Hepatic Bioactivation of Skin-Sensitizing Drugs to Immunogenic Reactive Metabolites

Abstract

The clinical use of some drugs, such as carbamazepine, phenytoin, and allopurinol, is often associated with adverse cutaneous reactions. The bioactivation of drugs into immunologically reactive metabolites by the liver is postulated to be the first step in initiating a downstream cascade of pathological immune responses. Current mechanistic understanding and the ability to predict such adverse drug cutaneous responses have been partly limited by the lack of appropriate cutaneous drug bioactivation experimental models. Although in vitro human liver models have been extensively investigated for predicting hepatotoxicity and drug–drug interactions, their ability to model the generation of antigenic reactive drug metabolites that are capable of eliciting immunological reactions is not well understood. Here, we employed a human progenitor cell (HepaRG)-derived hepatocyte model and established highly sensitive liquid chromatography-mass spectrometry analytical assays to generate and quantify different reactive metabolite species of three paradigm skin sensitizers, namely, carbamazepine, phenytoin, and allopurinol. We found that the generation of reactive drug metabolites by the HepaRG-hepatocytes was sensitive to the medium composition. In addition, a functional assay based on the activation of U937 myeloid cells into the antigen-presenting cell (APC) phenotype was established to evaluate the immunogenicity potential of the reactive drug metabolites produced by HepaRG-derived hepatocytes. We showed that the reactive drug metabolites of known skin sensitizers could significantly upregulate IL 8, IL 1β, and CD 86 expressions in U937 cells compared to the metabolites from a nonskin sensitizer (i.e., acetaminophen). Thus, the extent of APC activation by HepaRG-hepatocytes conditioned medium containing reactive drug metabolites can potentially be used to predict their skin sensitization potential.

Effects of <i>Moringa oleifera</i> Biochemical Constituents on Kidney, Liver and Brain of Wister Rats

“…When protective defenses are overwhelmed by excess toxicant insult, the effects of reactive intermediate species lead to deregulation of cell signaling pathways and dysfunction of biomolecules, leading to failure of target organelles and eventual cell death [18]. This is similar to the finding reported by [19], whose discovered biotransformation of certain xenobiotics to be among the causes of short-lived, unstable, highly reactive chemical species that can interact with functional biomolecules and potentially lead to adverse effects. The functional groups or structural motifs in xenobiotics that can produce harmful reactive intermediates have been defined as 'toxicophores' [20].…”

Integration of a microfluidic multicellular coculture array with machine learning analysis to predict adverse cutaneous drug reactions

“…HepaRG progenitor cells (Biopredic International, France) were maintained and differentiated as previously described. 14,24 Briefly, HepaRG progenitor cells were maintained in HepaRG growth medium for 14 days before switching to adaptive medium to initiate the differentiation process. The adaptive media was prepared by mixing growth medium and differentiation medium at 1 : 1 ratio.…”

“…U937 cells were cultured as previously described. 14,15 Briefly, U937 cells were maintained in RPMI1640 medium (ThermoFisher Scientific, USA) supplemented with 2 mM glutamine (ThermoFisher Scientific, USA), 10 mM HEPES (Gibco, USA), 1 mM sodium pyruvate (Gibco, USA), 100 U mL supplemented with 10% heat-inactivated FBS (Hyclone, GE Healthcare, USA) and 1% penicillin-streptomycin (Gibco). Cells were passaged at around 70-80% confluence prior to use.…”

“…liver bioactivation and APC activation), which can be independently measured with simplistic cellular assays, provides a more plausible approach to screen a large panel of drugs and identify potential skin sensitizers. 14,15 The proposed MCA was developed on a compartmentalized microfluidic platform. Individual cell types can be maintained in their dedicated culture environments (i.e.…”

“…culture media and configurations) within separate compartments, which were connected by micro-channel networks to allow the cells to interact via soluble diffusible factors, such as drug metabolites. 15 Here, the MCA was designed to have quadruple compartments to house 4 cell types, namely HepaRG-derived hepatocyte spheroids (HHS) as the drug bioactivation model because they exhibit similar Cytochrome P450 (CYP450) metabolic activities to primary human hepatocytes; 16,17 U937 monocytes as the APC activation model because they can undergo phenotypic changes from monocytes to APC-like cells upon exposure to dermal skin sensitizers; 18,19 and lastly, human keratinocytes (HaCaT) and primary dermal fibroblasts were selected to detect keratinocyte-specific FasL-mediated cell apoptosis since HaCaT expressed FasL 20 but not in dermal fibroblast. 21 The device was able to simultaneously capture 5 different readouts in a single experiment, which were integrated using a machine learning algorithm to classify and visualize the drugs as skin sensitizers or non-skin sensitizers.…”