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Being critical mediators of liver homeostasis, connexins and their channels are frequently involved in liver toxicity. In the current paper, specific attention is paid to actions of hepatotoxic drugs on these communicative structures. In a first part, an overview is provided on the structural, regulatory and functional properties of connexin-based channels in the liver. In the second part, documented effects of acetaminophen, hypolipidemic drugs, phenobarbital and methapyriline on connexin signaling are discussed. Furthermore, the relevance of this subject for the fields of clinical and in vitro toxicology is demonstrated. Relevance for patients: The role of connexin signaling in drug-induced hepatotoxicity may be of high clinical relevance, as it offers perspectives for the therapeutic treatment of such insults by interfering with connexin channel opening.

The neutral red uptake assay is a cell viability assay that allows in vitro quantification of xenobiotic-induced cytotoxicity. The assay relies on the ability of living cells to incorporate and bind neutral red, a weak cationic dye, in lysosomes. As such, cytotoxicity is expressed as a concentration-dependent reduction of the uptake of neutral red after exposure to the xenobiotic under investigation. The neutral red uptake assay is mainly used for hazard assessment in in vitro toxicology applications. This method has also been introduced in regulatory recommendations as part of 3T3-NRU-phototoxicity-assay, which was regulatory accepted in all EU member states in 2000 and in the OECD member states in 2004 as a test guideline (TG 432). The present protocol describes the neutral red uptake assay using the human hepatoma cell line HepG2, which is often employed as an alternative in vitro model for human hepatocytes. As an example, the cytotoxicity of acetaminophen and acetyl salicylic acid is assessed.

Inclusive ep double differential cross sections for neutral current deep inelastic scattering are measured with the H1 detector at HERA. The data were taken with a lepton beam energy of 27.6 GeV and two proton beam energies of Ep=460 and 575 GeV corresponding to centre-of-mass energies of 225 and 252 GeV, respectively. The measurements cover the region of 6.5×10-4≤x≤0.65 for 35≤Q2≤800 GeV 2 up to y=0.85 . The measurements are used together with previously published H1 data at Ep=920 GeV and lower Q2 data at Ep=460 , 575 and 920 GeV to extract the longitudinal proton structure function FL in the region 1.5≤Q2≤800 GeV 2 .

Food contact materials (FCM) are estimated to be the largest source of food contamination. Apart from plastics, the most commonly used FCM are made of printed paper and board. Unlike their plastic counterparts, these are not covered by a specific European regulation. Several contamination issues have raised concerns towards potential adverse health effects caused by exposure to substances migrating from printed paper and board FCM. In the current study, an inventory combining the substances which may be used in printed paper and board FCM, was created. More than 6000 unique compounds were identified, the majority (77%) considered non-evaluated in terms of potential toxicity. Based on a preliminary study of their physicochemical properties, it is estimated that most of the non-evaluated single substances have the potential to migrate into the food and become bioavailable after oral intake. Almost all are included in the FACET tool, indicating that their use in primary food packaging has been confirmed by industry. Importantly, 19 substances are also present in one of the lists with substances of concern compiled by the European Chemicals Agency (ECHA). To ensure consumer safety, the actual use of these substances in printed paper and board FCM should be investigated urgently. (C) 2016 Elsevier Inc. All rights reserved.

SummaryBecause of their critical role as goalkeepers of hepatic homeostasis, gap junctions are frequent targets in liver disease. This concept has been demonstrated on many occasions in the light of hepatocarcinogenesis. Relatively little focus has been put on the fate of gap junctions in other liver pathologies, including hepatitis, liver fibrosis and cirrhosis, cholestasis and hepatic ischemia and reperfusion injury. The present paper provides an in-depth description of the multiple changes in expression, localization and function of connexins, the molecular constituents of gap junctions. The use of connexins as biomarkers and therapeutic targets in liver disease is also illustrated.

Tissue homeostasis is driven by a myriad of extracellular, intracellular and intercellular signaling networks. Direct communication between neighboring cells is typically controlled by gap junctions (Decrock et al. 2009; Nielsen et al. 2012; Vinken et al. 2011). Gap junctions mediate the intercellular diffusion of small and hydrophilic substances, including cyclic adenosine monophosphate, adenosine triphosphate, inositol triphosphate, glutathione, glutamate, glucose and several ions (Alexander and Goldberg 2003) (Fig. 1a). This flux is denoted as gap junctional intercellular communication (GJIC) and is considered as a key mechanism in the maintenance of tissue functioning (Decrock et al. 2009; Nielsen et al. 2012; Vinken et al. 2011). Over the last decades, GJIC has been shown indispensable for the establishment of metabolic or electrical intercellular coupling in all vital organs, such as the brain (Eugenin et al. 2012), the heart (Kurtenbach et al. 2014) and the liver (Vinken et al. 2008). Gap junctions are formed by the docking of two hemichannels of adjacent cells, which in turn are composed of six connexin proteins. At present, 21 different connexins have been identified in humans, all which are expressed in a cell-specific way (Kar et al. 2012; Nielsen et al. 2012). They are named based upon their molecular weight. Thus, the most widespread connexin species has a molecular mass of 43 kDa and hence is called Cx43. Connexin proteins share a common molecular structure consisting of four transmembrane domains, two extracellular loops, one cytosolic loop, one cytosolic aminotail and one cytosolic carboxytail (Fig. 1b) (Decrock et al. 2009; Nielsen et al. 2012). Fig. 1 a Gap junctions consist of two connexin hemichannels of adjacent cells and mediate intercellular communication. Connexin hemichannels also provide a pathway for communication, namely between the cytosol and the extracellular environment. Similar messengers ... Although considered as merely structural precursors of gap junctions for a long time, an abundance of reports published in the last few years shows that connexin hemichannels as such can provide a pathway for cellular communication, albeit between the cytosol of individual cells and their extracellular environment and not between adjacent cells as is the case for GJIC. Nonetheless, the messengers that are conveyed through connexin hemichannels are very similar to those involved in GJIC, including adenosine triphosphate, nicotinamide dinucleotide, glutamate, glutathione, prostaglandin, sodium and calcium ions (Fig. 1a) (Chandrasekhar and Bera 2012; Decrock et al. 2009; Kar et al. 2012). Furthermore, connexin hemichannels are regulated by mechanisms that equally affect gap junctions, yet an identical factor can have opposing effects on the two channel types, such as shown for certain inflammatory triggers (De Vuyst et al. 2007; Retamal et al. 2007). In line with this notion, connexin hemichannels, unlike their full channel counterparts, display a low open probability. In fact, connexin hemichannels seem to be preferably activated by pathological stimuli, including ischemia–reperfusion insults and oxidative stress, and thereby drive processes like cell death and inflammation (Chandrasekhar and Bera 2012; Decrock et al. 2009; Kar et al. 2012). For this very reason, connexin hemichannels are sometimes considered as “pathological pores” (Decrock et al. 2009). This concept is clearly relevant to toxicologists, as several toxic substances open up connexin hemichannels. For example, ultrafine carbon black particles activate Cx43-based hemichannels in astrocytes, which may represent a novel mechanism of neurotoxicity (Wei et al. 2014). Similarly, cobalt–chromium nanoparticles can damage human fibroblasts by a mechanism involving transmission of adenosine triphosphate and signaling through connexin hemichannels (Bhabra et al. 2009). The connexin research field has been surrounded by a lot of controversy in the last few years. Specifically, the concept of (dys) functional connexin hemichannels has been debated heavily on several occasions (Saez and Leybaert 2014; Spray et al. 2006). A major reason for this impediment is the ubiquitous lack of tools and technologies to distinguish between the different channel types, in casu between gap junctions and connexin hemichannels (Bodendiek and Raman 2010; Iyyathurai et al. 2013). Classical strategies, such as the use of RNA interference-based technologies, genetically modified animals or even antibodies, are indeed not applicable, as they target connexins, which are the shared building stones of gap junctions and connexin hemichannels. Furthermore, most, if not all, of the routinely used gap junction inhibitors, including long-chain alcohol substances, anaesthetic substances, the glycyrrhetinic acid derivative carbenoxolone and the fenamate family of blockers, equally suppress connexin hemichannel activity (Bodendiek and Raman 2010). Great expectations now lie with peptides that reproduce sequences in the cytosolic loop regions of connexins, as they suppress connexin hemichannel activity without influencing GJIC (Iyyathurai et al. 2013). Among those, a very prominent one is Gap19, a peptide that inhibits Cx43-based hemichannels while leaving the corresponding gap junctions unaffected (Abudara et al. 2014; Wang et al. 2013). In fact, Gap19 was found to reduce experimentally induced cell death in a mouse model of cardiac ischemia–reperfusion (Wang et al. 2013). This area is still in its infancy, but upon further exploration, it can be expected that this will also open new perspectives for the field of toxicology. Indeed, such specific connexin hemichannel inhibitors are not only interesting experimental tools to shed light onto yet unraveled toxicological mechanisms of action, but they may also form the basis for the development of antidotes pertinent for clinical use.

Adverse outcome pathways (AOPs) are novel tools in toxicology and human risk assessment with broad potential. AOPs are designed to provide a clear-cut mechanistic representation of toxicological effects that span over different layers of biological organization. AOPs share a common structure consisting of a molecular initiating event, a series of key events connected by key event relationships, and an adverse outcome. Development and evaluation of AOPs ideally complies with guidelines issued by the Organization for Economic Cooperation and Development. AOP frameworks have yet been proposed for major types of drug-induced injury, especially in the liver, including steatosis, fibrosis, and cholestasis. These newly postulated AOPs can serve a number of purposes pertinent to safety assessment of drugs, in particular the establishment of quantitative structure-activity relationships, the development of novel in vitro toxicity screening tests, and the elaboration of prioritization strategies.

Connexins and their channels are involved in the control of all aspects of the cellular life cycle, ranging from cell growth to cell death, by mediating extracellular, intercellular and intracellular communication. These multifaceted aspects of connexin-related cellular signaling obviously require strict regulation. While connexin channel activity is mainly directed by posttranslational modifications, connexin expression as such is managed by classical cis/trans mechanisms. Over the past few years, it has become clear that connexin production is equally dictated by epigenetic actions. This paper provides an overview of the role of major determinants of the epigenome, including DNA methylation, histone acetylation and microRNA species, in connexin expression.

Homeostasis relies on the intimate interplay between extracellular, intracellular and intercellular signaling networks. Direct communication between adjacent cells is mediated by gap junctions [1-3]. They allow the intercellular diffusion of small (i.e. less than 1 kDa) and hydrophilic substances, including adenosine triphosphate (ATP), cyclic adenosine monophosphate, inositol triphosphate, glucose, glutathione, glutamate and several ions, like sodium, calcium and potassium (Fig. 1) [4]. This flux is called gap junctional intercellular communication (GJIC) and is considered as a basic mechanism in the maintenance of tissue functioning [1-3]. Over the last decades, GJIC has been shown indispensable for the establishment of metabolic or electrical intercellular coupling in all vital organs, such as the brain [5], the heart [6] and the liver [7], to name a few.

Drug-induced liver injury is a prominent reason for premarketing and postmarketing drug withdrawal and can be manifested in a number of ways, such as cholestasis, steatosis, and fibrosis. The mechanisms driving these toxicological processes have been well characterized and have been emdedded in adverse outcome pathway frameworks in recent years. This review evaluates these constructs and simultaneously illustrates their use in the preclinical testing of drug-induced liver injury.

The aim of this research is to inform about the different meanings street youth give tothe use of inhalants, in the city of La Paz -Bolivia. The study draws on two sources:visual and written material produced by them in creative workshops and subsequentdialogues with participants. Through a collaborative data collection methodology,preliminary findings were presented and discussed with participants to deepen in themeaning construction of inhalant use. We found meanings are neither static norgeneralizable, but dynamic and particular to each participant´s experience. Moreover,meanings are constructed in relation to different physical and social spaces with whichthey interact –such as welfare institutions, street groups and society in general- to resistsocial exclusion. We argue that greater interaction with social contexts different fromthe street can affect the frequency of consumption, decreasing the use of this kind ofdrugs. Further research is needed to confirm this latter point, in order to beimplemented in intervention designs.

Les ateliers multi-média tels que les auteurs les ont conçus investissent à la fois l'expression corporelle, l'activité ludique, graphique et narrative en tant qu'outils médiateurs de la vie psychique. Les ateliers se sont déroulés sur une période variant de un à deux ans, de façon hebdomadaire, et avaient comme objectif principal de soulager l'agitation et le débordement moteurs et de relancer les processus de mentalisation et de symbolisation chez les enfants. Avant de proposer une brève évaluation des effets du dispositif de soin en reprenant l'évolution de plusieurs indicateurs de l'expression corporelle et de l'activité symbolique, les auteurs présentent les raisons principales du placement en Service d'Accueil Spécialisé de la Petite Enfance.

Adverse outcome pathways (AOPs) are novel tools in toxicology and human risk assessment with broad potential. AOPs are designed to provide a clear-cut mechanistic representation of critical toxicological effects that span over different layers of biological organization. AOPs share a common structure consisting of a molecular initiating event, a series of intermediate steps and key events, and an adverse outcome. Development of AOPs ideally complies with OECD guidelines. This also holds true for AOP evaluation, which includes consideration of the Bradford Hill criteria for weight-of-evidence assessment and meeting a set of key questions defined by the OECD. Elaborate AOP frameworks have yet been proposed for chemical-induced skin sensitization, cholestasis, liver fibrosis and liver steatosis. These newly postulated AOPs can serve a number of ubiquitous purposes, including the establishment of (quantitative) structure-activity relationships, the development of novel in vitro toxicity screening tests and the elaboration of prioritization strategies.

Previous chapters in this volume pointed to the diversity of theories and approaches within narrative research, and showed the creativity of narrative researchers to integrate or incorporate this diversity in their studies. However, Squire (this volume) has noticed earlier that this diversity also seems to force narrative research to deal with two "incommensurable" theories of human subjectivity and selfhood. Starting with a condense review of the field of tension between both theories, this chapter demonstrates narrative practice based on a recently developed perspective, that was inspired by the rhizome thinking introduced by the French writers Gilles Deleuze and Felix Guattari (Sermijn, De Vlieger & Loots, 2008). We illustrate and discuss how this perspective can be applied in practicing narrative research, by presenting a case-study on life narratives in a post-conflict community in Northern Uganda.

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Direct intercellular communication mediated by gap junctions constitutes a major regulatory platform in the control of hepatic homeostasis. Hepatocellular gap junctions are composed of two hemichannels of adjacent cells which are built up by connexin proteins, in casu Cx32. Dr. Mathieu Vinken, postdoctoral research fellow at the Department of Toxicology of the Free University Brussels-Belgium, was one of the first investigators to demonstrate that hepatic connexin expression is controlled by epigenetic mechanisms. In particular, he found that inhibitors of histone deacetylase enzymes enhance Cx32 production and gap junction activity in cultures of primary hepatocytes, a finding that is of importance for liver-based in vitro modelling. Dr. Mathieu Vinken's recent work is focussed on the elucidation of the role of connexin proteins and their channels in the hepatocyte life cycle. Specific attention is paid to apoptosis in this context, whereby it has yet been found that Cx32 hemichannels control the terminationof induced cell death in cultures of primary hepatocytes. Overall, Dr. Mathieu Vinken's research can be considered as an important contribution to the field hepatic connexin physiology.

During our daily life, we are exposed to a variety of chemical substances and products including cosmetics and their ingredients. When they are brought on the EU market, they must be safe for human health. This safety is traditionally guaranteed through quantitative risk assessment which is not only based on human exposure data, but in particular on testing results derived from experimental animals, assessing both local and whole organism systemic toxicity. For local and short-term toxicity, the development of 3R (Refinement, Reduction and Replacement)—alternative methods had some important success. A number of toxicological endpoints, including skin corrosion, skin irritation, eye irritation, skin sensitization, phototoxicity, dermal absorption, acute toxicity, and in vitro genotoxicity/mutagenicity testing could be covered, although not all with replacement and stand-alone alternatives (EU 2010). Refinement and reduction are, for example at the basis of the local lymph node assay (LLNA), used for skin sensitization testing; for eye irritation only some screening tests for strong irritants are available. For long-term and systemic toxicity testing, consuming in fact the largest number of experimental animals, nearly no alternatives are available and the time required for their scientific development is a huge question mark. Exactly here lies the problem! In the EU, the cosmetic legislation was made the most stringent one of the world by bringing in a rigid time frame through testing and marketing bans (EU 1976 amended by EU 2003). This political decision has positive and negative sides. Positive is the fact that over the last years the number of efforts, made by all stakeholders involved (academic world, industry, animal welfare, and legislative bodies) substantially improved. Also collaboration towards a common goal gained importance and financial resources in the field became more available. A negative outcome, however, is that this politically driven initiative does not take into account the scientific feasibility of the timely and complex development of alternatives to guarantee human safety. Personally spoken, I see as one of the most limiting factors for further progress in the cosmetic field, the way Europe sticks to replacement methods only. This deliberate choice ‘‘kills’’ reduction and refinement efforts. As such, progress made in the practical application of tiered approaches combining in vitro and in vivo methodology, and of read across and weight of evidence strategies, is ignored to a major extend. This is difficult to accept as these strategies became internationally accepted to come to a more balanced risk assessment and at the same time save animal lives. Also very recently, a panel of European experts reported on the current status and future prospects of non-animal methods for cosmetic testing (Adler et al. 2011, this issue). They concluded that specific timelines were impossible to give for these areas in which most animals are consumed, namely for reproductive toxicity, repeated dose toxicity, carcinogenicity, and toxicokinetics. Interesting to note is that they also concluded that significant advances were made in reducing the number of animals in tests by refinement strategies and by the use of nonanimal methods alongside animal tests. From March 2013, however, this is no longer possible. V. Rogiers (&) Department of Toxicology, Center for Pharmaceutical Research, Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, 1090 Brussels, Belgium e-mail: vrogiers@vub.ac.be

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