Development of an adverse outcome pathway network for nephrotoxicity.

Adverse outcome pathways (AOPs) were introduced in modern toxicology to provide evidence-based representations of the events and processes involved in the progression of toxicological effects across varying levels of the biological organisation to better facilitate the safety assessment of chemicals. AOPs offer an opportunity to address knowledge gaps and help to identify novel therapeutic targets. They also aid in the selection and development of existing and new in vitro and in silico test methods for hazard identification and risk assessment of chemical compounds. However, many toxicological processes are too intricate to be captured in a single, linear AOP. As a result, AOP networks have been developed to aid in the comprehension and placement of associated events underlying the emergence of related forms of toxicity-where complex exposure scenarios and interactions may influence the ultimate adverse outcome. This study utilised established criteria to develop an AOP network that connects thirteen individual AOPs associated with nephrotoxicity (as sourced from the AOP-Wiki) to identify several key events (KEs) linked to various adverse outcomes, including kidney failure and chronic kidney disease. Analysis of the modelled AOP network and its topological features determined mitochondrial dysfunction, oxidative stress, and tubular necrosis to be the most connected and central KEs. These KEs can provide a logical foundation for guiding the selection and creation of in vitro assays and in silico tools to substitute for animal-based in vivo experiments in the prediction and assessment of chemical-induced nephrotoxicity in human health.

Putative adverse outcome pathways for silver nanoparticle toxicity on mammalian male reproductive system: a literature review.

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 mechanisms 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-eight studies published between 2005 and 2022 were selected to identify the mechanisms of Ag NPs impact on reproductive function in human 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.

Identification of molecular initiating events (MIE) using chemical database analysis and nuclear receptor activity assays for screening potential inhalation toxicants.

An adverse outcome pathway (AOP) framework can facilitate the use of alternative assays in chemical regulations by providing scientific evidence. Previously, an AOP, peroxisome proliferative-activating receptor gamma (PPARγ) antagonism that leads to pulmonary fibrosis, was developed. Based on a literature search, PPARγ inactivation has been proposed as a molecular initiating event (MIE). In addition, a list of candidate chemicals that could be used in the experimental validation was proposed using toxicity database and deep learning models. In this study, the screening of environmental chemicals for MIE was conducted using in silico and in vitro tests to maximize the applicability of this AOP for screening inhalation toxicants. Initially, potential inhalation exposure chemicals that are active in three or more key events were selected, and in silico molecular docking was performed. Among the chemicals with low binding energy to PPARγ, nine chemicals were selected for validation of the AOP using in vitro PPARγ activity assay. As a result, rotenone, triorthocresyl phosphate, and castor oil were proposed as PPARγ antagonists and stressor chemicals of the AOP. Overall, the proposed tiered approach of the database-in silico-in vitro can help identify the regulatory applicability and assist in the development and experimental validation of AOP.

In vitro activity of a panel of per- and polyfluoroalkyl substances (PFAS), fatty acids, and pharmaceuticals in peroxisome proliferator-activated receptor (PPAR) alpha, PPAR gamma, and estrogen receptor assays.

Data demonstrate numerous per- and polyfluoroalkyl substances (PFAS) activate peroxisome proliferator-activated receptor alpha (PPARα), however, additional work is needed to characterize PFAS activity on PPAR gamma (PPARγ) and other nuclear receptors. We utilized in vitro assays with either human or rat PPARα or PPARγ ligand binding domains to evaluate 16 PFAS (HFPO-DA, HFPO-DA-AS, NBP2, PFMOAA, PFHxA, PFOA, PFNA, PFDA, PFOS, PFBS, PFHxS, PFOSA, EtPFOSA, and 4:2, 6:2 and 8:2 FTOH), 3 endogenous fatty acids (oleic, linoleic, and octanoic), and 3 pharmaceuticals (WY14643, clofibrate, and the metabolite clofibric acid). We also tested chemicals for human estrogen receptor (hER) transcriptional activation. Nearly all compounds activated both PPARα and PPARγ in both human and rat ligand binding domain assays, except for the FTOH compounds and PFOSA. Receptor activation and relative potencies were evaluated based on effect concentration 20% (EC20), top percent of max fold induction (pmaxtop), and area under the curve (AUC). HFPO-DA and HFPO-DA-AS were the most potent (lowest EC20, highest pmaxtop and AUC) of all PFAS in rat and human PPARα assays, being slightly less potent than oleic and linoleic acid, while NBP2 was the most potent in rat and human PPARγ assays. Only PFHxS, 8:2 and 6:2 FTOH exhibited hER agonism >20% pmax. In vitro measures of human and rat PPARα and PPARγ activity did not correlate with oral doses or serum concentrations of PFAS that induced increases in male rat liver weight from the National Toxicology Program 28-d toxicity studies. Data indicate that both PPARα and PPARγ activation may be molecular initiating events that contribute to the in vivo effects observed for many PFAS.

Adverse outcome pathway (AOP): α2u-globulin nephropathy and kidney tumors in male rats.

The National Research Council's vision of using adverse outcome pathways (AOPs) as a framework to assist with toxicity assessment for regulatory requirements of chemical assessment has continued to gain traction since its release in 2007. The need to expand the AOP knowledge base has gained urgency, with the U.S. Environmental Protection Agency's directive to eliminate reliance on animal toxicity testing by 2035. To meet these needs, our goal was to elucidate the AOP for male-rat-specific kidney cancer. Male-rat-specific kidney tumors occur through the ability of structurally diverse substances to induce α2u-globulin nephropathy (α2u-N), a well-studied mode of action (MoA) not relevant in humans that results in kidney tumor formation in male rats. An accepted AOP may help facilitate the differentiation from other kidney tumors MoAs. Following identification and review of relevant in vitro and in vivo literature, both the MIE and subsequent KEs were identified. Based on the weight of evidence from the various resources, the confidence in this AOP is high. Uses of this AOP include hazard identification, development of in vitro assays to determine if the MoA is through α2u-N and not relevant to humans resulting in decreased use of animals, and regulatory applications.

Nuclear Receptor and Stress Response Pathways Associated with Antineoplastic Agent-Induced Diarrhea.

In severe cases, antineoplastic agent-induced diarrhea may be life-threatening; therefore, it is necessary to determine the mechanism of toxicity and identify the optimal management. The mechanism of antineoplastic agent-induced diarrhea is still unclear but is often considered to be multifactorial. The aim of this study was to determine the molecular initiating event (MIE), which is the initial interaction between molecules and biomolecules or biosystems, and to evaluate the MIE specific to antineoplastic agents that induce diarrhea. We detected diarrhea-inducing drug signals based on adjusted odds ratios using the Food and Drug Administration Adverse Event Reporting System. We then used the quantitative structure-activity relationship platform of Toxicity Predictor to identify potential MIEs that are specific to diarrhea-inducing antineoplastic agents. We found that progesterone receptor antagonists were potential MIEs associated with diarrhea. The findings of this study may help improve the prediction and management of antineoplastic agent-induced diarrhea.

Towards a science-based testing strategy to identify maternal thyroid hormone imbalance and neurodevelopmental effects in the progeny - part II: how can key events of relevant adverse outcome pathways be addressed in toxicological assessments?

The current understanding of thyroid-related adverse outcome pathways (AOPs) with adverse neurodevelopmental outcomes in mammals has been reviewed. This served to establish if standard rodent toxicity test methods and in vitro assays allow identifying thyroid-related modes-of-action potentially leading to adverse neurodevelopmental outcomes, and the human relevance of effects - in line with the European Commission's Endocrine Disruptor Criteria. The underlying hypothesis is that an understanding of the key events of relevant AOPs provides insight into differences in incidence, magnitude, or species sensitivity of adverse outcomes. The rodent studies include measurements of serum thyroid hormones, thyroid gland pathology and neurodevelopmental assessments, but do not directly inform on specific modes-of-action. Opportunities to address additional non-routine parameters reflecting critical events of AOPs in toxicological assessments are presented. These parameters appear relevant to support the identification of specific thyroid-related modes-of-action, provided that prevailing technical limitations are overcome. Current understanding of quantitative key event relationships is often weak, but would be needed to determine if the triggering of a molecular initiating event will ultimately result in an adverse outcome. Also, significant species differences in all processes related to thyroid hormone signalling are evident, but the biological implications thereof (including human relevance) are often unknown. In conclusion, careful consideration of the measurement (e.g. timing, method) and interpretation of additional non-routine parameters is warranted. These findings will be used in a subsequent paper to propose a testing strategy to identify if a substance may elicit maternal thyroid hormone imbalance and potentially also neurodevelopmental effects in the progeny.

The steroid receptor coactivator 1 (SRC1) and 3 (SRC3) recruitment as a novel molecular initiating event of 4-n-nonylphenol in estrogen receptor α-mediated pathways.

Recently, the action of steroid receptor coactivators (SRCs) has been recognized to be an important molecular initiating event (MIE) in estrogenic adverse outcome pathways (AOPs). However, the role of SRCs in the molecular mechanisms of many highly concerned environmental estrogens remains poorly understood. In this study, the widely studied environmental estrogen, 4-n-nonylphenol (4-n-NP), was used as a typical pollutant to study SRCs recruitment in its estrogenic effects. In MCF7 cell proliferation (E-SCREEN) assay and MVLN cell assay, 4-n-NP showed significant estrogenic potency that involved an increase in estrogen receptor α (ERα), SRC1 and SRC3 transcript levels. Moreover, 4-n-NP was found to induce estrogen response element (ERE)-mediated activity via ERα in MVLN cells. To investigate the mechanism by which SRCs recruitment is induced by 4-n-NP-ERα, a coactivators recruitment assay was performed, and the results showed that 4-n-NP-ERα recruited both SRC1 and SRC3, whereas it failed to recruit SRC2. Similarly, it had no interaction with SRC2 in the ERα-SRC2 two-hybrid yeast assay. This is the first report to investigate the novel MIE of SRCs recruitment in 4-n-NP-ERα-induced estrogenicity. Overall, our results suggest that the action of 4-n-NP on estrogenic effects involves the following MIEs: the activation of ERα, the recruitment of SRC1 and SRC3, and the induction of ERE-mediated activity. The findings also provide valuable insights into the MIE associated with the different SRCs that are recruited in the adverse outcome pathways of environmental estrogens.

In Silico Molecular Docking and In Vivo Validation with Caenorhabditis elegans to Discover Molecular Initiating Events in Adverse Outcome Pathway Framework: Case Study on Endocrine-Disrupting Chemicals with Estrogen and Androgen Receptors.

Molecular docking is used to analyze structural complexes of a target with its ligand for understanding the chemical and structural basis of target specificity. This method has the potential to be applied for discovering molecular initiating events (MIEs) in the Adverse Outcome Pathway framework. In this study, we aimed to develop in silico⁻in vivo combined approach as a tool for identifying potential MIEs. We used environmental chemicals from Tox21 database to identify potential endocrine-disrupting chemicals (EDCs) through molecular docking simulation, using estrogen receptor (ER), androgen receptor (AR) and their homology models in the nematode Caenorhabditis elegans (NHR-14 and NHR-69, respectively). In vivo validation was conducted on the selected EDCs with C. elegans reproductive toxicity assay using wildtype N2, nhr-14, and nhr-69 loss-of-function mutant strains. The chemicals showed high binding affinity to tested receptors and showed the high in vivo reproductive toxicity, and this was further confirmed using the mutant strains. The present study demonstrates that the binding affinity from the molecular docking potentially correlates with in vivo toxicity. These results prove that our in silico⁻in vivo combined approach has the potential to be applied for identifying MIEs. This study also suggests the potential of C. elegans as useful in the in vivo model for validating the in silico approach.

Tributyltin: Advancing the Science on Assessing Endocrine Disruption with an Unconventional Endocrine-Disrupting Compound.

Tributyltin (TBT) has been recognized as an endocrine disrupting chemical (EDC) for several decades. However, only in the last decade, was its primary endocrine mechanism of action (MeOA) elucidated-interactions with the nuclear retinoid-X receptor (RXR), peroxisome proliferator-activated receptor γ (PPARγ), and their heterodimers. This molecular initiating event (MIE) alters a range of reproductive, developmental, and metabolic pathways at the organism level. It is noteworthy that a variety of MeOAs have been proposed over the years for the observed endocrine-type effects of TBT; however, convincing data for the MIE was provided only recently and now several researchers have confirmed and refined the information on this MeOA. One of the most important lessons learned from years of research on TBT concerns apparent species sensitivity. Several aspects such as the rates of uptake and elimination, chemical potency, and metabolic capacity are all important for identifying the most sensitive species for a given chemical, including EDCs. For TBT, much of this was discovered by trial and error, hence important relationships and important sensitive taxa were not identified until several decades after its introduction to the environment. As recognized for many years, TBT-induced responses are known to occur at very low concentrations for molluscs, a fact that has more recently also been observed in fish species. This review explores the MeOA and effects of TBT in different species (aquatic molluscs and other invertebrates, fish, amphibians, birds, and mammals) according to the OECD Conceptual Framework for Endocrine Disruptor Testing and Assessment (CFEDTA). The information gathered on biological effects that are relevant for populations of aquatic animals was used to construct Species Sensitivity Distributions (SSDs) based on No Observed Effect Concentrations (NOECs) and Lowest Observed Effect Concentrations (LOECs). Fish appear at the lower end of these distributions, showing that they are as sensitive as molluscs, and for some species, even more sensitive. Concentrations in the range of 1 ng/L for water exposure (10 ng/g for whole-body burden) have been shown to elicit endocrine-type responses, whereas mortality occurs at water concentrations ten times higher. Current screening and assessment methodologies as compiled in the OECD CFEDTA are able to identify TBT as a potent endocrine disruptor with a high environmental risk for the original use pattern. If those approaches had been available when TBT was introduced to the market, it is likely that its use would have been regulated sooner, thus avoiding the detrimental effects on marine gastropod populations and communities as documented over several decades.

DTNI: a novel toxicogenomics data analysis tool for identifying the molecular mechanisms underlying the adverse effects of toxic compounds.

Unravelling gene regulatory networks (GRNs) influenced by chemicals is a major challenge in systems toxicology. Because toxicant-induced GRNs evolve over time and dose, the analysis of global gene expression data measured at multiple time points and doses will provide insight in the adverse effects of compounds. Therefore, there is a need for mathematical methods for GRN identification from time-over-dose-dependent data. One of the current approaches for GRN inference is Time Series Network Identification (TSNI). TSNI is based on ordinary differential equations (ODE), describing the time evolution of the expression of each gene, which is assumed to be dependent on the expression of other genes and an external perturbation (i.e. chemical exposure). Here, we present Dose-Time Network Identification (DTNI), a method extending TSNI by including ODE describing how the expression of each gene evolves with dose, which is supposed to depend on the expression of other genes and the exposure time. We also adapted TSNI in order to enable inclusion of time-over-dose-dependent data from multiple compounds. Here, we show that DTNI outperforms TSNI in inferring a toxicant-induced GRN. Moreover, we show that DTNI is a suitable method to infer a GRN dose- and time-dependently induced by a group of compounds influencing a common biological process. Applying DTNI on experimental data from TG-GATEs, we demonstrate that DTNI provides in-depth information on the mode of action of compounds, in particular key events and potential molecular initiating events. Furthermore, DTNI also discloses several unknown interactions which have to be verified experimentally.

A weight of evidence assessment approach for adverse outcome pathways.

The adverse outcome pathway (AOP) is a framework to mechanistically link molecular initiating events to adverse biological outcomes. From a regulatory perspective, it is of crucial importance to determine the confidence for the AOP in question as well as the quality of data available in supporting this evaluation. A weight of evidence approach has been proposed for this task, but many of the existing frameworks for weight of evidence evaluation are qualitative and there is not clear guidance regarding how weight of evidence should be calculated for an AOP. In this paper we advocate the use of a subject matter expertise driven approach for weight of evidence evaluation based on criteria and metrics related to data quality and the strength of causal linkages between key events. As a demonstration, we notionally determine weight of evidence scores for two AOPs: Non-competitive ionotropic GABA receptor antagonism leading to epileptic seizures, and Antagonist-binding and stabilization of a co-repressor to the peroxisome proliferator-activated receptor α (PPARα) signaling complex ultimately causing starvation-like weight loss.

Putative adverse outcome pathways relevant to neurotoxicity.

The Adverse Outcome Pathway (AOP) framework provides a template that facilitates understanding of complex biological systems and the pathways of toxicity that result in adverse outcomes (AOs). The AOP starts with an molecular initiating event (MIE) in which a chemical interacts with a biological target(s), followed by a sequential series of KEs, which are cellular, anatomical, and/or functional changes in biological processes, that ultimately result in an AO manifest in individual organisms and populations. It has been developed as a tool for a knowledge-based safety assessment that relies on understanding mechanisms of toxicity, rather than simply observing its adverse outcome. A large number of cellular and molecular processes are known to be crucial to proper development and function of the central (CNS) and peripheral nervous systems (PNS). However, there are relatively few examples of well-documented pathways that include causally linked MIEs and KEs that result in adverse outcomes in the CNS or PNS. As a first step in applying the AOP framework to adverse health outcomes associated with exposure to exogenous neurotoxic substances, the EU Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) organized a workshop (March 2013, Ispra, Italy) to identify potential AOPs relevant to neurotoxic and developmental neurotoxic outcomes. Although the AOPs outlined during the workshop are not fully described, they could serve as a basis for further, more detailed AOP development and evaluation that could be useful to support human health risk assessment in a variety of ways.

Nuclear Receptors and Stress Response Pathways Associated with the Development of Oral Mucositis Induced by Antineoplastic Agents.

Oral mucositis (OM) is one of the common adverse events associated with cancer treatment that decreases the quality of life and affects treatment outcomes. However, the medications used to manage OM are generally only palliative, and our knowledge of the syndrome is limited. The etiology of the syndrome is thought to be complex and multifactorial. We investigated the trends and characteristics of OM and estimated molecular initiating events (MIEs) associated with the development of the syndrome using the FDA Adverse Event Reporting System. The study of trends and characteristics suggested that OM is significantly more likely to occur in females and nonelderly patients and is likely to be induced by protein kinase inhibitors such as afatinib and everolimus. Next, we used Toxicity Predictor, an in-house quantitative structure-activity relationship system, to estimate OM-associated MIEs. The results revealed that the agonist activity of the human pregnane X receptor, thyroid-stimulating hormone-releasing hormone receptor, and androgen receptor may be associated with OM development. Our study findings are expected to help avoid the risk of OM induction during the drug discovery process and clinical use of antineoplastic agents.

In vitro screening of per- and polyfluorinated substances (PFAS) for interference with seven thyroid hormone system targets across nine assays.

The US Environmental Protection Agency is evaluating the ecological and toxicological effects of per- and polyfluorinated chemicals. A number of perfluorinated chemicals have been shown to impact the thyroid axis in vivo suggesting that the thyroid hormone system is a target of these chemicals. The objective of this study was to evaluate the activity of 136 perfluorinated chemicals at seven key molecular initiating events (MIE) within the thyroid axis using nine in vitro assays. The potential MIE targets investigated are Human Iodothyronine Deiodinase 1 (hDIO1), Human Iodothyronine Deiodinase 2 (hDIO2), Human Iodothyronine Deiodinase 3 (hDIO3), Xenopus Iodothyronine Deiodinase (xDIO3); Human Iodotyrosine Deiodinase (hIYD), Xenopus Iodotyrosine Deiodinase (xIYD), Human Thyroid Peroxidase (hTPO); and the serum binding proteins Human Transthyretin (hTTR) and Human Thyroxine Binding Globulin (hTBG). Of the 136 PFAS chemicals tested, 85 had sufficient activity to produce a half-maximal effect concentration (EC50) in at least one of the nine assays. In general, most of these PFAS chemicals did not have strong potency towards the seven MIEs examined, apart from transthyretin binding, for which several PFAS had potency similar to the respective model inhibitor. These data sets identify potentially active PFAS chemicals to prioritize for further testing in orthogonal in vitro assays and at higher levels of biological organization to evaluate their capacity for altering the thyroid hormone system and causing potential adverse health and ecological effects.

Bioactivity assessment of organophosphate flame retardants via a dose-dependent yeast functional genomics approach.

Organophosphate flame retardants (OPFRs) have been widely detected in multiple environment media and have many adverse effects with complex toxicity mechanisms. However, the early molecular responses to OPFRs have not been fully elucidated, thereby making it difficult to assess their risks accurately. In this work, we systematically explored the point of departure (POD) of biological pathways at genome-wide level perturbed by 14 OPFRs with three substituents (alkyl, halogen, and aryl) using a dose-dependent functional genomics approach in Saccharomyces cerevisiae at 24 h exposure. Firstly, our results demonstrated that the overall biological potency at gene level (PODDRG20) ranged from 0.013 to 35.079 µM for 14 OPFRs, especially the tributyl phosphate (TnBP) exhibited the strongest biological potency with the least PODDRG20. Secondly, we found that structural characteristics of carbon number and logKow were significantly negatively correlated with POD, and carbon number and logKow also significantly affected lipid metabolism associated processes. Thirdly, these early biological pathways of OPFRs toxification were found to be involved in lipid metabolism, oxidative stress, DNA damage, MAPK signaling pathway, and amino acid and carbohydrate metabolism, among which the lipid metabolism was the most sensitive molecular response perturbed by most OPFRs. More importantly, we identified one resistant mutant strain with knockout of ERG2 (YMR202W) gene participated in steroid biosynthesis pathway, which can serve as a key yeast strain of OPFRs toxification. Overall, our study demonstrated an effective platform for accurately assessing OPFRs risks and provided a basis for further green OPFRs development.

Putative adverse outcome pathways of the male reproductive toxicity derived from toxicological studies of perfluoroalkyl acids.

Adverse outcome pathway (AOP) as a conceptual framework is a powerful tool in the field of toxicology to connect seemingly discrete events at different levels of biological organizations into an organized pathway from molecular interactions to whole organism toxicity. Based on numerous toxicological studies, eight AOPs for reproductive toxicity have been endorsed by the Organization for Economic Co-operation and Development (OECD) Task Force on Hazard Assessment. We have conducted a literature survey on the mechanistic studies on male reproductive toxicity of perfluoroalkyl acids (PFAAs), a class of global environmental contaminants with high persistence, bioaccumulation and toxicity. Using the AOP development strategy, five new AOPs for male reproductive toxicity were proposed here, namely (1) changes in membrane permeability leading to reduced sperm motility, (2) disruption of mitochondrial function leading to sperm apoptosis, (3) decreased gonadotropin-releasing hormone (GnRH) expression in hypothalamus leading to reduced testosterone production in male rats, (4) activation of the p38 signaling pathway leading to disruption of BTB in mice, (5) inhibition of p-FAK-Tyr407 activity leading to the destruction of BTB. The molecular initiating events in the proposed AOPs are different from those in the endorsed AOPs, which are either receptor activation or enzyme inhibition. Although some of the AOPs are still incomplete, they can serve as a building block upon which full AOPs can be developed and applied to not only PFAAs but also other chemical toxicants with male reproductive toxicity.

Delving into the molecular initiating event of cadmium toxification via the dose-dependent functional genomics approach in Saccharomyces cerevisiae.

Determining dose-response relationship is essential for comprehensively revealing chemical-caused effects on organisms. However, uncertainty and complexity of gene/protein interactions cause the inability of traditional toxicogenomic methods (e.g., transcriptomics, proteomics and metabolomics) to effectively establish the direct relationship between chemical exposure and genes. In this work, we built an effective dose-dependent yeast functional genomics approach, which can clearly identify the direct gene-chemical link in the process of cadmium (Cd) toxification from a genome-wide scale with wide range concentrations (0.83, 2.49, 7.48, 22.45, 67.34, 202.03 and 606.1 µM). Firstly, we identified 220 responsive strains, and found that 142, 110, 91, 34, 8, 0 and 0 responsive strains can be respectively modulated by seven different Cd exposure concentrations ranging from high to low. Secondly, our results demonstrated that these genes induced by the high Cd exposure were mainly enriched in the process of cell autophagy, but ones caused by the low Cd exposure were primarily involved in oxidative stress. Thirdly, we found that the top-ranked GO biological processes with the lowest point of departure (POD) were transmembrane transporter complex and mitochondrial respiratory chain complex III, suggesting that mitochondrion might be the toxicity target of Cd. Similarly, nucleotide excision repair was ranked first in KEGG pathway with the least POD, indicating that this dose-dependent functional genomics approach can effectively detect the molecular initiating event (MIE) of cadmium toxification. Fourthly, we identified four key mutant strains (RIP1, QCR8, CYT1 and QCR2) as biomarkers for Cd exposure. Finally, the dose-dependent functional genomics approach also performed well in identifying MIE for additional genotoxicity chemical 4-nitroquinoline-1-oxide (4-NQO) data. Overall, our study developed a dose-dependent functional genomics approach, which is powerful to delve into the MIE of chemical toxification and is beneficial for guiding further chemical risk assessment.

Characterizing temporal variability and repeatability of dose-dependent functional genomics approach for evaluating triclosan toxification.

Dose-dependent functional genomics approach has shown great advantage in identifying the molecular initiating event (MIE) of chemical toxification and yielding point of departure (POD) at genome-wide scale. However, POD variability and repeatability derived from experimental design (settings of dose, replicate number, and exposure time) has not been fully determined. In this work, we evaluated POD profiles perturbed by triclosan (TCS) using dose-dependent functional genomics approach in Saccharomyces cerevisiae at multiple time points (9 h, 24 h and 48 h). The full dataset (total 9 concentrations with 6 replicates per treatment) at 9 h was subsampled 484 times to generate subsets of 4 dose groups (Dose A - Dose D with varied concentration range and spacing) and 5 replicate numbers (2 reps - 6 reps). Firstly, given the accuracy of POD and the experimental cost, the POD profiles from 484 subsampled datasets demonstrated that the Dose C group (space narrow at high concentrations and wide dose range) with three replicates was best choice at both gene and pathway levels. Secondly, the variability of POD was found to be relatively robustness and stability across different experimental designs, but POD was more dependent on the dose range and interval than the number of replicates. Thirdly, MIE of TCS toxification was identified to be the glycerophospholipid metabolism pathway at all-time points, supporting the ability of our approach to accurately recognize MIE of chemical toxification at both short- and long-term exposure. Finally, we identified and validated 13 key mutant strains involved in MIE of TCS toxification, which could serve as biomarkers for TCS exposure. Taken together, our work evaluated the repeatability of dose-dependent functional genomics approach and the variability of POD and MIE of TCS toxification, which will benefit the experimental design for future dose-dependent functional genomics study.

Prediction of Molecular Initiating Events for Adverse Outcome Pathways Using High-Throughput Identification of Chemical Targets.

The impact of exposure to multiple chemicals raises concerns for human and environmental health. The adverse outcome pathway method offers a framework to support mechanism-based assessment in environmental health starting by describing which mechanisms are triggered upon interaction with different stressors. The identification of the molecular initiating event and the molecular interaction between a chemical and a protein target is still a challenge for the development of adverse outcome pathways. The cellular response to chemical exposure studied with omics could not directly identify the protein targets. However, recent mass spectrometry-based methods are offering a proteome-wide identification of protein targets interacting with s but unrevealing a molecular initiating event from a set of targets is still dependent on available knowledge. Here, we directly coupled the target identification findings from the proteome integral solubility alteration assay with an analytical hierarchy process for the prediction of a prioritized molecular initiating event. We demonstrate the applicability of this combination of methodologies with a test compound (TCDD), and it could be further studied and integrated into AOPs. From the eight protein targets identified by the proteome integral solubility alteration assay after analyzing 2824 human hepatic proteins, the analytical hierarchy process can select the most suitable protein for an AOP. Our combined method solves the missing links between high-throughput target identification and prediction of the molecular initiating event. We anticipate its utility to decipher new molecular initiating events and support more sustainable methodologies to gain time and resources in chemical assessment.