Primary Human Hepatocytes, But not HepG2 or Balb/c 3T3 Cells, Efficiently Metabolize Salinomycin and Are Resistant to Its Cytotoxicity
Abstract
:1. Introduction
2. Results
2.1. Cytotoxicity of Salinomycin and its Combinations with Tiamulin or Prednisolone
2.2. Metabolism of SAL
3. Discussion
4. Materials and Methods
4.1. Chemicals and Reagents
4.2. Cell Cultures
4.2.1. PHH and Culture Conditions
4.2.2. Cell lines and Culture Conditions
4.3. Exposure to Drugs
4.4. Cytotoxicity Assessment
4.4.1. MTT Assay
4.4.2. NRU Assay
4.4.3. TPC Assay
4.4.4. LDH Leakage Assay
4.5. Analysis of Drug Interactions
4.6. Determination of SAL Metabolites
4.6.1. Sample Preparation
4.6.2. LC-MS/MS Determination
4.6.3. Data Analysis
4.7. Statistical Analysis
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
References
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Sample Availability: Not available. |
Cell Model | Assay | Time | SAL | SAL + T1 | SAL + T10 | SAL + P |
---|---|---|---|---|---|---|
PHH | MTT | 12 h | >25 | >25 | N/A* | >25 |
24 h | >25 | >25 | N/A | >25 | ||
NRU | 12 h | 20.4 ± 1.9 a | 4.4 ± 2.3 b | N/A | 8.2 ± 2.5 c | |
24 h | 19.8 ± 1.6 a | 2.2 ± 0.6 b | N/A | 5.2 ± 1.2 c | ||
TPC | 12 h | >25 | >25 | N/A | >25 | |
24 h | >25 | >25 | N/A | >25 | ||
LDH | 12 h | 12.9 ± 3.2 a | 8.7 ± 1.3 b | N/A | 12.5 ± 0.7 a | |
24 h | 11.3 ± 0.7 a | 5.9 ± 2.4 b | N/A | 11.5 ± 2.2 a | ||
HepG2 | MTT | 24 h | 15.4 ± 1.7 a | 13.5 ± 1.1 a | 11.0 ± 1.9 b | >25 |
48 h | 12.9 ± 1.4 abc | 12.8 ± 0.2 a | 10.5 ± 1.7 b | 14.9 ± 1.5 c | ||
NRU | 24 h | 0.6 ± 0.2 a | <0.39 | <0.39 | 0.7 ± 0.1 a | |
48 h | <0.39 | <0.39 | <0.39 | <0.39 | ||
TPC | 24 h | 13.3 ± 2.5 a | 12.6 ± 1.7 a | 4.6 ± 0.6 b | 10.5 ± 1.5 a | |
48 h | 11.3 ± 1.3 a | 9.6 ± 0.7 a | 3.0 ± 0.1 b | 4.9 ± 1.2 c | ||
LDH | 24 h | >25 | 18.5 ± 1.1 b | 18.4 ± 1.1 b | >25 | |
48 h | 13.8 ± 0.2 a | 12.1 ± 1.3 ac | 5.6 ± 0.4 b | 11.4 ± 1.1 c | ||
Balb/c 3T3 | MTT | 24 h | 10.4 ± 1.4 a | 4.5 ± 0.7 b | 4.9 ± 0.7 b | 16.4 ± 1.5 c |
48 h | 4.3 ± 0.9 a | 3.7 ± 0.7 ac | 2.3 ± 0.4 b | 2.6 ± 0.5 bc | ||
NRU | 24 h | 0.6 ± 0.2 | <0.39 | <0.39 | <0.39 | |
48 h | <0.39 | <0.39 | <0.39 | <0.39 | ||
TPC | 24 h | 7.9 ± 1.1 a | 3.9 ± 0.7 b | 3.1 ± 0.7 b | 12.3 ± 2.0 c | |
48 h | 1.4 ± 0.5 ac | 0.9 ± 0.2 a | 0.5 ± 0.1 b | 1.8 ± 0.3 c | ||
LDH | 24 h | >25 | >25 | >25 | >25 | |
48 h | 23.1 ± 0.8 a | 22.6 ± 1.5 a | 19.1 ± 1.6 a | 24.8 ± 1.2 a |
ID | Relative RT a | Biotransformation Route | Ratio to Control b | % of Signal of All Metabolites in PHH | Detected In | ||||
---|---|---|---|---|---|---|---|---|---|
SAL | SAL + P | SAL + T | HepG2 | Balb/c 3T3 | |||||
M1 | 0.501 | Trihydroxylation | 1424 | 1.64 | 1.40 | 0.61 | |||
M2 | 0.552 | Dihydroxylation | 5357 | 4.00 | 4.00 | 2.59 | Yes, P c | ||
M3 | 0.563 | Dihydroxylation | 468 | 1.09 | 1.05 | 0.59 | |||
M4 | 0.573 | Dihydroxylation | 414 | 0.83 | 0.40 | 0.42 | |||
M5 | 0.617 | Dihydroxylation | ∞ d | 0.20 | 0.20 | 0.13 | |||
M6 | 0.621 | Dihydroxylation | 6167 | 15.36 | 14.18 | 9.59 | Yes, P | ||
M7 | 0.644 | Dihydroxylation | ∞ | 0.27 | 0.25 | 0.16 | |||
M8 | 0.656 | Dihydroxylation | 408 | 1.08 | 1.12 | 0.97 | |||
M9 | 0.670 | Dihydroxylation | 490 | 1.10 | 1.10 | 1.02 | |||
M10 | 0.682 | Hydroxylation + Demethylathion | ∞ | 0.05 | 0.05 | 0.05 | |||
M11 | 0.691 | Demethylathion | 11.3 | 0.04 | 0.04 | 0.03 | |||
M12 | 0.703 | Hydroxylation | 190 | 48.1 | 49.9 | 52.0 | Yes | ||
M13 | 0.709 | Dihydroxylation | 155 | 0.22 | 0.22 | 0.31 | |||
M14 | 0.722 | Hydroxylation | ∞ | 23.9 | 23.6 | 28.4 | Yes | ||
M15 | 0.733 | Dihydroxylation | 155 | 0.17 | 0.18 | 0.28 | |||
M16 | 0.789 | Dihydroxylation | 29.6 | 0.19 | 0.22 | 0.24 | |||
M17 | 0.801 | Hydroxylation | 59.4 | 1.33 | 1.39 | 1.86 | |||
M18 | 0.851 | Demethylathion | 48.5 | 0.04 | 0.22 | 0.32 | |||
M19 | 0.866 | Dihydroxylation | 14.0 | 0.29 | 0.33 | 0.28 | |||
M20 | 0.956 | Dihydroxylation | 3.00 | 0.12 | 0.13 | 0.12 |
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Radko, L.; Olejnik, M.; Posyniak, A. Primary Human Hepatocytes, But not HepG2 or Balb/c 3T3 Cells, Efficiently Metabolize Salinomycin and Are Resistant to Its Cytotoxicity. Molecules 2020, 25, 1174. https://doi.org/10.3390/molecules25051174
Radko L, Olejnik M, Posyniak A. Primary Human Hepatocytes, But not HepG2 or Balb/c 3T3 Cells, Efficiently Metabolize Salinomycin and Are Resistant to Its Cytotoxicity. Molecules. 2020; 25(5):1174. https://doi.org/10.3390/molecules25051174
Chicago/Turabian StyleRadko, Lidia, Małgorzata Olejnik, and Andrzej Posyniak. 2020. "Primary Human Hepatocytes, But not HepG2 or Balb/c 3T3 Cells, Efficiently Metabolize Salinomycin and Are Resistant to Its Cytotoxicity" Molecules 25, no. 5: 1174. https://doi.org/10.3390/molecules25051174