Human liver microsomes, or HLMs, play a key role in drug metabolism because they contain important cytochrome P450 (CYP) enzymes that are critical in oxidation and hydrolysis processes. We hypothesized that the variability in CYP enzyme concentration and activity among individuals will contribute to differences in metabolic capacity. In this study, my goal was to characterize HLMs from a bank of individual donors with different genetic backgrounds. I assessed their drug-metabolizing capabilities, with an emphasis on CYP-mediated pathways. I used bicinchoninic acid assays (BCA) to quantify protein content, carbon monoxide heme spectra (P450 spectra) to determine CYP enzyme concentrations, and a cytochrome c reduction assay to measure P450 reductase activity. For conducting these experiments, we used HLM samples from five different individuals from the UW Human Liver Bank. From the first subset of samples, I found that the ratio of CYP protein to total protein content varied across individuals with different clinical presentations. The differences among individuals in our findings emphasize the importance of HLM characterization in preclinical drug trials to understand the need for the personalization of pharmacological treatments. The implementation of such characterization can have a tremendous effect on predictions of drug responses and optimization of drug dosages, ultimately improving drug efficacy and safety.

Cytochrome P450 enzymes (CYPs) are a family of proteins that play an integral part in drug metabolism. An example is CYP3A4, a specialized protein within the CYP family, involved in the oxidation of small drugs and toxins, like cannabinoids, to allow for their removal from the body. Understanding this reaction is important in evaluating how different expression levels of CYP3A4 in different individuals affect the efficiency of drug metabolism. In this project, I studied drug binding to recombinant, purified, CYP3A4 protein. The binding assays of CYP3A4 allow evaluation of the quality of the purified protein. Ultraviolet-visible spectroscopy (UV-Vis) was used to quantify the protein, and spectral binding through drug titration was used to characterize the binding affinities of different substrates. Drug titration enables us to observe the amount of ligand bound to the protein at various concentrations, displayed as a binding curve from which we can determine the spectral binding constant (Ks). The CYP active site contains a heme group. Upon binding, the ligand replaces the water molecule that is originally docked in the active site. This transfer reaction shifts the heme spin state and appears on the UV-Vis spectra as an increasing absorbance at the 380 nm wavelength and a decreasing absorbance at 420 nm. Inhibitors would have the reverse spectrum. In vitro studies with purified protein have several benefits when investigating protein function, such as simplifying the experimental system and reducing the limitations of complicated sample preparations from living organisms. Having a well-defined assay to determine recombinant 3A4 protein quality will contribute to the value of further in vitro activity and pharmacokinetic studies with this protein. 

Liver fatty acid binding protein (FABP1) is highly expressed in the liver, kidney, and gut and is known for its role in binding endogenous lipids. FABP1 has also been shown to bind drugs and modulate metabolism in the liver. A high frequency single nucleotide polymorphism (SNP T94A) in FABP1 is shown to correlate with nonalcoholic fatty liver disease. We hypothesize that this SNP also affects drug binding. To evaluate drug-FABP1 binding, I measure equilibrium dissociation constants (K_ds) by fluorescent displacement assays for both FABP1 wild-type and T94A using two fluorescent probes, 11-(dansylamino)undecanoic acid (DAUDA) and 8-anilino-1-naphthalenesulfonic acid (ANS). FABP1 has a large binding pocket that can accommodate 2 ligands simultaneously in a ‘high affinity’ and ‘low affinity’ binding site. When DAUDA-FABP1 or ANS-FABP1 are titrated with a drug, a drug-FABP1-probe ternary complex is formed rather than the probe being fully displaced. This complicates data analysis and suggests that endogenous lipids may change the affinity of drugs for FABP1. Therefore, I use multiple fluorescent probes with different binding affinity to obtain drug K_d values. I use singular value decomposition (SVD) to isolate individual fluorescent components from the overall observed fluorescence spectra. I then estimate drug and probe K_ds for FABP1 T94A and T94T by fitting the fluorescent change due to binding to dynamic models in COPASI software. From forward and reverse titrations, DAUDA K_d for FABP1 wild-type was found to be 0.194 µM while ANS binds more weakly (K_d = 1.38 µM). From DAUDA displacement assays, diclofenac was found to have a K_d of 3.90 µM for wild-type FABP1 and 3.78 µM for T94A. I anticipate measurement of K_ds for 8 other drugs using both DAUDA and ANS in the coming months. The developed methods will enable evaluation of FABP1’s role in drug disposition.

Acetaminophen (APAP) is a widely used over-the-counter drug known for its analgesic and antipyretic properties. Several clinical factors can influence how APAP is absorbed, distributed, metabolized and excreted from the body (i.e., its pharmacokinetics (PK)). APAP is metabolized into several metabolites, including APAP-glucuronide, APAP-sulfate, APAP-cysteine, and APAP-N-acetylcysteine. Therefore, accurately determining plasma concentrations of APAP and its metabolites is crucial for understanding how individuals metabolize APAP and environmental influences on APAP PK. To address this, I am reproducing a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay to determine concentrations of APAP and its metabolites in plasma. I analyzed plasma samples collected from seven healthy volunteers following oral administration of 500 mg of APAP. To prepare the samples, I added 70 μL of diluted internal standard mix into the tubes containing 30 μL of plasma. Samples were centrifuged and the supernatants were transferred clean tubes and dried down with nitrogen gas. Samples were reconstituted in mobile phase for analysis using LC-MS/MS. I analyzed the LC-MS/MS data to calculate the plasma concentration vs. time curves for each analyte. I used Phoenix WinNonlin to estimate key PK parameters, including peak concentration (Cmax), time of peak concentration (Tmax), clearance, half-life, volume of distribution, and area under the curve (AUC). This LC-MS/MS assay provides a platform for precise quantification of APAP and its metabolites, and will be essential for our lab’s future studies on the impact of the gut microbiome on APAP’s PK.

Chronic kidney disease is projected to be the fifth leading cause of death worldwide by 2040. Chronic kidney disease of unknown etiology (CKDu) makes up 70% of CKD cases in places such as India, Mexico, and Sri Lanka, largely through environmental factors. Ochratoxin A (OTA) accumulates in the kidney and is a nephrotoxic mycotoxin that contaminates grain products such as wheat, rice, beer, and most plant-based foods. Chronic OTA exposure has been linked to CKDu in rural agricultural areas, such as Sri Lanka. A prominent family of cell membrane transporters, Organic Anion Transporters (OATs), are one of the main drug transporter families in the kidney. Previous work in our lab elucidated that OAT1/3 and 4 are major OTA transporters. Certain antioxidants, found in common plant-based food products like green tea, coffee, and certain vegetables have been studied to reduce OTA-mediated nephrotoxicity. However, since our preliminary data indicate OAT transporter-dependent uptake into the kidney, we aim to test the competitive inhibition effect of OAT-substrate antioxidants in preventing kidney accumulation of OTA. Potential inhibitors include epicatechin gallate, miquelianan, caffeic acid, luteolin, and myricetin. Competitive inhibition in individuals consuming these products along with OTA exposure could lead to decreased uptake of OTA into the kidney, mitigating toxicity. Our preliminary uptake experiment with those inhibitors indicates that miquelianan reduces OAT3 mediated uptake of OTA by 48%. We will next assess the inhibition potential of miquelianan on OTA with an IC50 curve via mass spectrometry analysis. This study will provide evidence for a potential new mechanism of antioxidant amelioration of kidney toxicity to OTA exposure.

Ochratoxin-A (OTA) is a ubiquitous food contaminant linked to nephrotoxicity and carcinogenicity. Yet, its exposure risk and metabolic pathway in humans remain poorly understood. This research aims to investigate the intrinsic clearance of OTA in the human liver and to identify cytochrome P450 (CYP450) isozyme(s) responsible for its biotransformation. I employed a substrate depletion assay on OTA-treated human liver microsomes and used ultraviolet–visible spectroscopy to determine the kinetic parameters of clearance rates. To identify specific CYP450 isozyme(s) involved in metabolism, a parallel substrate depletion assay was conducted with recombinant CYP450 supersomes at defined intervals. Findings from this study reveal human susceptibility to OTA-induced toxicity and offers insight to our understanding to the hepatic metabolism of this widespread dietary toxin. Future research will explore human proximal-tubule specific OTA bioactivation, ultimately guiding regulatory decisions and public health interventions to reduce OTA-associated health risks.

The metabolite of vitamin A, retinoic acid (RA), plays a critical role in regulating cell differentiation in mammals. RA and its metabolites exist as different geometric isoforms (all-trans, 13-cis, 9-cis, 4-oxo-all-trans, 4-oxo-13-cis .). All-trans-RA is the biologically active isomer, and 13-cis-RA is found as the drug “Accutane” used to treat severe acne. Cellular retinoic acid binding proteins are evolutionarily conserved intracellular proteins that regulate RA tissue concentrations. The all-trans isomer is known to bind tightly to CRABP1 and 2, but little is known about the binding of the other four isomers and metabolites. No data on the binding affinities of the 4-oxo isomers is available. I hypothesize that the RA and 4-oxo-RA isomers that have not been extensively researched, have different binding affinities between the two CRABPs. To test this hypothesis, I use fluorescence spectroscopy coupled with single value decomposition (SVD) analysis and stopped-flow analysis to measure the equilibrium dissociation constant (Kd), association rate constant (kon), and dissociation rate constant (koff) for retinoid binding to CRABPs. My current data generated by the fluorescence spectroscopy method shows that binding affinities of the tested retinoids are comparable between CRABP 1 and 2, except for 13-cis-RA which bound CRABP2 significantly more tightly than CRABP1. (CRABP1 Kd  = 609 nM, CRABP2 Kd  = 70.5 nM) . All-trans-RA (atRA) has the tightest binding to both CRABP 1 and 2, (CRABP1 Kd  = 0.51 nM, CRABP2 Kd  = 0.73 nM), followed by 4-oxo-atRA, (0.39nM, 1.4 nM), 9-cis-RA, (61.5 nM, 96.2 nM), and finally 4-oxo-13-cis (779.5 nM, 743.6 nM) with the lowest binding affinity. These relationships will be further investigated using the stopped-flow method.

In vitro models (cells in a dish) are a powerful tool in toxicology, allowing for advanced research in biological mechanisms while decreasing our reliance on in vivo animal models. Reproductive development is a critical endpoint in toxicology and requires a large number of animals, making reproductive studies a priority for in vitro alternatives. The current in vitro testis models are insufficient to recapitulate human reproductive development as they still rely on cells from laboratory rodents due to low human testis tissue availability and the need to capture dynamic developmental stages. To address this, I am developing an in vitro model that recapitulates human spermatogonia development to generate human primordial germ cell-like cells (hPGCLCs) using two induced pluripotent stem cell (iPSC) lines. This approach relies on spontaneous differentiation of the iPSCs using an extracellular matrix overlay. My pilot experiments did not robustly differentiate; therefore, I adapted the protocol to first induce incipient mesoderm-like cells (iMeLCs), which are primed for differentiation to hPGCLCs. I observed distinct cell morphological differences in the iMeLCs relative to control iPSCs using phase-contrast microscopy and found increased expression of Vimentin in the iMeLCs using immunocytochemistry. I am completing additional experiments to visualize expression of the mesoderm marker Brachyury, proliferative marker ki67, and primordial germ cell markers ki67 and SOX17. Using these iMeLCs I will follow the overlay protocol to derive hPGCLCs. I will assess the hPGCLC phenotype using flow cytometry for TFAP2C, a marker of PGCs. The hPGCLCs will then be cocultured with primary testis tissue to drive development towards spermatogonia-like cells (SpLCs), determined by expression of DDX4. The primary tissue will include our labs standard rodent model, as well as human tissue from collaborators at the UW Male Fertility Lab. Developing a fully human in vitro model system will be a powerful tool to study infertility.

Organs-on-a-chip (OOAC) are biomimetic systems that replicate the physiological environments of human organs at a micro-scale. They are gaining industry acceptance due to their ability to control critical parameters including shear stress, concentration gradients, and cell-biofluid interactions. By mimicking the behavior of human organs, OOACs are transforming how pharmacokinetics, physiological, and toxicological studies are performed, offering a more relevant model than animal-based studies. Our studies focus on how drugs and toxins affect the human kidney, a crucial organ for processing medications and filtering out harmful compounds. A key component of kidney OOACs is a hydrogel, which provides a structural scaffold and a biological substrate for cells. The hydrogel consists of rat tail Collagen I (Col-I) and specialized cell culture media (PTEC and 199 (10x)). The media mimics the extracellular fluids that surround kidney cells in the body, providing a more realistic environment for cell growth/interaction. Collagen IV (Col-IV) is the most abundant protein in kidney tissue but lacks structural rigidity. A combination of these materials is crucial for achieving a more accurate representation of kidney structure and function. While adding more matrix to the hydrogel improves the model’s ability to replicate the native environment, it is challenging to maintain structural stability, hence the need for a stabilizing agent. The aim of this project is to determine the proximal tubule epithelial cell (PTEC) viability of a mixed collagen I and IV matrix. At this stage, we have shifted from determining optimal collagen ratios to evaluating cell viability. By refining these models with optimized kidney extracellular matrices, the Kelly-Yeung lab aims to develop OOAC systems that better predict how drugs, toxins, and diseases impact human kidneys. This progress will lead to more effective and personalized treatments, as well as a reduction in reliance on animal testing.