Description: We report here a novel experimental system, cryopreserved MetMax human hepatocytes (MMHHs), for in vitro drug metabolism studies. MMHHs consist of cofactor-supplemented permeabilized cryopreserved human hepatocytes. The use procedures for MMHHs are significantly simplified from that for conventional cryopreserved human hepatocytes (CCHHs): 1) storage at –80°C instead of in liquid nitrogen and 2) usage directly after thawing without centrifugation and microscopic evaluation of cell density and viability and cell density adjustment. In this study, we compared MMHHs and CCHHs in CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2D6, CYP2E1, CYP3A4, CYP2J2, monoamine oxidase A, aldehyde oxidase, flavin-containing monooxygenase, UDP-glucuronyl transferase, SULT, N -acetyltransferase 1, and acetaminophen glutathione (GSH) conjugation activities based on liquid chromatography–tandem mass spectrometry quantification of substrate metabolism. MMHHs were prepared from CCHHs consisting of hepatocytes pooled from 10 individual donors. The drug metabolizing enzyme activities of both CCHHs and MMHHs were cell concentration and time dependent, with specific activities of MMHHs ranging from 27.2% (carboxylesterase 2) to 234.2% (acetaminophen GSH conjugation) of that for CCHHs. As observed in CCHHs, sequential oxidation and conjugation was observed in MMHHs for coumarin, 7-ethoxycoumarin, and acetaminophen. 7-Hydroxycoumarin conjugation results showed that metabolic pathways in MMHHs could be selected via the choice of cofactors, with glucuronidation but not sulfation observed in the presence of UDP-glucuronic acid and not 3-phosphoadenosine-5-phosphosulfate, and vice versa. Results with noncytotoxic and cytotoxic concentrations of acetaminophen showed that drug metabolism was compromised in CCHHs but not in MMHHs. Our results suggest that the MMHHs system represents a convenient and robust in vitro experimental system for the evaluation of drug metabolism.

Description: Chimeric mouse liver models are useful in vivo tools for human drug metabolism studies; however, liver integrity and the microcirculation remain largely uninvestigated. Hence, we conducted liver perfusion studies to examine these attributes in FRGN [ Fah(–/–) , Rag2(–/–) , and Il2rg(–/–), NOD strain] livers (control) and chimeric livers repopulated with mouse (mFRGN) or human (hFRGN) hepatocytes. In single-pass perfusion studies (2.5 ml/min), outflow dilution profiles of noneliminated reference indicators ( 51 Cr-RBC, 125 I-albumin, 14 C-sucrose, and 3 H-water) revealed preservation of flow-limited distribution and reduced water and albumin spaces in hFRGN livers compared with FRGN livers, a view supported microscopically by tightly packed sinusoids. With prograde and retrograde perfusion of harmol (50 µ M) in FRGN livers, an anterior sulfation (Sult1a1) over the posterior distribution of glucuronidation (Ugt1a1) activity was preserved, evidenced by the 42% lower sulfation-to-glucuronidation ratio (HS/HG) and 14% higher harmol extraction ratio (E) upon switching from prograde to retrograde flow. By contrast, zonation was lost in mFRGN and hFRGN livers, with HS/HG and E for both flows remaining unchanged. Remnant mouse genes persisted in hFRGN livers (10%–300% those of FRGN). When hFRGN livers were compared with human liver tissue, higher UGT1A1 and MRP2, lower MRP3, and unchanged SULT1A1 and MRP4 mRNA expression were observed. Total Sult1a1/SULT1A1 protein expression in hFRGN livers was higher than that of FRGN livers, consistent with higher harmol sulfate formation. The composite data on humanized livers suggest a loss of zonation, lack of complete liver humanization, and persistence of murine hepatocyte activities leading to higher sulfation.

Description: The endogenous ligand-activated aryl hydrocarbon receptor (AHR) plays an important role in numerous biologic processes. As the known number of AHR-mediated processes grows, so too does the importance of determining what endogenous AHR ligands are produced, how their production is regulated, and what biologic consequences ensue. Consequently, our studies were designed primarily to determine whether ER – /PR – /Her2 – breast cancer cells have the potential to produce endogenous AHR ligands and, if so, how production of these ligands is controlled. We postulated that: 1) malignant cells produce tryptophan-derived AHR ligand(s) through the kynurenine pathway; 2) these metabolites have the potential to drive AHR-dependent breast cancer migration; 3) the AHR controls expression of a rate-limiting kynurenine pathway enzyme(s) in a closed amplification loop; and 4) environmental AHR ligands mimic the effects of endogenous ligands. Data presented in this work indicate that primary human breast cancers, and their metastases, express high levels of AHR and tryptophan-2,3-dioxygenase (TDO); representative ER – /PR – /Her2 – cell lines express TDO and produce sufficient intracellular kynurenine and xanthurenic acid concentrations to chronically activate the AHR. TDO overexpression, or excess kynurenine or xanthurenic acid, accelerates migration in an AHR-dependent fashion. Environmental AHR ligands 2,3,7,8-tetrachlorodibenzo[p]dioxin and benzo[a]pyrene mimic this effect. AHR knockdown or inhibition significantly reduces TDO2 expression. These studies identify, for the first time, a positive amplification loop in which AHR-dependent TDO2 expression contributes to endogenous AHR ligand production. The net biologic effect of AHR activation by endogenous ligands, which can be mimicked by environmental ligands, is an increase in tumor cell migration, a measure of tumor aggressiveness.

Description: We report in this work successful isolation and cryopreservation of enterocytes from human small intestine. The enterocytes were isolated by enzyme digestion of the intestinal lumen, followed by partial purification via differential centrifugation. The enterocytes were cryopreserved directly after isolation without culturing to maximize retention of in vivo drug-metabolizing enzyme activities. Post-thaw viability of the cryopreserved enterocytes was consistently over 80% based on trypan blue exclusion. Cryopreserved enterocytes pooled from eight donors (four male and four female) were evaluated for their metabolism of 14 pathway-selective substrates: CYP1A2 (phenacetin hydroxylation), CYP2A6 (coumarin 7-hydroxylation), CYP2B6 (bupropion hydroxylation), CYP2C8 (paclitaxel 6 α -hydroxylation), CYP2C9 (diclofenac 4-hydroxylation), CYP2C19 ( S -mephenytoin 4-hydroxylation), CYP2D6 (dextromethorphan hydroxylation), CYP2E1 (chlorzoxazone 6-hydroxylation), CYP3A4 (midazolam 1'-hydroxylation and testosterone 6 β -hydroxylation), CYP2J2 (astemizole O-demethylation), UDP-glucuronosyltransferase (UGT; 7-hydroxycoumarin glucuronidation), sulfotransferase (SULT; 7-hydroxycoumarin sulfation), and carboxylesterase 2 (CES2; irinotecan hydrolysis) activities. Quantifiable activities were observed for CYP2C8, CYP2C9, CYP2C19, CYP2E1, CYP3A4, CYPJ2, CES2, UGT, and SULT, but not for CYP1A2, CYP2A6, CYP2B6, and CYP2D6. Enterocytes from all 24 donors were then individually evaluated for the quantifiable drug metabolism pathways. All demonstrated quantifiable activities with the expected individual variations. Our results suggest that cryopreserved human enterocytes represent a physiologically relevant and convenient in vitro experimental system for the evaluation of intestinal metabolism, akin to cryopreserved human hepatocytes for hepatic metabolism.

Description: We report here a novel in vitro enteric experimental system, cryopreserved human intestinal mucosa (CHIM), for the evaluation of enteric drug metabolism, drug-drug interaction, drug toxicity, and pharmacology. CHIM was isolated from the small intestines of four human donors. The small intestines were first dissected into the duodenum, jejunum, and ileum, followed by collagenase digestion of the intestinal lumen. The isolated mucosa was gently homogenized to yield multiple cellular fragments, which were then cryopreserved in a programmable liquid cell freezer and stored in liquid nitrogen. After thawing and recovery, CHIM retained robust cytochrome P450 (P450) and non-P450 drug-metabolizing enzyme activities and demonstrated dose-dependent induction of transcription of CYP24A1 (approximately 300-fold) and CYP3A4 (approximately 3-fold) by vitamin D 3 as well as induction of CYP3A4 (approximately 3-fold) by rifampin after 24 hours of treatment. Dose-dependent decreases in cell viability quantified by cellular ATP content were observed for naproxen and acetaminophen, with higher enterotoxicity observed for naproxen, consistent with that observed in humans in vivo. These results suggest that CHIM may be a useful in vitro experimental model for the evaluation of enteric drug properties, including drug metabolism, drug-drug interactions, and drug toxicity.