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Xenobiotica
the fate of foreign compounds in biological systems
Volume 44, 2014 - Issue 5
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General Xenobiochemistry

In vitro metabolism of thyroxine by rat and human hepatocytes

Vicki M. RichardsonPharmacokinetics Branch, Integrated Systems Toxicology Division, National Health and Environmental Effects Research Laboratory, Office of Research and Development, US Environmental Protection Agency Research Triangle Park, NCUSA;Curriculum in Toxicology, University of North Carolina Chapel Hill, NCUSACorrespondence[email protected]
,
Stephen S. FergusonLife Technologies Durham, NCUSA
,
Yusupha M. SeyPharmacokinetics Branch, Integrated Systems Toxicology Division, National Health and Environmental Effects Research Laboratory, Office of Research and Development, US Environmental Protection Agency Research Triangle Park, NCUSA
&
Michael J. DeVitoNational Toxicology Program, National Institute of Environmental Health Sciences Research Triangle Park, NCUSA
Pages 391-403 | Received 31 Jul 2013, Accepted 19 Sep 2013, Published online: 31 Oct 2013

Figures & data

Table 1. Liver microsomes and cytosol.

Table 2. Donor information for human hepatocytes.

Figure 1. Representative chromatogram of T4 and its metabolites in medium of SCRH. The graph shows metabolites [iodide (-I), T4G, T4S, T3 and rT3] in medium of SCRH following a 24-h incubation with 5.0 µM [125I]-T4 on Culture Day 6. Peaks were separated using UPLC. Fractions of eluent were collected and analyzed by gamma spectroscopy.

Figure 1. Representative chromatogram of T4 and its metabolites in medium of SCRH. The graph shows metabolites [iodide (-I), T4G, T4S, T3 and rT3] in medium of SCRH following a 24-h incubation with 5.0 µM [125I]-T4 on Culture Day 6. Peaks were separated using UPLC. Fractions of eluent were collected and analyzed by gamma spectroscopy.

Figure 2. Comparison of hepatic and intestinal T4G formation rates in microsomes and hepatic T4S formation rates in cytosols from rats and humans. Data are expressed as pmol/mg protein/min (mean). Data represent the average of duplicate experiments with variability of <30%; T4 conjugates were generated following an incubation with 4 μM [125I]-T4. Samples in (a) are pooled liver and intestine microsomes and were obtained from XenoTech. Samples in (b) are pooled and individual liver microsomes and were obtained from CellzDirect.

Figure 2. Comparison of hepatic and intestinal T4G formation rates in microsomes and hepatic T4S formation rates in cytosols from rats and humans. Data are expressed as pmol/mg protein/min (mean). Data represent the average of duplicate experiments with variability of <30%; T4 conjugates were generated following an incubation with 4 μM [125I]-T4. Samples in (a) are pooled liver and intestine microsomes and were obtained from XenoTech. Samples in (b) are pooled and individual liver microsomes and were obtained from CellzDirect.

Figure 3. T4 metabolite levels in media during time in culture. SCH arrive on Culture Day 2 and are untreated. SCH were incubated with 0.05 µM [125I]-T4 (rat) or 0.1 µM [125I]-T4 (human) for 24 h on Culture Days 3, 4, 5 or 6. Metabolites were separated using the established UPLC method. Metabolites analyzed are (a) T4G, (b) T4S and (c) T3 + rT3. Data are expressed as pmol/mg cellular protein [mean (human hepatocytes)] or [mean ± SD (rat hepatocytes)]. Human hepatocyte data represent the average of duplicates in a single experiment. Human hepatocytes are from two donors (Hu8092 and Hu8096). Rat hepatocytes are from three donors (n = 3).

Figure 3. T4 metabolite levels in media during time in culture. SCH arrive on Culture Day 2 and are untreated. SCH were incubated with 0.05 µM [125I]-T4 (rat) or 0.1 µM [125I]-T4 (human) for 24 h on Culture Days 3, 4, 5 or 6. Metabolites were separated using the established UPLC method. Metabolites analyzed are (a) T4G, (b) T4S and (c) T3 + rT3. Data are expressed as pmol/mg cellular protein [mean (human hepatocytes)] or [mean ± SD (rat hepatocytes)]. Human hepatocyte data represent the average of duplicates in a single experiment. Human hepatocytes are from two donors (Hu8092 and Hu8096). Rat hepatocytes are from three donors (n = 3).

Figure 4. Accumulation of [125I]-derived radioactivity in rat and human hepatocytes. SCH were incubated with 0.00005 µM (5000 cpm) per well [125I]-T4 on Culture Day 6. The accumulation of [125I]-T4 was determined over time (1–5 min). Data represent the average of duplicates in a single experiment. Individual hepatocyte donor values are presented. Data are expressed as percentage of dose. The lines represent the linear regression of the data (1–5 min). The curved lines are the non-linear regression of the data (1–30 min). Rat hepatocytes are from two donors (n = 2) and human hepatocytes are from three donors (n = 3).

Figure 4. Accumulation of [125I]-derived radioactivity in rat and human hepatocytes. SCH were incubated with 0.00005 µM (5000 cpm) per well [125I]-T4 on Culture Day 6. The accumulation of [125I]-T4 was determined over time (1–5 min). Data represent the average of duplicates in a single experiment. Individual hepatocyte donor values are presented. Data are expressed as percentage of dose. The lines represent the linear regression of the data (1–5 min). The curved lines are the non-linear regression of the data (1–30 min). Rat hepatocytes are from two donors (n = 2) and human hepatocytes are from three donors (n = 3).

Figure 5. T4 metabolite levels in media during incubation time. SCH were incubated with 0.05 µM [125I]-T4 (rat) or 0.1 µM [125I]-T4 (human) for on Culture Day 6. Metabolite appearance were determined over time (4–24 h). Metabolites were separated using the established UPLC method. Metabolites analyzed are (a) T4G, (b) T4S and (c) T3 + rT3. Data are expressed as pmol/mg cellular protein (mean). Data represent the average of duplicates in a single experiment. Limits of detection = 0.5 pmol/mg cellular protein. BLQ = below limits of quantitation.

Figure 5. T4 metabolite levels in media during incubation time. SCH were incubated with 0.05 µM [125I]-T4 (rat) or 0.1 µM [125I]-T4 (human) for on Culture Day 6. Metabolite appearance were determined over time (4–24 h). Metabolites were separated using the established UPLC method. Metabolites analyzed are (a) T4G, (b) T4S and (c) T3 + rT3. Data are expressed as pmol/mg cellular protein (mean). Data represent the average of duplicates in a single experiment. Limits of detection = 0.5 pmol/mg cellular protein. BLQ = below limits of quantitation.

Table 3. Mass balance of T4 and T4 metabolites after exposure of rat hepatocytes on Culture Day 6 to 0.05 µM [125I]-T4.

Table 4. Mass balance of T4 and T4 metabolites after exposure of human hepatocytes on Culture Day 6 to 0.1 µM [125I]-T4.

Figure 6. [125I]-T4 clearance in the media of rat (a) and human (b) hepatocytes. SCH were incubated with 0.05 µM [125I]-T4 (rat) or 0.1 µM [125I]-T4 (human) on Culture Day 6. The accumulation of [125I]-T4 was determined over time (4–24 h). Data represent the average of duplicates in a single experiment. Data are expressed as percentage of T4 remaining in media. The lines represent the linear regression of the data.

Figure 6. [125I]-T4 clearance in the media of rat (a) and human (b) hepatocytes. SCH were incubated with 0.05 µM [125I]-T4 (rat) or 0.1 µM [125I]-T4 (human) on Culture Day 6. The accumulation of [125I]-T4 was determined over time (4–24 h). Data represent the average of duplicates in a single experiment. Data are expressed as percentage of T4 remaining in media. The lines represent the linear regression of the data.

Figure 7. Metabolite levels in media of hepatocytes exposed to [125I]-T4 on Culture Day 6 for 24 h. (a) Rat hepatocytes were incubated with 0.05–100 µM [125I]-T4 and (b) human hepatocytes were incubated with 0.1–100 µM [125I]-T4 for 24 h. Data are expressed as pmol/mg cellular protein (mean). Data represent the average of duplicate experiments. The lines represent the linear regression of the data. Limits of detection = 0.5 pmol/mg cellular protein; BLQ = below limits of quantitation. n = 2 for rat hepatocytes and human hepatocytes (Hu8092 and Hu8096).

Figure 7. Metabolite levels in media of hepatocytes exposed to [125I]-T4 on Culture Day 6 for 24 h. (a) Rat hepatocytes were incubated with 0.05–100 µM [125I]-T4 and (b) human hepatocytes were incubated with 0.1–100 µM [125I]-T4 for 24 h. Data are expressed as pmol/mg cellular protein (mean). Data represent the average of duplicate experiments. The lines represent the linear regression of the data. Limits of detection = 0.5 pmol/mg cellular protein; BLQ = below limits of quantitation. n = 2 for rat hepatocytes and human hepatocytes (Hu8092 and Hu8096).

Figure 8. Comparison of metabolites in the media of SCRH and SCHH following treatment with PCB 153. Hepatocytes were incubated with 0.1% DMSO (control), 0.3, 3 or 30 µM PCB 153 for 72 h starting on Culture Day 3. Hepatocytes are then incubated for 24 h on Culture Day 6 with 0.05 µM (rat) or 0.1 µM (human) [125I]-T4. Metabolites were separated using the established UPLC method. Metabolites analyzed are (a) T4G, (b) T4S and (c) T3 + rT3. Data represent the average of duplicate experiments. Data are expressed as pmol/mg cellular protein (mean ± SD for rat hepatocytes and mean for human hepatocytes); Limits of detection = 0.4 pmol/mg cellular protein; BLQ = below limits of quantitation. n = 2 for rat hepatocytes. Human hepatocytes are from two donors (Hu8092 and Hu8096).

Figure 8. Comparison of metabolites in the media of SCRH and SCHH following treatment with PCB 153. Hepatocytes were incubated with 0.1% DMSO (control), 0.3, 3 or 30 µM PCB 153 for 72 h starting on Culture Day 3. Hepatocytes are then incubated for 24 h on Culture Day 6 with 0.05 µM (rat) or 0.1 µM (human) [125I]-T4. Metabolites were separated using the established UPLC method. Metabolites analyzed are (a) T4G, (b) T4S and (c) T3 + rT3. Data represent the average of duplicate experiments. Data are expressed as pmol/mg cellular protein (mean ± SD for rat hepatocytes and mean for human hepatocytes); Limits of detection = 0.4 pmol/mg cellular protein; BLQ = below limits of quantitation. n = 2 for rat hepatocytes. Human hepatocytes are from two donors (Hu8092 and Hu8096).

Table 5. Intrinsic clearance of [125I]-T4 following treatment with PCB 153.

 

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