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ABSTRACT:We identified human UDP-glucuronosyltransferase (UGT) isoforms responsible for producing dihydrotestosterone (DHT) diglucuronide, a novel glucuronide in which the second glucuronosyl moiety is attached at the C2 position of the first glucuronosyl moiety, leading to diglucuronosyl conjugation of a single hydroxyl group of DHT at the C17 position. Incubation of the DHT monoglucuronide with 12 cDNA-expressed recombinant human UGT isoforms and uridine 5-diphosphoglucuronic acid resulted in a low but measurable DHT diglucuronidation activity primarily with UGT1A8, a gastrointestinal UGT, and to a lesser extent with UGT1A1 and UGT1A9. In contrast, the activity of DHT monoglucuronidation was high and was found in UGT2B17, UGT2B15, UGT1A8, and UGT1A4 in descending order. Among the 12 UGT isoforms tested, only UGT1A8 was capable of producing DHT diglucuronide from DHT. The kinetics of DHT diglucuronidation by microsomes from human liver and intestine fitted the Michaelis-Menten model, and the V max /K m value for the intestinal microsomes was approximately 4 times greater than that for the liver microsomes.

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Mer proto-oncogene tyrosine kinase (MerTK), expressed in the retinal pigment epithelium (RPE), regulates the phagocytosis of shed photoreceptor outer segments. To investigate the influence of dosing time on MerTK inhibitor UNC569-induced retinal toxicity, UNC569 at 100 mg/kg was orally administered to male mice at 2 different Zeitgeber times (ZT5.5 or ZT22) for 28 days. Electron microscopy was conducted at ZT2 after the final dosing. Additionally, the visual cycle components (11-cis-retinal, all-trans-retinal, all-trans-retinol, and 11-cis-retinol), which play an important role in maintaining retinal homeostasis, were quantified by liquid chromatography/mass spectrometry/mass spectrometry. Under electron microscopic examination, the number of phagosomes and phagolysosomes in the RPE increased in both the ZT5.5 and ZT22 administered groups, while endoplasmic reticulum dilatation in the RPE and chromatin aggregation of photoreceptor nuclei were observed only in the ZT22 administered group. No change was observed in any of the visual cycle components. These results suggest that the timing of the dosing in relation to the physiological MerTK phosphorylation affected the severity of changes in the RPE, leading to the apoptosis of the photoreceptor cells.