Adverse Outcome Pathways as Tools to Assess Drug-Induced Toxicity

Abstract

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.

Keywords: AOP; Cholestasis; Drug safety; Fibrosis; Steatosis.

Figure 1. Generic structure of an AOP.

Each AOP consists of two anchors, namely the molecular initiating event (MIE), which refers to the interaction of a chemical with a biological system at the molecular level, and the adverse outcome (AO), which is the actual apical toxicological endpoint. The entire response matrix between the MIE and AO is filled with key events (KEs), which represent changes in the biological state that are both measurable and essential to the progression of a defined biological perturbation leading to a specific AO. Subsequent KEs are connected by key event relationships (KERs), defining a link between both KEs and that facilitate inference or extrapolation of the state of the downstream KE from the known, measured or predicted state of the upstream KE (adapted from (10, 11)).

Figure 2. AOP for drug-induced liver steatosis.

Activation of the liver X receptor (LXR), which is the MIE (blue), induces a number of transcriptional changes, including activation of the expression of carbohydrate response element binding protein (ChREBP), sterol response element binding protein 1c (SREBP-1c), fatty acid synthase (FAS) and stearoyl-coenzyme A desaturase 1 (SCD1). As a result, de novo synthesis of fatty acids is enhanced in the liver. At the same time, fatty acid translocase (CD36) production is upregulated, which mediates increased hepatic influx of fatty acids from peripheral tissues. All together, these intermediate steps drive accumulation of triglycerides, which is considered a key event (dark green). At the organelle level, this evokes cytoplasm displacement, distortion of the nucleus and mitochondrial disruption. This ultimately burgeons into the appearance of fatty liver cells (orange) and further into the clinical diagnosis of liver steatosis (red) (adapted from (20)).

Figure 3. AOP for drug-induced liver fibrosis.

The MIE (blue) is considered protein alkylation and covalent protein binding in the liver. This serves as a trigger to provoke hepatocyte injury, including apoptosis, which in turn activates Kupffer cells. As a result, transforming growth factor beta 1 (TGF-β1) expression is induced, which is a key factor for stellate cell activation. The latter goes hand in hand with the occurrence of inflammation and oxidative stress. The different events at the cellular level (green) are interconnected in several ways. The overall end result is accumulation of collagen and changes in the extracellular matrix composition in the liver (orange), which becomes clinically manifested as the AO, namely liver fibrosis (red) (adapted from (20)).

Figure 4. AOP for drug-induced cholestasis.

The response matrix between the MIE (dark blue) and AO (red), the inhibition of the bile salt export pump (BSEP) and cholestasis, respectively, spans over the cellular and organ levels. Identified KEs (dark green) include the accumulation of bile, the induction of oxidative stress and inflammation, and the activation of the nuclear receptors pregnane X receptor (PXR), farnesoid X receptor (FXR) and constitutive androstane receptor (CAR). Together with a number of intermediate steps, these KEs drive both a deteriorative cellular response (yellow), which underlies directly caused cholestatic injury, and an adaptive cellular response (purple), which is aimed at counteracting the primary cholestatic insults. Direct inducing and inhibiting effects are indicated with green and red arrows, respectively. Secondary inducing and inhibiting effects of oxidative stress and/or inflammation are indicated with blue and orange arrows, respectively (31) (5′-NT, 5′-nucleotidase; ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; CYP2B10/3A4/7A1, cytochrome P450 2B10/3A4/7A1; GGT, gamma-glutamyl transpeptidase; MPP, mitochondrial permeability pore; MRP2/3, multidrug resistance-associated protein 2/3; NTCP, sodium/taurocholate cotransporter; OATP1B1, organic anion transporter 1B1; OSTα/β organic solute transporter α/β; SHP, small heterodimeric partner; SULT2A1, dehydroepiandrosterone sulfotransferase; UGT2B4, uridine 5′-diphosphate-glucuronosyltransferase 2B4).