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Ribavirin, an antiretroviral agent targeting the hepatitis C virus, causes male reproductive toxicity. This study investigated the mechanism of ribavirin transport at the blood-testis barrier (BTB). In vivo mouse integration plot analysis after intravenous administration revealed that the net influx clearance of [ 3 H]ribavirin in the testis was 3.6-fold greater than that of [ 14 C]D-mannitol, a paracellular transport marker, implying transcellular transport of ribavirin across the BTB. Moreover, [ 3 H]ribavirin uptake by TM4 cells, mouse-derived Sertoli cells, was time- and concentration-dependent, with a K m value of 2.49 mM. S-[(4-nitrophenyl)methyl]-6-thioinosine, an inhibitor of Na + -independent equilibrative nucleoside transporters (ENTs), strongly inhibited the [ 3 H]ribavirin uptake by TM4 cells at 100 µM. Compared to the uptake of [ 3 H]adenosine, a typical endogenous nucleoside, [ 3 H]ribavirin uptake was relatively similar to ENT2 transport. [ 3 H]Ribavirin uptake was also observed in mouse ENT2-expressing Xenopus laevis oocytes, and gene silencing via the transfection of ENT2 small interfering RNA significantly reduced the [ 3 H]ribavirin transport into TM4 cells by 13%. Taken together, these results suggest that ENT2 partially contributes to ribavirin transport at the BTB., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Cerebral creatine deficiency syndromes (CCDS) are neurodevelopmental disorders caused by a decrease in creatine levels in the central nervous system (CNS) due to functional mutations in creatine synthetic enzymes or creatine transporter (CRT/SLC6A8). Although SLC6A8 mutations have been reported to be the most frequent cause of CCDS, sufficient treatment for patients with CCDS harboring SLC6A8 mutations has not yet been achieved. This study aimed to elucidate the molecular mechanism of SLC6A8 dysfunction caused by the c. 1699T > C missense mutation, which is thought to induce dysfunction through an unidentified mechanism. A study on SLC6A8-expressing oocytes showed that the c.1699T > C mutation decreased creatine uptake compared to that in wild-type (WT) oocytes. In addition, a kinetics study of creatine uptake revealed that the c.1699T > C mutation reduced the maximum uptake rate but not Michaelis-Menten constant. In contrast, the c.1699T > C mutation did not attenuate SLC6A8 protein levels or alter its cellular localization. Based on the SLC6A8 structure in the AlphaFold protein structure database, it is possible that the c.1699T > C mutation alters the interaction between the S567 and Y143 residues of SLC6A8, leading to decreased creatine transport function. These findings contribute to the understanding of the pathology of CCDS and to the development of strategies for CCDS treatment.