosis in human colon cancer cells mediated through p53. Nanomedicine (Lond). 2013;8(8):1307–22. doi:10.2217/NNM.12.176.
Shi J, Sun X, Lin Y, Zou X, Li Z, Liao Y, et al. Endothelial cell injury and dysfunction induced by silver nanoparticles through oxidative stress via IKK/NF-κB pathways. Biomaterials. 2014;35(24):6657–66. doi:10.1016/j.biomaterials.2014.04.093.
Wang X, Sharma RK, Sikka SC, Thomas AJ, Falcone T, Agarwal A. Oxidative stress is associated with increased apoptosis leading to spermatozoa DNA damage in patients with male factor infertility. Fertil Steril. 2003;80(3):531–5.
Muratori M, Tamburrino L, Marchiani S, Cambi M, Olivito B, Azzari C, et al. Investigation on the Origin of Sperm DNA Fragmentation: Role of Apoptosis, Immaturity and Oxidative Stress. Mol Med. 2015;21(1):109–22. doi:10.2119/molmed.2014.00158.
Evans EPP, Scholten JTM, Mzyk A, Reyes-San-Martin C, Llumbet AE, Hamoh T, et al. Male subfertility and oxidative stress. Redox Biol. 2021;46:102071.
Cheng X, Zhang W, Ji Y, Meng J, Guo H, Liu J, et al. Revealing silver cytotoxicity using Au nanorods/Ag shell nanostructures: disrupting cell membrane and causing apoptosis through oxidative damage. RSC Adv. 2013;3(7):2296. doi:10.1039/c2ra23131j.
AOP-Wiki. AOP322: Alkylation of DNA leading to reduced sperm count. https://aopwiki.org/aops/322.
Fathi N, Hoseinipanah SM, Alizadeh Z, Assari MJ, Moghimbeigi A, Mortazavi M, et al. The effect of silver nanoparticles on the reproductive system of adult male rats: A morphological, histological and DNA integrity study. Adv Clin Exp Med. 2018;28(3):299–305. doi:10.17219/acem/81607.
Zirkin BR, Papadopoulos V. Leydig cells: formation, function, and regulation†. Biol Reprod. 2018;99(1):101–11. doi:10.1093/biolre/ioy059.
Baki ME, Miresmaili SM, Pourentezari M, Amraii E, Yousefi V, Spenani HR, et al. Effects of silver nano-particles on sperm parameters, number of Leydig cells and sex hormones in rats. Iran J Reprod Med. 2014;12(2):139–44.
Park TJ, Song KY, Sohn SH, Lim IK. Marked inhibition of testosterone biosynthesis by the hepatotoxin nodularin due to apoptosis of Leydig cells. Mol Carcinog. 2002;34(3):151–63.
Orazizadeh M, Khorsandi L, Absalan F, Hashemitabar M, Daneshi E. Effect of beta-carotene on titanium oxide nanoparticles-induced testicular toxicity in mice. J Assist Reprod Genet. 2014;31(5):561–8. doi:10.1007/s10815-014-0184-5.
Korytowski W, Pilat A, Schmitt JC, Girotti AW. Deleterious cholesterol hydroperoxide trafficking in steroidogenic acute regulatory (StAR) protein-expressing MA-10 Leydig cells: implications for oxidative stress-impaired steroidogenesis. J Biol Chem. 2013;288(16):11509–19. doi:10.1074/JBC.M113.452151.
Tong MH, Christenson LK, Song WC. Aberrant Cholesterol Transport and Impaired Steroidogenesis in Leydig Cells Lacking Estrogen Sulfotransferase. Endocrinology. 2004;145(5):2487–97. doi:10.1210/EN.2003-1237.
Papadopoulos V, Miller WL. Role of mitochondria in steroidogenesis. Best Pract Res Clin Endocrinol Metab. 2012;26(6):771–90. doi:10.1016/j.beem.2012.05.002.
Valerio-García RC, Carbajal-Hernández AL, Martínez-Ruíz EB, Jarquín-Díaz VH, Haro-Pérez C, Martínez-Jerónimo F. Exposure to silver nanoparticles produces oxidative stress and affects macromolecular and metabolic biomarkers in the goodeid fish Chapalichthys pardalis. Sci Total Environ. 2017;583:308–18. doi:10.1016/j.scitotenv.2017.01.070.
Dziendzikowska K, Krawczyńska A, Oczkowski M, Królikowski T, Brzóska K, Lankoff A, et al. Progressive effects of silver nanoparticles on hormonal regulation of reproduction in male rats. Toxicol Appl Pharmacol. 2016;313:35–46. doi:10.1016/j.taap.2016.10.013.
Dziendzikowska K, Wilczak J, Grodzicki W, Gromadzka-Ostrowska J, Węsierska M, Kruszewski M. Coating-Dependent Neurotoxicity of Silver Nanoparticles—An In Vivo Study on Hippocampal Oxidative Stress and Neurosteroids. Int J Mol Sci. 2022;23(3):1365. doi:10.3390/ijms23031365.
Hadrup N, Loeschner K, Mortensen A, Sharma AK, Qvortrup K, Larsen EH, et al. The similar neurotoxic effects of nanoparticulate and ionic silver in vivo and in vitro. Neurotoxicology. 2012;33(3):416–23. doi:10.1016/j.neuro.2012.04.008.
De Matteis V, Malvindi MA, Galeone A, Brunetti V, De Luca E, Kote S, et al. Negligible particle-specific toxicity mechanism of silver nanoparticles: The role of Ag+ ion release in the cytosol. Nanomedicine Nanotechnology, Biol Med. 2015;11(3):731–9. doi:10.1016/j.nano.2014.11.002.
AOP-Wiki. AOP18: PPARα activation in utero leading to impaired fertility in males. https://aopwiki.org/aops/18.
Nepelska M, Odum J, Munn S. Adverse Outcome Pathway: Peroxisome Proliferator-Activated Receptor α Activation and Reproductive Toxicity—Development and Application in Assessment of Endocrine Disruptors/Reproductive Toxicants. Appl Vitr Toxicol. 2017;3(3):234–49. doi:10.1089/aivt.2017.0004.
Park EJ, Bae E, Yi J, Kim Y, Choi K, Lee SH, et al. Repeated-dose toxicity and inflammatory responses in mice by oral administration of silver nanoparticles. Environ Toxicol Pharmacol. 2010;30(2):162–8.
Lee J, Kim Y, Song K, Ryu H, Sung J, Park J, et al. Biopersistence of silver nanoparticles in tissues from Sprague-Dawley rats. Part Fibre Toxicol. 2013;10(1). doi:10.1186/1743-8977-10-36.
Castellini C, Ruggeri S, Mattioli S, Bernardini G, Macchioni L, Moretti E, et al. Long-term effects of silver nanoparticles on reproductive activity of rabbit buck. Syst Biol Reprod Med. 2014;60(3):143–50. doi:10.3109/19396368.2014.891163.
Sleiman HK, Romano RM, Oliveira CA De, Romano MA. Effects of Prepubertal Exposure to Silver Nanoparticles on Reproductive Parameters in Adult Male Wistar Rats. J Toxicol Environ Heal Part A. 2013;76(17):1023–32. doi:10.1080/15287394.2013.831723.
Iftikhar M, Noureen A, Uzair M, Jabeen F, Abdel Daim M, Cappello T. Perspectives of Nanoparticles in Male Infertility: Evidence for Induced Abnormalities in Sperm Production. Int J Environ Res Public Health. 2021;18(4):1758. doi:10.3390/ijerph18041758.
AOP-Wiki. AOP64:Glucocorticoid Receptor (GR) Mediated Adult Leydig Cell Dysfunction Leading to Decreased Male Fertility. https://aopwiki.org/aops/64.
AOP-Wiki. AOP323:PPARalpha Agonism Impairs Fish Reproduction. https://aopwiki.org/aops/323.
AOP-Wiki. AOP444:Ionizing radiation leads to reduced reproduction in Eisenia fetida via reduced spermatogenesis and cocoon hatchability. https://aopwiki.org/aops/444.
Spinu N, Bal-Price A, Cronin MTD, Enoch SJ, Madden JC, Worth AP. Development and analysis of an adverse outcome pathway network for human neurotoxicity. Arch Toxicol. 2019;93(10):2759–72. doi:10.1007/s00204-019-02551-1.
Coccini T. Gene Expression Changes in Rat Liver and Testes after Lung Instillation of a Low Dose of Silver Nanoparticles. J Nanomed Nanotechnol. 2014;05(05).
Elespuru R, Pfuhler S, Aardema MJ, Chen T, Doak SH, Doherty A, et al. Genotoxicity Assessment of Nanomaterials: Recommendations on Best Practices, Assays, and Methods. Toxicol Sci. 2018;164(2):391–416. doi:10.1093/toxsci/kfy100.
OECD. Test No. 476: In Vitro Mammalian Cell Gene Mutation Tests using the Hprt and xprt genes. https://www.oecd-ilibrary.org/environment/test-no-476-in-vitro-mammalian-cell-gene-mutation-tests-using-the-hprt-and-xprt-genes_9789264264809-en. OECD Guidelines for the Testing of Chemicals. OECD; 2016. (OECD Guidelines for the Testing of Chemicals, Section 4). doi:10.1787/9789264264809-en.
OECD. Test No. 490: In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene. https://www.oecd-ilibrary.org/environment/test-no-490-in-vitro-mammalian-cell-gene-mutation-tests-using-the-thymidine-kinase-gene_9789264264908-en. OECD Guidelines for the Testing of Chemicals. OECD; 2016. (OECD Guidelines for the Testing of Chemicals, Section 4). doi:10.1787/9789264264908-en.
OECD. Test No. 487: In Vitro Mammalian Cell Micronucleus Test. https://www.oecd-ilibrary.org/environment/test-no-487-in-vitro-mammalian-cell-micronucleus-test_9789264264861-en. OECD Guidelines for the Testing of Chemicals. OECD; 2016. (OECD Guidelines for the Testing of Chemicals, Section 4). doi:10.1787/9789264264861-en.
OECD. Test No. 473: In Vitro Mammalian Chromosomal Aberration Test. https://www.oecd-ilibrary.org/environment/test-no-473-in-vitro-mammalian-chromosomal-aberration-test_9789264264649-en. OECD Guidelines for the Testing of Chemicals. OECD; 2016. (OECD Guidelines for the Testing of Chemicals, Section 4). doi:10.1787/9789264264649-en.
OECD. Test No. 489: In Vivo Mammalian Alkaline Comet Assay. https://www.oecd-ilibrary.org/environment/test-no-489-in-vivo-mammalian-alkaline-comet-assay_9789264264885-en. OECD Guidelines for the Testing of Chemicals. OECD; 2016. (OECD Guidelines for the Testing of Chemicals, Section 4). doi:10.1787/9789264264885-en.
OECD. Test No. 488: Transgenic Rodent Somatic and Germ Cell Gene Mutation Assays. https://www.oecd-ilibrary.org/environment/test-no-488-transgenic-rodent-somatic-and-germ-cell-gene-mutation-assays_9789264203907-en. OECD Guidelines for the Testing of Chemicals. OECD; 2022. (OECD Guidelines for the Testing of Chemicals, Section 4). doi:10.1787/9789264203907-en.
OECD. Test No. 474: Mammalian Erythrocyte Micronucleus Test. https://www.oecd-ilibrary.org/environment/test-no-474-mammalian-erythrocyte-micronucleus-test_9789264224292-en. OECD Guidelines for the Testing of Chemicals. OECD; 2014. doi:10.1787/9789264224292-en.
OECD. Test No. 475: Mammalian Bone Marrow Chromosomal Aberration Test. OECD Guidelines for the Testing of Chemicals. OECD Publishing; 2014.
Ravanat JL, Breton J, Douki T, Gasparutto D, Grand A, Rachidi W, et al. Radiation-mediated formation of complex damage to DNA: a chemical aspect overview. Br J Radiol. 2014;87(1035). doi:10.1259/BJR.20130715.
Zhang XF, Gurunathan S, Kim JH. Effects of silver nanoparticles on neonatal testis development in mice. Int J Nanomedicine. 2015;10:6243–56.
OECD. Test No. 456: H295R Steroidogenesis Assay. https://www.oecd-ilibrary.org/environment/test-no-456-h295r-steroidogenesis-assay_9789264122642-en. OECD; 2022. p. 1–10. (OECD Guidelines for the Testing of Chemicals, Section 4). doi:10.1787/9789264122642-en.
Taieb J, Mathian B, Millot F, Patricot MC, Mathieu E, Queyrel N, et al. Testosterone Measured by 10 Immunoassays and by Isotope-Dilution Gas Chromatography–Mass Spectrometry in Sera from 116 Men, Women, and Children. Clin Chem. 2003;49(8):1381–95. doi:10.1373/49.8.1381.
Paduch DA, Brannigan RE, Fuchs EF, Kim ED, Marmar JL, Sandlow JI. The Laboratory Diagnosis of Testosterone Deficiency. Urology. 2014;83(5):980–8. doi:10.1016/j.urology.2013.12.024.
ECVAM. EURL ECVAM Database on Alternative Methods to Animal Experimentation (DB-ALM) | EU Science Hub. https://ec.europa.eu/jrc/en/scientific-tool/database-alternative-methods-animal-experimentation.
Gliga AR, Skoglund S, Odnevall Wallinder I, Fadeel B, Karlsson HL. Size-dependent cytotoxicity of silver nanoparticles in human lung cells: the role of cellular uptake, agglomeration and Ag release. Part Fibre Toxicol. 2014;11(1):11. doi:10.1186/1743-8977-11-11.
Hamilton RF, Buckingham S, Holian A. The Effect of Size on Ag Nanosphere Toxicity in Macrophage Cell Models and Lung Epithelial Cell Lines Is Dependent on Particle Dissolution. Int J Mol Sci 2014, Vol 15, Pages 6815-6830. 2014;15(4):6815–30. doi:10.3390/IJMS15046815.
Lafuente D, Garcia T, Blanco J, Sánchez DJ, Sirvent JJ, Domingo JL, et al. Effects of oral exposure to silver nanoparticles on the sperm of rats. Reprod Toxicol. 2016;60:133–9. doi:10.1016/j.reprotox.2016.02.007.
Miresmaeili SM, Halvaei I, Fesahat F, Fallah A, Nikonahad N, Taherinejad M. Evaluating the role of silver nanoparticles on acrosomal reaction and spermatogenic cells in rat. Iran J Reprod Med. 2013;11(5):423–30.
Gromadzka-Ostrowska J, Dziendzikowska K, Lankoff A, Dobrzyńska M, Instanes C, Brunborg G, et al. Silver nanoparticles effects on epididymal sperm in rats. Toxicol Lett. 2012;214(3):251–8. doi:10.1016/j.toxlet.2012.08.028.
Pourali P, Nouri M, Ameri F, Heidari T, Kheirkhahan N, Arabzadeh S, et al. Histopathological study of the maternal exposure to the biologically produced silver nanoparticles on different organs of the offspring. Naunyn Schmiedebergs Arch Pharmacol. 2020;393(5):867–78. doi:10.1007/s00210-019-01796-y.

No competing interests reported.

AdditionalFile1.docx
Additional file 1 (.docx): List of the 48 studies selected for the putative AOP construction

Download PDF

Journal Publication

published 05 Jan, 2023

Read the published version in Particle and Fibre Toxicology →

Editorial decision: Major revision
07 Nov, 2022
Reviews received at journal
03 Nov, 2022
Reviewers agreed at journal
31 Oct, 2022
Reviewers invited by journal
31 Oct, 2022
Editor assigned by journal
26 Oct, 2022
Submission checks completed at journal
24 Oct, 2022
First submitted to journal
17 Oct, 2022

You are reading this latest preprint version

Putative adverse outcome pathway for silver nanoparticle toxicity on male reproductive system: A literature review

Status:

Journal Publication

Version 1

Abstract

Figures

1. Background

2. Methods

2.1 Literature search

2.2 Quality assessment

2.3 Database analysis to identify potential KEs

3. Results And Discussion

3.1. Quality assessment of the identified studies

3.2. Dataset evaluation

3.3. Potential molecular initiating events

3.4. Identification and selection of key events

3.5. Network of AOPs

3.6. Experimental methods for assessing the KEs

Conclusion

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1