Sub-lethal toxicity of five disinfection by-products on microalgae determined by flow cytometry - Lines of evidence for adverse outcome pathways.

Standardised tests are often used to determine the ecotoxicity of chemicals and focus mainly on one or a few generic endpoints (e.g. mortality, growth), but information on the sub-cellular processes leading to these effects remain usually partial or missing. Flow cytometry (FCM) can be a practical tool to study the physiological responses of individual cells (such as microalgae) exposed to a stress via the use of fluorochromes and their morphology and natural autofluorescence. This work aimed to assess the effects of five chlorine-based disinfection by-products (DBPs) taken individually on growth and sub-cellular endpoints of the green microalgae Raphidocelis subcapitata. These five DBPs, characteristic of a chlorinated effluent, are the following monochloroacetic acid (MCAA), dichloroacetic acid (DCAA), trichloroacetic acid (TCAA), bromochloroacetic acid (BCAA) and 1,1-dichloropropan-2-one (1,1-DCP). Results showed that 1,1-DCP had the strongest effect on growth inhibition (EC50 = 1.8 mg.L-1), followed by MCAA, TCAA, BCAA and DCAA (EC50 of 10.1, 15.7, 27.3 and 64.5 mg.L-1 respectively). Neutral lipid content, reactive oxygen species (ROS) formation, red autofluorescence, green autofluorescence, size and intracellular complexity were significantly affected by the exposure to the five DBPs. Only mitochondrial membrane potential did not show any variation. Important cellular damages (>10%) were observed for only two of the chemicals (BCAA and 1,1-DCP) and were probably due to ROS formation. The most sensitive and informative sub-lethal parameter studied was metabolic activity (esterase activity), for which three types of response were observed. Combining all this information, an adverse outcome pathways framework was proposed to explain the effect of the targeted chemicals on R. subcapitata. Based on these results, both FCM sub-cellular analysis and conventional endpoint of algal toxicity were found to be complementary approaches.

Application of the adverse outcome pathway concept for investigating developmental neurotoxicity potential of Chinese herbal medicines by using human neural progenitor cells in vitro.

Adverse outcome pathways (AOPs) are organized sequences of key events (KEs) that are triggered by a xenobiotic-induced molecular initiating event (MIE) and summit in an adverse outcome (AO) relevant to human or ecological health. The AOP framework causally connects toxicological mechanistic information with apical endpoints for application in regulatory sciences. AOPs are very useful to link endophenotypic, cellular endpoints in vitro to adverse health effects in vivo. In the field of in vitro developmental neurotoxicity (DNT), such cellular endpoints can be assessed using the human "Neurosphere Assay," which depicts different endophenotypes for a broad variety of neurodevelopmental KEs. Combining this model with large-scale transcriptomics, we evaluated DNT hazards of two selected Chinese herbal medicines (CHMs) Lei Gong Teng (LGT) and Tian Ma (TM), and provided further insight into their modes-of-action (MoA). LGT disrupted hNPC migration eliciting an exceptional migration endophenotype. Time-lapse microscopy and intervention studies indicated that LGT disturbs laminin-dependent cell adhesion. TM impaired oligodendrocyte differentiation in human but not rat NPCs and activated a gene expression network related to oxidative stress. The LGT results supported a previously published AOP on radial glia cell adhesion due to interference with integrin-laminin binding, while the results of TM exposure were incorporated into a novel putative, stressor-based AOP. This study demonstrates that the combination of phenotypic and transcriptomic analyses is a powerful tool to elucidate compounds' MoA and incorporate the results into novel or existing AOPs for a better perception of the DNT hazard in a regulatory context.

Application of the adverse outcome pathway to identify molecular changes in prenatal brain programming induced by IUGR: Discoveries after EGCG exposure.

Following a multi-disciplinary approach integrating information from several experimental models we have collected new evidence supporting, expanding and redesigning the AOP "Disrupted laminin/int-ß1 interaction leading to decreased cognitive function". Investigations in vitro in rabbit and rat neurospheres and in vivo in mice exposed to EGCG (epigallocatechin-gallate) during neurodevelopment are combined with in vitro evaluations in neural progenitor cells overexpressing int-ß1 and literature information from int-ß1 deficiency models. We have discovered for the first time that neural progenitor cells from intrauterine growth restricted (IUGR) animals overexpress int-ß1 at gene and protein level and due to this change in prenatal brain programming they respond differently than control neurospheres to the exposure of EGCG, a compound triggering neural progenitor cell migration alterations. We have also identified that EGCG developmental exposure has deleterious effects on neuronal branching and arborization in vitro and in vivo. Our results warn that a thorough developmental neurotoxicity characterization of this and other catechin-based food supplements is needed before recommending their consumption during pregnancy.

An adverse outcome pathway-based approach to assess aurantio-obtusin-induced hepatotoxicity.

Cassiae semen (CS), a traditional Chinese medicine, has various bioactivities in preclinical and clinical practice. Aurantio-obtusin (AO) is a major anthraquinone (AQ) ingredient derived from CS, and has drawn public concerns over its potential hepatotoxicity. We previously found that AO induces hepatic necroinflammation by activating NOD-like receptor protein 3 inflammasome signaling. However, the mechanisms contributing to AO-motivated hepatotoxicity remain unclear. Herein, we evaluated hepatotoxic effects of AO on three liver cell lines by molecular and biochemical analyses. We found that AO caused cell viability inhibition and biochemistry dysfunction in the liver cells. Furthermore, AO elevated reactive oxygen species (ROS), followed by mitochondrial dysfunction (decreases in mitochondrial membrane potential and adenosine triphosphate) and apoptosis (increased Caspase-3, Cleaved caspase-3, Cytochrome c and Bax expression, and decreased Bcl-2 expression). We also found that AO increased the lipid peroxidation (LPO) and enhanced ferroptosis by activating cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA)-cAMP response element-binding (CREB) pathway (increases in PKA, p-CREB, acyl-CoA synthetase long chain family member 4). Based on these results, we used an AOP framework to explore the mechanisms underlying AO's hepatotoxicity. It starts from molecular initiating event (ROS), and follows two critical toxicity pathways (i.e., mitochondrial dysfunction-mediated apoptosis and LPO-enhanced ferroptosis) over a series of key events (KEs) to the adverse outcome of hepatotoxicity. The results of an assessment confidence in the adverse outcome pathway (AOP) framework supported the evidence concordance in dose-response, temporal and incidence relationships between KEs in AO-induced hepatotoxicity. This study's findings offer a novel toxicity pathway network for AO-caused hepatotoxicity.

Review of Knowledge of Uranium-Induced Kidney Toxicity for the Development of an Adverse Outcome Pathway to Renal Impairment.

An adverse outcome pathway (AOP) is a conceptual construct of causally and sequentially linked events, which occur during exposure to stressors, with an adverse outcome relevant to risk assessment. The development of an AOP is a means of identifying knowledge gaps in order to prioritize research assessing the health risks associated with exposure to physical or chemical stressors. In this paper, a review of knowledge was proposed, examining experimental and epidemiological data, in order to identify relevant key events and potential key event relationships in an AOP for renal impairment, relevant to stressors such as uranium (U). Other stressors may promote similar pathways, and this review is a necessary step to compare and combine knowledge reported for nephrotoxicants. U metal ions are filtered through the glomerular membrane of the kidneys, then concentrate in the cortical and juxtaglomerular areas, and bind to the brush border membrane of the proximal convoluted tubules. U uptake by epithelial cells occurs through endocytosis and the sodium-dependent phosphate co-transporter (NaPi-IIa). The identified key events start with the inhibition of the mitochondria electron transfer chain and the collapse of mitochondrial membrane potential, due to cytochrome b5/cytochrome c disruption. In the nucleus, U directly interacts with negatively charged DNA phosphate, thereby inducing an adduct formation, and possibly DNA strand breaks or cross-links. U also compromises DNA repair by inhibiting zing finger proteins. Thereafter, U triggers the Nrf2, NF-κB, or endoplasmic reticulum stress pathways. The resulting cellular key events include oxidative stress, DNA strand breaks and chromosomal aberrations, apoptosis, and pro-inflammatory effects. Finally, the main adverse outcome is tubular damage of the S2 and S3 segments of the kidneys, leading to tubular cell death, and then kidney failure. The attribution of renal carcinogenesis due to U is controversial, and specific experimental or epidemiological studies must be conducted. A tentative construction of an AOP for uranium-induced kidney toxicity and failure was proposed.

AOP Report: Inhibition of Chitin Synthase 1 Leading to Increased Mortality in Arthropods.

Arthropods (including insects, crustaceans, and arachnids) rely on the synthesis of chitin to complete their life cycles (Merzendorfer 2011). The highly conserved chitin synthetic process and the absence of this process in vertebrates make it an exploitable target for pest management and veterinary medicines (Merzendorfer 2013; Junquera et al. 2019). Susceptible, nontarget organisms, such as insects and aquatic invertebrates, exposed to chitin synthesis inhibitors may suffer population declines, which may have a negative impact on ecosystems and associated services. Hence, it is important to properly identify, prioritize, and regulate relevant chemicals posing potential hazards to nontarget arthropods. The need for a more cost-efficient and mechanistic approach in risk assessment has been clearly evident and triggered the development of the adverse outcome pathway (AOP) framework (Ankley et al. 2010). An AOP links a molecular initiating event (MIE) through key events (KEs) to an adverse outcome. The mechanistic understanding of the underlying toxicological processes leading to a regulation-relevant adverse outcome is necessary for the utilization of new approach methodologies (NAMs) and efficient coverage of wider chemical and taxonomic domains. In the last decade, the AOP framework has gained traction and expanded within the (eco)toxicological research community. However, there exists a lack of mature invertebrate AOPs describing molting defect-associated mortality triggered by direct inhibition of relevant enzymes in the chitin biosynthetic pathway (chitin synthesis inhibitors) or interference with associated endocrine systems by environmental chemicals (endocrine disruptors). Arthropods undergo molting to grow and reproduce (Heming 2018). This process is comprised of the synthesis of a new exoskeleton, followed by the exuviation of the old exoskeleton (Reynolds 1987). The arthropod exoskeleton (cuticle) can be divided into 2 layers, the thin and nonchitinous epicuticle, which is the outermost layer of the cuticle, and the underlying chitinous procuticle. A single layer of epithelial cells is responsible for the synthesis and secretion of both cuticular layers (Neville 1975). The cuticle protects arthropods from predators and desiccation, acts as a physical barrier against pathogens, and allows for locomotion by providing support for muscular function (Vincent and Wegst 2004). Because the procuticle mainly consists of chitin microfibrils embedded in a matrix of cuticular proteins supplemented by lipids and minerals in insects (Muthukrishnan et al. 2012) and crustaceans (Cribb et al. 2009; Nagasawa 2012), chitin is a determinant factor for the appropriate composition of the cuticle and successful molting (Cohen 2001). A detailed overview of the endocrine mechanisms regulating chitin synthesis is given in Supplemental Data, Figure S1. The shedding of the old exoskeleton in insects is mediated by a sequence of distinct muscular contractions, the ecdysis motor program (EMP; Ayali 2009; Song et al. 2017a). Like the expression of chitin synthase isoform 1 (CHS-1), the expression of peptide hormones regulating the EMP is also controlled by ecdysteroids (Antoniewski et al. 1993; Gagou et al. 2002; Ayali 2009). Cuticular chitin is polymerized from uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) by the transmembrane enzyme CHS-1, which is localized in the epithelial plasma membrane in insects (Locke and Huie 1979; Binnington 1985; Merzendorfer and Zimoch 2003; Merzendorfer 2006). Because crustaceans are also dependent on the synthesis of chitin, the underlying mechanisms are believed to be similar, although less is known about different CHS isoforms and their localization (Rocha et al. 2012; Qian et al. 2014; Uddowla et al. 2014; Harðardóttir et al. 2019). Disruption of either chitin synthesis or the upstream endocrine pathways can lead to lethal molting disruption (Arakawa et al. 2008; Merzendorfer et al. 2012; Song et al. 2017a, 2017b). In the case of chitin synthesis inhibition, molting disruption can be referred to as "premature molting." If ecdysis cannot be completed because of decreased chitin synthesis, the organism may not successfully molt. Even if ecdysis can be completed on inhibition of chitin synthesis, the organism may not survive because of the poor integrity of the new cuticle. These effects are observed in arthropods following molting, which fail to survive subsequent molts (Arakawa et al. 2008; Chen et al. 2008) or animals being stuck in their exuviae (Wang et al. 2019) and ultimately dying as a result of insufficient food or oxygen intake (Camp et al. 2014; Song et al. 2017a). The term "premature molting" is used to differentiate from the term "incomplete ecdysis," which describes inhibition of ecdysis on a behavioral level, namely through reduction of the EMP (Song et al. 2017a). The present AOP describes molting-associated mortality through direct inhibition of the enzyme CHS-1. It expands the small but increasing number of invertebrate AOPs that have relevance to arthropods, the largest phylum within the animal kingdom (Bar-On et al. 2018). The development of this AOP will be useful in further research and regulatory initiatives related to assessment of CHS inhibitors and identification of critical knowledge gaps and may suggest new strategies for ecotoxicity testing efforts. Environ Toxicol Chem 2021;40:2112-2120. © 2021 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Understanding COVID-19 through adverse outcome pathways - 2nd CIAO AOP Design Workshop.

The CIAO project (Modelling the Pathogenesis of COVID-19 using the Adverse Outcome Pathway framework) aims at a holistic assembly of knowledge to deliver a truly transdisciplinary description of the entire COVID-19 physiopathology starting with the initial contact with the SARS-CoV-2 virus and ending with one or several adverse outcomes, e.g., respiratory failure. On 27-28 January 2021, a group of 50+ scientists from numerous organizations around the world met in the 2nd CIAO AOP Design Workshop to discuss the depiction of the COVID-19 disease process as a series of key events (KEs) in a network of AOPs. During the workshop, 74 such KEs forming 13 AOPs were identified, covering COVID-19 manifestations that affect the respiratory, neurological, liver, cardiovascular, kidney and gastrointestinal systems. Modulating factors influencing the course and severity of the disease were also addressed, as was a possible extension of the investigations beyond purely biological phenomena. The workshop ended with the creation of seven working groups, which will further elaborate on the AOPs to be presented and discussed in the 3rd CIAO workshop on 28-29 April 2021.

Predicting the safety of medicines in pregnancy: A workshop report.

The framework for developmental toxicity testing has remained largely unchanged for over 50 years and although it remains invaluable in assessing potential risks in pregnancy, knowledge gaps exist, and some outcomes do not necessarily correlate with clinical experience. Advances in omics, in silico approaches and alternative assays are providing opportunities to enhance our understanding of embryo-fetal development and the prediction of potential risks associated with the use of medicines in pregnancy. A workshop organised by the Medicines and Healthcare products Regulatory Agency (MHRA), "Predicting the Safety of Medicines in Pregnancy - a New Era?", was attended by delegates representing regulatory authorities, academia, industry, patients, funding bodies and software developers to consider how to improve the quality of and access to nonclinical developmental toxicity data and how to use this data to better predict the safety of medicines in human pregnancy. The workshop delegates concluded that based on comparative data to date alternative methodologies are currently no more predictive than conventional methods and not qualified for use in regulatory submissions. To advance the development and qualification of alternative methodologies, there is a requirement for better coordinated multidisciplinary cross-sector interactions coupled with data sharing. Furthermore, a better understanding of human developmental biology and the incorporation of this knowledge into the development of alternative methodologies is essential to enhance the prediction of adverse outcomes for human development. The output of the workshop was a series of recommendations aimed at supporting multidisciplinary efforts to develop and validate these alternative methodologies.

Adverse outcome pathway for aminoglycoside ototoxicity in drug-resistant tuberculosis treatment.

Individuals treated for multidrug-resistant tuberculosis (MDR-TB) with aminoglycosides (AGs) in resource-limited settings often experience permanent hearing loss. However, AG ototoxicity has never been conceptually integrated or causally linked to MDR-TB patients' pre-treatment health condition. We sought to develop a framework that examines the relationships between pre-treatment conditions and AG-induced hearing loss among MDR-TB-infected individuals in sub-Saharan Africa. The adverse outcome pathway (AOP) approach was used to develop a framework linking key events (KEs) within a biological pathway that results in adverse outcomes (AO), which are associated with chemical perturbation of a molecular initiating event (MIE). This AOP describes pathways initiating from AG accumulation in hair cells, sound transducers of the inner ear immediately after AG administration. After administration, the drug catalyzes cellular oxidative stress due to overproduction of reactive oxygen species. Since oxidative stress inhibits mitochondrial protein synthesis, hair cells undergo apoptotic cell death, resulting in irreversible hearing loss (AO). We identified the following pre-treatment conditions that worsen the causal linkage between MIE and AO: HIV, malnutrition, aging, noise, smoking, and alcohol use. The KEs are: (1) nephrotoxicity, pre-existing hearing loss, and hypoalbuminemia that catalyzes AG accumulation; (2) immunodeficiency and antioxidant deficiency that trigger oxidative stress pathways; and (3) co-administration of mitochondrial toxic drugs that hinder mitochondrial protein synthesis, causing apoptosis. This AOP clearly warrants the development of personalized interventions for patients undergoing MDR-TB treatment. Such interventions (i.e., choosing less ototoxic drugs, scheduling frequent monitoring, modifying nutritional status, avoiding poly-pharmacy) will be required to limit the burden of AG ototoxicity.

Development of the Adverse Outcome Pathway (AOP): Chronic binding of antagonist to N-methyl-d-aspartate receptors (NMDARs) during brain development induces impairment of learning and memory abilities of children.

The Adverse Outcome Pathways (AOPs) are designed to provide mechanistic understanding of complex biological systems and pathways of toxicity that result in adverse outcomes (AOs) relevant to regulatory endpoints. AOP concept captures in a structured way the causal relationships resulting from initial chemical interaction with biological target(s) (molecular initiating event) to an AO manifested in individual organisms and/or populations through a sequential series of key events (KEs), which are cellular, anatomical and/or functional changes in biological processes. An AOP provides the mechanistic detail required to support chemical safety assessment, the development of alternative methods and the implementation of an integrated testing strategy. An example of the AOP relevant to developmental neurotoxicity (DNT) is described here following the requirements of information defined by the OECD Users' Handbook Supplement to the Guidance Document for developing and assessing AOPs. In this AOP, the binding of an antagonist to glutamate receptor N-methyl-d-aspartate (NMDAR) receptor is defined as MIE. This MIE triggers a cascade of cellular KEs including reduction of intracellular calcium levels, reduction of brain derived neurotrophic factor release, neuronal cell death, decreased glutamate presynaptic release and aberrant dendritic morphology. At organ level, the above mentioned KEs lead to decreased synaptogenesis and decreased neuronal network formation and function causing learning and memory deficit at organism level, which is defined as the AO. There are in vitro, in vivo and epidemiological data that support the described KEs and their causative relationships rendering this AOP relevant to DNT evaluation in the context of regulatory purposes.

Ninety-day post-operative morbidity and mortality using the National Cancer Institute's common terminology criteria for adverse events better describe post-operative outcome after cytoreductive surgery and hyperthermic intraperitoneal chemotherapy.

BACKGROUND: The post-operative morbidity and mortality after CRS-HIPEC has been widely evaluated. However, there is a major discrepancy between rates reported due to different metrics and time of analysis used. OBJECTIVE: To evaluate the legitimacy of 90-day morbidity and mortality based on the National Cancer Institute's Common Terminology Criteria for Adverse Events (NCI-CTCAE) v4.0 classification as criteria of quality for cytoreductive surgery combined with hyperthermic intraperitoneal chemotherapy (CRS-HIPEC). METHODS: A prospective database of all patients undergoing CRS-HIPEC for peritoneal carcinomatosis between 2004 and 2015 was queried for 90-day morbidity and mortality and survival. RESULTS: Among 881 patients, the 90-day major complication rate based on NCI-CTCAE classification and Clavien-Dindo's classification were 51% (n = 447 patients) and 25% (n = 222 patients), respectively. Among patients who presented with a 90-day complication based on the NCI-CTCAE classification, 50% (n = 225 patients) presented a medical complication not reported by Clavien-Dindo's classification. After surgery, 24 patients (2.7%) died of post-operative complications, for only 10 (42%) of them the death occurred within 30-day after surgery. Occurrence of major complication based on either NCI-CTCAE classification, Clavien-Dindo's classification or the medical complication not reported by Clavien-Dindo's classification all negatively impacts the overall survival. CONCLUSION: Among commonly reported morbidity's classification, 90-day morbidity based on NCI-CTCAE classification represents a legitimate metric of CRS-HIPEC quality. Post-operative morbidity after CRS-HIPEC should be reported using 90-day NCI-CTCAE classification.

Pneumotoxicity associated with immune checkpoint inhibitor therapies.

PURPOSE OF REVIEW: Immune checkpoint inhibitor therapies represent a new paradigm in cancer therapeutics, in which the targets are not the cancer cells, but the body's own immune system. Harnessing the immune system to better fight cancer has generated a unique spectrum of immune-related adverse events (IrAEs) that effect virtually every major organ system. Although lung involvement is less common than other forms of IrAEs, its consequences are potentially lethal. This review focuses on the evolving spectrum of lung toxicities associated with the two major classes of immune checkpoint inhibitor therapies, cytotoxic T-cell ligand-4, and programed cell death-1 (PDL-1). RECENT FINDINGS: Lung injury was not reported in the earliest clinical trials of immune checkpoint inhibitors. More recent studies, however, have described unique radiographic and clinical toxicity profiles that differ significantly from lung injury patterns associated with conventional cytotoxic therapies. The pathophysiologic mechanisms of immune-related lung injury, its radiographic and clinical disease spectrum, associated risk factors, and optimal treatment strategies remain poorly understood. SUMMARY: Adverse immune-mediated lung events are increasingly recognized as unique and potentially life-threatening sequelae of checkpoint inhibitor therapies. Early recognition of symptoms and radiographic abnormalities is essential to proper management and successful outcome.