Martin F. Wilks

Systems Toxicology: From Basic Research to Risk Assessment

Systems Toxicology is the integration of classical toxicology with quantitative analysis of large networks of molecular and functional changes occurring across multiple levels of biological organization. Society demands increasingly close scrutiny of the potential health risks associated with exposure to chemicals present in our everyday life, leading to an increasing need for more predictive and accurate risk-assessment approaches. Developing such approaches requires a detailed mechanistic understanding of the ways in which xenobiotic substances perturb biological systems and lead to adverse outcomes. Thus, Systems Toxicology approaches offer modern strategies for gaining such mechanistic knowledge by combining advanced analytical and computational tools. Furthermore, Systems Toxicology is a means for the identification and application of biomarkers for improved safety assessments. In Systems Toxicology, quantitative systems-wide molecular changes in the context of an exposure are measured, and a causal chain of molecular events linking exposures with adverse outcomes (i.e., functional and apical end points) is deciphered. Mathematical models are then built to describe these processes in a quantitative manner. The integrated data analysis leads to the identification of how biological networks are perturbed by the exposure and enables the development of predictive mathematical models of toxicological processes. This perspective integrates current knowledge regarding bioanalytical approaches, computational analysis, and the potential for improved risk assessment.

Improvement in Survival after Paraquat Ingestion Following Introduction of a New Formulation in Sri Lanka

Background Pesticide ingestion is a common method of self-harm in the rural developing world. In an attempt to reduce the high case fatality seen with the herbicide paraquat, a novel formulation (INTEON) has been developed containing an increased emetic concentration, a purgative, and an alginate that forms a gel under the acid conditions of the stomach, potentially slowing the absorption of paraquat and giving the emetic more time to be effective. We compared the outcome of paraquat self-poisoning with the standard formulation against the new INTEON formulation following its introduction into Sri Lanka. Methods and Findings Clinical data were prospectively collected on 586 patients with paraquat ingestion presenting to nine large hospitals across Sri Lanka with survival to 3 mo as the primary outcome. The identity of the formulation ingested after October 2004 was confirmed by assay of blood or urine samples for a marker compound present in INTEON. The proportion of known survivors increased from 76/297 with the standard formulation to 103/289 with INTEON ingestion, and estimated 3-mo survival improved from 27.1% to 36.7% (difference 9.5%; 95% confidence interval [CI] 2.0%–17.1%; p = 0.002, log rank test). Cox proportional hazards regression analyses showed an approximately 2-fold reduction in toxicity for INTEON compared to standard formulation. A higher proportion of patients ingesting INTEON vomited within 15 min (38% with the original formulation to 55% with INTEON, p < 0.001). Median survival time increased from 2.3 d (95% CI 1.2–3.4 d) with the standard formulation to 6.9 d (95% CI 3.3–10.7 d) with INTEON ingestion (p = 0.002, log rank test); however, in patients who did not survive there was a comparatively smaller increase in median time to death from 0.9 d (interquartile range [IQR] 0.5–3.4) to 1.5 d (IQR 0.5–5.5); p = 0.02. Conclusions The survey has shown that INTEON technology significantly reduces the mortality of patients following paraquat ingestion and increases survival time, most likely by reducing absorption.

Linking pesticide exposure and dementia: What is the evidence?

There has been a steep increase in the prevalence of dementia in recent decades, which has roughly followed an increase in pesticide use some decades earlier, a time when it is probable that current dementia patients could have been exposed to pesticides. This raises the question whether pesticides contribute to dementia pathogenesis. Indeed, many studies have found increased prevalence of cognitive, behavioral and psychomotor dysfunction in individuals chronically exposed to pesticides. Furthermore, evidence from recent studies shows a possible association between chronic pesticide exposure and an increased prevalence of dementia, including Alzheimers disease (AD) dementia. At the cellular and molecular level, the mechanism of action of many classes of pesticides suggests that these compounds could be, at least partly, accountable for the neurodegeneration accompanying AD and other dementias. For example, organophosphates, which inhibit acetylcholinesterase as do the drugs used in treating AD symptoms, have also been shown to lead to microtubule derangements and tau hyperphosphorylation, a hallmark of AD. This emerging association is of considerable public health importance, given the increasing dementia prevalence and pesticide use. Here we review the epidemiological links between dementia and pesticide exposure and discuss the possible pathophysiological mechanisms and clinical implications of this association.

International STakeholder NETwork (ISTNET): creating a developmental neurotoxicity (DNT) testing road map for regulatory purposes

Abstract A major problem in developmental neurotoxicity (DNT) risk assessment is the lack of toxicological hazard information for most compounds. Therefore, new approaches are being considered to provide adequate experimental data that allow regulatory decisions. This process requires a matching of regulatory needs on the one hand and the opportunities provided by new test systems and methods on the other hand. Alignment of academically and industrially driven assay development with regulatory needs in the field of DNT is a core mission of the International STakeholder NETwork (ISTNET) in DNT testing. The first meeting of ISTNET was held in Zurich on 23–24 January 2014 in order to explore the concept of adverse outcome pathway (AOP) to practical DNT testing. AOPs were considered promising tools to promote test systems development according to regulatory needs. Moreover, the AOP concept was identified as an important guiding principle to assemble predictive integrated testing strategies (ITSs) for DNT. The recommendations on a road map towards AOP-based DNT testing is considered a stepwise approach, operating initially with incomplete AOPs for compound grouping, and focussing on key events of neurodevelopment. Next steps to be considered in follow-up activities are the use of case studies to further apply the AOP concept in regulatory DNT testing, making use of AOP intersections (common key events) for economic development of screening assays, and addressing the transition from qualitative descriptions to quantitative network modelling.

The interplay between environmental and genetic factors in Parkinson's disease susceptibility: the evidence for pesticides.

Parkinsons disease (PD) is a common neurodegenerative disorder characterized by dopaminergic neuron loss in the substantia nigra. Several genetic and environmental factors have been implicated in the pathogenesis of PD. Single risk factors are likely to exert relatively minor effects, whereas their interaction may prove to be sufficient to cause PD. In the present review we summarize current knowledge from human genetic association studies regarding the interaction between gene polymorphisms and pesticide exposure in the risk of PD. A number of genetic association studies have investigated joint effects between genes and pesticides on PD risk. They have provided some evidence that genetic susceptibility either in metabolism, elimination and transport of pesticides or in the extent of mitochondrial dysfunction, oxidative stress and neuronal loss may predispose individuals to PD if they have been exposed to pesticides. These findings confirm the importance of considering pesticide-gene interactions in future studies in order to gain a better understanding of the pathogenic mechanisms of PD.

Changes in the concentrations of creatinine, cystatin C and NGAL in patients with acute paraquat self-poisoning

An increase in creatinine >3 μmol/L/h has been suggested to predict death in patients with paraquat self-poisoning and the value of other plasma biomarkers of acute kidney injury has not been assessed. The aim of this study was to validate the predictive value of serial creatinine concentrations and to study the utility of cystatin C and neutrophil gelatinase-associated lipocalin (NGAL) as predictors of outcome in patients with acute paraquat poisoning. The rate of change of creatinine (dCr/dt) and cystatin C (dCyC/dt) concentrations were compared between survivors and deaths. Receiver-operating characteristic (ROC) curves were constructed to determine the best threshold for predicting death. Paraquat was detected in 20 patients and 7 of these died between 18 h and 20 days post-ingestion. The dCr/dt ROC curve had an area of 0.93 and the cut-off was >4.3 μmol/L/h (sensitivity 100%, specificity 85%, likelihood ratio 7). The dCyC/dt ROC curve had an area of 0.97 and the cutoff was >0.009 mg/L/h (sensitivity 100%, specificity 91%, likelihood ratio 11). NGAL did not separate survivors from deaths. Death due to acute paraquat poisoning is associated with changes in creatinine and cystatin concentrations. Further validation of these measurements is needed before they can be adopted in guiding intensive treatments.

Neurodevelopmental and neurobehavioural effects of polybrominated and perfluorinated chemicals: a systematic review of the epidemiological literature using a quality assessment scheme.

Concerns over effects of halogenated persistent environmental contaminants on the developing brain have been expressed for many years, and human biomonitoring has confirmed that low-level, prenatal and/or postnatal exposure of children to these chemicals is ubiquitous. Over the last decade there have been increasing reports in the epidemiological literature of the potential association of exposure to polybromo diphenylethers (PBDEs) and perfluorinated chemicals (PFCs) with neurodevelopmental and/or neurobehavioural effects in infants and children, such as adverse birth outcomes, cognitive deficits, developmental delay and attention deficit hyperactivity disorders (ADHD). However, direct evidence from epidemiology studies has been limited and contradictory. Given the general lack of comparability across studies in terms of design, conduct, methodology and reporting, we developed a checklist-type quality assessment scheme based on the STROBE guidelines and the proposed HONEES criteria, and conducted a systematic review of the epidemiological peer-reviewed literature published since 2006 on neurodevelopmental and/or neurobehavioural effects following prenatal and postnatal exposure to PBDEs and PFCs. We rated 7 of the 18 studies that met our inclusion criteria as being of high quality, 7 of moderate quality and 4 of low quality. Frequently observed shortcomings were the lack of consideration of confounding factors; uncertainties regarding exposure characterization; inadequate sample size; the lack of a clear dose-response; and the representativeness/generalizability of the results. Collectively, the epidemiological evidence does currently not support a strong causal association between PBDEs and PFCs and adverse neurodevelopmental and neurobehavioural outcomes in infants and children. However, despite their limitations, the studies raise questions that require further investigation through hypothesis-driven studies using more harmonized study designs and methodologies, more detailed exposure assessments and repeated testing with larger study populations.

Escitalopram causes fewer seizures in human overdose than citalopram.

Context. Seizures are a recognized complication of acute overdose with the racemic (1:1 ratio of R- and S-enantiomers) selective serotonin reuptake inhibitor antidepressant citalopram. Objective. We tested the hypothesis that escitalopram (the therapeutically active S‐enantiomer of citalopram) causes fewer seizures in overdose than citalopram at comparable doses of the S-enantiomer. Methods. Multicenter retrospective review of cases with citalopram and escitalopram overdose reported to German, Austrian, and Swiss Poisons Centers between 1997 and 2006. Results. 316 citalopram and 63 escitalopram cases were analyzed. Somnolence, nausea, vomiting, tachycardia, QT prolongation, and tremor occurred with similar frequency in both groups. There was a striking difference in the frequency of single and multiple seizures: 43 cases (13.5%) in the citalopram group and 1 case (1.6%) with a single seizure in the escitalopram group (p = 0.0065). Discussion and conclusions. At comparable ingested doses of the S-enantiomer, the symptom profile for citalopram and escitalopram intoxications is similar except for seizures that occur more frequently in citalopram than in escitalopram poisoning.

Perspectives for integrating human and environmental risk assessment and synergies with socio-economic analysis

For more than a decade, the integration of human and environmental risk assessment (RA) has become an attractive vision. At the same time, existing European regulations of chemical substances such as REACH (EC Regulation No. 1907/2006), the Plant Protection Products Regulation (EC regulation 1107/2009) and Biocide Regulation (EC Regulation 528/2012) continue to ask for sector-specific RAs, each of which have their individual information requirements regarding exposure and hazard data, and also use different methodologies for the ultimate risk quantification. In response to this difference between the vision for integration and the current scientific and regulatory practice, the present paper outlines five medium-term opportunities for integrating human and environmental RA, followed by detailed discussions of the associated major components and their state of the art. Current hazard assessment approaches are analyzed in terms of data availability and quality, and covering non-test tools, the integrated testing strategy (ITS) approach, the adverse outcome pathway (AOP) concept, methods for assessing uncertainty, and the issue of explicitly treating mixture toxicity. With respect to exposure, opportunities for integrating exposure assessment are discussed, taking into account the uncertainty, standardization and validation of exposure modeling as well as the availability of exposure data. A further focus is on ways to complement RA by a socio-economic assessment (SEA) in order to better inform about risk management options. In this way, the present analysis, developed as part of the EU FP7 project HEROIC, may contribute to paving the way for integrating, where useful and possible, human and environmental RA in a manner suitable for its coupling with SEA.

OECD/EFSA workshop on developmental neurotoxicity (DNT): The use of non-animal test methods for regulatory purposes

311 cluding: neural proliferation, differentiation, migration, neurite outgrowth, synaptogenesis, myelin formation, and neural network formation and function. Many of these human cell-based assays have been used to study small numbers of chemicals (n < 15; e.g., Harrill et al., 2011; He et al., 2012; Rempel et al., 2015; Baumann et al., 2016; Brown et al., 2016) or to derive mechanistic information for limited numbers of chemicals (e.g., Gassmann et al., 2010; Balmer et al., 2012; Balmer and Leist, 2014; Barenys et al., 2016). Only a few have been utilized to screen larger numbers (n > 15) of compounds (e.g., Stiegler et al., 2011; Zimmer et al., 2012; Culbreth et al., 2012; McConnell et al., 2012; Krug et al., 2013; Valdivia et al., 2014; Mundy et al., 2015; Hoelting et al., 2016; Nyffeler et al., 2016). On the scientific premise that alternative methods are available and can be assembled into a larger DNT screening battery, a joint OECD/EFSA workshop was held in Brussels on October 18 and 19, 2016 that aimed to facilitate the use of such methods in regulatory decision making. Specific objectives of this workshop were: 1. Development of a consensus that the proposed testing battery of alternative DNT methods is ready to be applied right now, and could be used in a fit-for-purpose manner for either screening and prioritization, or as a first starting point to conduct targeted testing in a tiered testing approach in the process of hazard identification and characterization for specific chemical risk assessment. 2. Identification of the next steps necessary to encourage the regulatory use of the alternative methods depending on their level of readiness. 3. Outline what could become an integrated approach to testing and assessment (IATA) for the purposes of screening and prioritization or hazard assessment. The meeting was co-chaired by Ellen Fritsche (Leibniz Research Institute for Environmental Medicine, IUF) and Kevin Crofton (US Environmental Protection Agency, US EPA). Meeting participants and their affiliations are reported in the supplementary file at https://doi.org/10.14573/altex.1701171s. Scientists from 15 countries across the world, representing stakeholders from regulatory agencies, non-governmental organizations (NGOs), academia and industry, reached a consensus that current data requirements for in vivo developmental neurotoxicity (DNT) testing are not sufficient to screen and characterize potentially hazardous compounds. In addition, there was agreement on the need to develop a standardized in vitro testing battery to generate additional data on the effects of chemicals on the developing nervous system. The need for more effective DNT screening is driven by the scientific fact that the developing nervous system might be more sensitive to exposures to some chemical classes of hazardous substances. In addition, recent societal concerns have been raised linking the rise in children’s neurodevelopmental impairments (e.g., learning disabilities) to chemical exposures. Despite a clear deficit in knowledge concerning DNT effects, only approximately 140 in vivo guideline studies (according to OECD 426 & EPA OPPTS 870.630) have been conducted to date, leaving a huge data gap on the DNT potential of chemicals within the universe of thousands of compounds present in industrial, agricultural and consumer products. This deficit is mainly due to the fact that currently accepted guideline studies are at present not mandatory data requirements and are extremely timeand costintensive. Additionally, they can result in methodological and scientific uncertainties. This includes the challenges in extrapolation of findings from rats to humans that result from timing differences in brain development, toxicokinetics, and inherent difficulties in the use of non-homologous functional tests (Tsuji and Crofton, 2012; Dorman et al., 2001; Kaufmann, 2003). For these reasons, DNT has been regarded as an area in need of the development of alternative methods in order to establish a timeand cost-efficient predictive testing strategy. A series of workshops held over the past decade (Lein et al., 2007; Crofton et al., 2011; Bal-Price et al., 2012, 2015a) have fostered the development of in vitro assays or methods using alternative model organisms that assess the impact of chemicals on cellular processes critical to normal brain development, inMeeting report