Adverse outcome pathway development from protein alkylation to liver fibrosis

Abstract

In modern toxicology, substantial efforts are undertaken to develop alternative solutions for in vivo toxicity testing. The adverse outcome pathway (AOP) concept could facilitate knowledge-based safety assessment of chemicals that does not rely exclusively on in vivo toxicity testing. The construction of an AOP is based on understanding toxicological processes at different levels of biological organisation. Here, we present the developed AOP for liver fibrosis and demonstrate a linkage between hepatic injury caused by chemical protein alkylation and the formation of liver fibrosis, supported by coherent and consistent scientific data. This long-term process, in which inflammation, tissue destruction, and repair occur simultaneously, results from the complex interplay between various hepatic cell types, receptors, and signalling pathways. Due to the complexity of the process, an adequate liver fibrosis cell model for in vitro evaluation of a chemical's fibrogenic potential is not yet available. Liver fibrosis poses an important human health issue that is also relevant for regulatory purposes. An AOP described with enough mechanistic detail might support chemical risk assessment by indicating early markers for downstream events and thus facilitating the development of an in vitro testing strategy. With this work, we demonstrate how the AOP framework can support the assembly and coherent display of distributed mechanistic information from the literature to support the use of alternative approaches for prediction of toxicity. This AOP was developed according to the guidance document on developing and assessing AOPs and its supplement, the users' handbook, issued by the Organisation for Economic Co-operation and Development.

Keywords: Adverse outcome pathway (AOP); Alternatives to animal testing; Liver fibrosis; Risk assessment; Systems toxicology.

Fig. 1

A schematic representation of the adverse outcome pathway (AOP). An AOP starts with a molecular initiating event in which a chemical interacts with a biological target (anchor 1) leading to a sequential series of intermediate key events at different levels of biological organisation to produce an adverse outcome with relevance to risk assessment (anchor 2)

Fig. 2

Graphic representation of the adverse outcome pathway from protein alkylation to liver fibrosis. The molecular initiating event (MIE) is protein alkylation, leading to structural and functional cell injury and cell death, the first key event (KE). Injured and apoptotic hepatocytes activate Kupffer cells, the next KE along the pathway. Activated KCs are the main source of TGF-β1, the most potent pro-fibrogenic cytokine. TGF-β1 expression causes the next KE, stellate cell activation, which leads to progressive collagen accumulation that together with changes in extracellular matrix (ECM) composition signifies the KE on tissue level. Collagen bands progress further to bridging fibrosis, finally affecting the whole organ. Full arrows represent direct KERs that link two adjacent KEs. The dotted line represents an indirect KER that bridges some of the KEs in the pathway

Fig. 3

Network of molecular events triggered during development of liver fibrosis. Molecular mechanisms, feed-back, and feed-forward loops as well as inter-relationships between individual key events are presented. The central line of events, marked by thick black frames, represents the developed AOP, as shown in Fig. 2. Violet boxes correspond to MIE and AO, blue boxes to molecular processes, and green boxes to various cell types involved in fibrogenesis. Orange ovals represent molecular mediators. α-SMA alpha-smooth muscle actin, CTGF connective tissue growth factor, DAMPs damage-associated molecular patterns, FasL Fas Ligand, GM-CSF granulocyte macrophage colony-stimulating factor, HNE-4 hydroxynonenal, IFNγ interferon gamma, iNOS nitric oxide synthase, MAPK mitogen-activated protein kinases, MCP-1 monocyte chemoattractant protein-1, MMPs metalloproteinases, N ₂ O ₃ peroxinitrite, NF-κB nuclear factor kappaB, NO nitric oxide, NOX NADH oxidase, P450 cytochrome P450, PDGF platelet-derived growth factor, PPARγ peroxisome proliferator-activated receptor-gamma, ROS reactive oxygen species, CSFs colony-stimulating factors, TGF-β1 Transforming growth factor beta1, TIMP-1 tissue inhibitor of metalloproteinases-1, TNFα tumor necrosis factor alpha, TLRs toll-like receptors, TRAIL TNF-related apoptosis-inducing ligand, and VEGF vascular endothelial growth factor (colour figure online)

Fig. 4

Graphic representation of the adverse outcome pathway describing the linkage between hepatic injury caused by protein alkylation and the formation of liver fibrosis, including the display of chronic inflammation and oxidative stress, thus illustrating their interrelationships with the various KEs