Article Figures & Data
Figures
- Fig. 1.
Concentration of ETV in fetal and maternal plasma at 0.25, 1.0, and 4.0 hours postdosing with 0.833 µg/g of ETV. Data are expressed as means ± S.D., n = 8.
- Fig. 2.
(A) mRNA expressions of relevant transporters in BeWo cells treated with or without 20 μM of forskolin for 48 hours. Compared with mRNA expression in BeWo cells treated without forskolin, *P < 0.05, **P < 0.01, ***P < 0.001; time-dependent accumulation of ETV (5 µM) in activated or nonactivated BeWo cells at 4°C or 37°C (B); ETV accumulation in activated or nonactivated BeWo cells at concentrations up to 2000 μM for 2 minutes at 4°C or 37°C; and the comparison of ETV (5 or 20 μM) uptake in BeWo cell treated with or without forskolin (C). All data represent means ± S.D. of two independent experiments in triplicate.
- Fig. 3.
Effects of SLC (A) and ABC transporter inhibitors (B) on the ETV accumulation in BeWo cells. Carnosine, carnitine, and salicylic acid were used as the inhibitors of PEPT2, OCTN2, and monocarboxylate transporter, whereas verapamil or GF120918, MK571, and Ko143 were use as the inhibitors of P-gp, MRP2, and BCRP, respectively. Compared with nonactivated BeWo cells treated with ETV group, *P < 0.05 and ***P < 0.001. Compared with activated BeWo cells treated with ETV group, ###P < 0.001. Compared with the control, △P < 0.05, △△P < 0.01, △△△P < 0.001. (C) Accumulation of rhodamine 123 (5 μM, 1 hour) in nonactivated BeWo cells. Compared with the control, *P < 0.05, **P < 0.01. (D) Accumulation of ETV (5 μM, 1 hour) in LLC-PK1-hBCRP and LLC-PK1 (mock) cells. Compared with the mock cells, ***P < 0.001; compared with LLC-PK1-hBCRP treated with ETV group, ##P < 0.01. All data represent means ± S.D. from three independent experiments conducted in triplicate.
- Fig. 4.
(A) Effect of general inhibitors (100 μM of adenosine and cytidine) of NTs, specific inhibitors of ENT or CNT (NBTI and 200 μM of phlorizin), and Na+-free medium (Na+ was replaced by N-methyl-d-glucamine) on the accumulation of ETV (5 μM, 2 minutes) in BeWo cells treated with or without 20 μM of forskolin for 48 hours. Compared with nonactivated BeWo cells treated with ETV group, ***P < 0.001. Compared with activated BeWo cells treated with ETV group, #P < 0.05, ###P < 0.001. Compared with the control, △△P < 0.01, △△△P < 0.001. (B) Effect of NBTI and Na+ on the accumulation of adefovir, emtricitabine, and tenofovir (20 µM, 2 minute) in nonactivated BeWo cells. Compared with control, *P < 0.05, ***P < 0.001. All data represent means ± S.D. from three independent experiments conducted in triplicate.
- Fig. 5.
Interaction of ETV with hCNT2/3. The MDCK cells transiently expressing hCNT2 or hCNT3 were verified by functional activity with the accumulation of guanosine (A) and mRNA expression level (B). Compared with mock cells, ***P < 0.001; compared with the accumulation without inhibitor, ###P < 0.001. Accumulation of ETV (10 μM, 5 minutes) in MDCK-hCNT2 (C) or MDCK-hCNT3 cells (D) was compared with that in mock cells. Phlorizin (200 µM) was used as an inhibitor of hCNT2/3. Concentration-dependent profiles of ETV uptake in MDCK-hCNT2 (E) or MDCK-hCNT3 cells (F) and Eadie-Hofstee plot. All data represent means ± S.D. from two independent experiments conducted in triplicate.
- Fig. 6.
Accumulations of probe substrates in mock cells and HEK293-hOAT4 (A) or MDCK-hOCT3 cells (C) in the absence or presence of ETV or probenecid (OAT4 inhibitor) or D22 (OCT3 inhibitor). Accumulation of ETV in mock cells and HEK293-hOAT4 (B) or MDCK-hOCT3 cells (D) with or without probenecid or D22. Compared with the accumulation in mock cells, ***P < 0.001; compared with the accumulation without inhibitor, ##P < 0.01 and ###P < 0.001. All data represent means ± S.D. from three independent experiments conducted in triplicate.
- Fig. 7.
Effects of SLC (A) and ABC (B) transporter inhibitors on the accumulation of ETV in PHTCs. Compared with the accumulation without inhibitors, *P < 0.05, **P < 0.01, ***P < 0.001. mRNA expressions level of SLC (C) and ABC (D) transporters in PHTCs. Data represent mean ± S.D. from three independent experiments conducted in triplicate.
- Fig. 8.
Schematic diagram of the placental transfer of ETV. CNT2/3, ENT1/2, and OCTN2 contributed to the ETV uptake from maternal circulation to trophoblast cells, whereas OCT3 contributed the ETV efflux from trophoblast cells to fetal circulation, and BCRP, MRP2, and P-gp might be involved in the efflux of ETV from trophoblast cells to maternal circulation.
Tables
Gene Direction Sequence(5′→3′) Product Length bp hENT1 Forward CAGAATGTGTCCTTGGTCACT 512 Reverse ATGATAACAGCACAGGCTGTG hENT2 Forward CCTCCGTCTGCTTCATCAACT 470 Reverse CTGGAAGACAGTGAAGACTGA hCNT1 Forward TGGAAGGTCTGGGACATGGAGAA 612 Reverse ATGATGCTTTGAGCAGGCAA hCNT2 Forward AAGAAGTAGAGCCTGAGGGAA 386 Reverse AACCAAGGAGACTCCTGCAAA hCNT3 Forward GAGAACGAGAACACATCAGGA 438 Reverse CCAGAACCAATGGCTGTTTAG hOCT3 Forward TCGCTCTGTTCAGGTCTGTG 115 Reverse TGGATGCCAGGATACCAAAG hOCTN1 Forward CGGAATATTGCCATAATGACC 72 Reverse CAGAGCAAAGTAACCCACTGAG hOCTN2 Forward GCAGCATCCTGTCTCCCTAC 91 Reverse GCTGTCAGGATGGTCAGACTT hOAT4 Forward CTGTGGAAAGTACCTCGCTCT 120 Reverse CTTGAAGTCGCCCAACTCG hBCRP Forward CCACTCCCACTGAGATTGAGA 73 Reverse TGCGTTCCTAAATCCTACCC hP-gp Forward GAAATTTAGAAGATCTGATGTCAAACA 110 Reverse ACTGTAATAATAGGCATACCTGGTCA hMRP2 Forward AGTGAATGACATCTTCACGTTTG 63 Reverse CTTGCAAAGGAGATCAGCAA hGADPH Forward GCACCGTCAAGGCTGAGAAC 138 Reverse TGGTGAAGACGCCAGTGGA
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