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    Mean ± SD cell viability for canine primary hepatocytes that were incubated with azathioprine (A), 6-mercaptopurine (B), or 6-thioguanine (C) at each of 6 concentrations (0.469, 0.937, 1.875, 3.750, 7.500, and 15.000 μmol/L) for 24 (black bars), 48 (gray bars), or 72 (white bars) hours. Each thiopurine-concentration combination was replicated in triplicate for incubation periods of 24 and 72 hours and in each of 6 wells for the 48-hour incubation period, and results are reported as the percentage of cell viability for the thiopurine-treated cells, compared with the cell viability for control cells that were incubated with a 0.1% dimethyl sulfoxide solution without a thiopurine. *Value differs significantly (P < 0.05) from the corresponding value for the control cells.

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    Mean ± SD LDH activity for canine primary hepatocytes that were incubated with azathioprine (A), 6-mercaptopurine (B), or 6-thioguanine (C) at each of 3 concentrations (0.937, 3.750, and 15.000 μmol/L) and positive control cells (non–thiopurine-treated hepatocytes that were treated with LDH lysis buffer solution to induce maximum cell lysis) for 24 (black bars), 48 (gray bars), or 72 (white bars) hours. Each thiopurine-concentration combination was replicated in triplicate for incubation periods of 24 and 72 hours and in each of 6 wells for the 48-hour incubation period, and results are reported as the percentage of LDH activity for the thiopurine-treated or positive control cells, compared with the LDH activity for negative control cells that were incubated with a 0.1% dimethyl sulfoxide solution without a thiopurine. *Value differs significantly (P < 0.05) from the corresponding value for the negative control cells.

  • 1. Bradford K, Shih DQ. Optimizing 6-mercaptopurine and azathioprine therapy in the management of inflammatory bowel disease. World J Gastroenterol 2011; 17: 41664173.

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  • 2. Petit E, Langouet S, Akhdar H, et al. Differential toxic effects of azathioprine, 6-mercaptopurine and 6-thioguanine on human hepatocytes. Toxicol In Vitro 2008; 22: 632642.

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  • 3. Menor C, Fernández-Moreno MD, Fueyo JA, et al. Azathioprine acts upon rat hepatocyte mitochondria and stress-activated protein kinases leading to necrosis: protective role of N-acetyl-l-cysteine. J Pharmacol Exp Ther 2004; 311: 668676.

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  • 4. Lee AU, Farrell GC. Mechanism of azathioprine-induced injury to hepatocytes: roles of glutathione depletion and mitochondrial injury. J Hepatol 2001; 35: 756764.

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  • 5. Tapner MJ, Jones BE, Wu WM, et al. Toxicity of low dose azathioprine and 6-mercaptopurine in rat hepatocytes. Roles of xanthine oxidase and mitochondrial injury. J Hepatol 2004; 40: 454463.

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  • 6. Lennard L. Implementation of TPMT testing. Br J Clin Pharmacol 2014; 77: 704714.

  • 7. Shipkova M, Franz J, Abe M, et al. Association between adverse effects under azathioprine therapy and inosine triphosphate pyrophosphatase activity in patients with chronic inflammatory bowel disease. Ther Drug Monit 2011; 33: 321328.

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  • 8. Haglund S, Taipalensuu J, Peterson C, et al. IMPDH activity in thiopurine-treated patients with inflammatory bowel disease—relation to TPMT activity and metabolite concentrations. Br J Clin Pharmacol 2008; 65: 6977.

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  • 9. Zabala-Fernández W, Barreiro-de Acosta M, Echarri A, et al. A pharmacogenetics study of TPMT and ITPA genes detects a relationship with side effects and clinical response in patients with inflammatory bowel disease receiving azathioprine. J Gastrointestin Liver Dis 2011; 20: 247253.

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  • 10. Dubinsky MC, Lamothe S, Yang HY, et al. Pharmacogenomics and metabolite measurement for 6-mercaptopurine therapy in inflammatory bowel disease. Gastroenterology 2000; 118: 705713.

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  • 11. Favrot C, Reichmuth P, Olivry T. Treatment of canine atopic dermatitis with azathioprine: a pilot study. Vet Rec 2007; 160: 520521.

  • 12. Fernández-Checa JC, Colell A, Garcia-Ruiz C. S-adenosyl-l-methionine and mitochondrial reduced glutathione depletion in alcoholic liver disease. Alcohol 2002; 27: 179183.

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  • 13. McGill MR, Jaeschke H. Metabolism and disposition of acetaminophen: recent advances in relation to hepatotoxicity and diagnosis. Pharm Res 2013; 30: 21742187.

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  • 14. Li M, Yuan H, Li N, et al. Identification of interspecies difference in efflux transporters of hepatocytes from dog, rat, monkey and human. Eur J Pharm Sci 2008; 35: 114126.

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  • 15. Gibco hepatocytes information sheet. Available at: www.lifetechnologies.com/us/en/home/life-science/drugdiscovery/adme-tox/gibco-hepatocytes/dog-hepatocytes.html. Accessed Oct 24, 2014.

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  • 16. Lennard L. The clinical pharmacology of 6-mercaptopurine. Eur J Clin Pharmacol 1992; 43: 329339.

  • 17. Lennard L, Welch JC, Lilleyman JS. Thiopurine drugs in the treatment of childhood leukaemia: the influence of inherited thiopurine methyltransferase activity on drug metabolism and cytotoxicity. Br J Clin Pharmacol 1997; 44: 455461.

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  • 18. Worth WS. Azathioprine effect on normal canine liver and kidney function. Toxicol Appl Pharmacol 1968; 12: 16.

  • 19. Wallisch K, Trepanier LA. Incidence, timing, and risk factors of azathioprine hepatotoxicosis in dogs. J Vet Intern Med 2015; 29: 513518.

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Effects of azathioprine, 6-mercaptopurine, and 6-thioguanine on canine primary hepatocytes

Kathleen E. LaDuke1, Sarah Ehling DVM, Dr Med Vet2, John M. Cullen DVM, PhD3,4, and Wolfgang Bäumer DVM, Dr Med Vet Habil5,6
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  • 1 Department of Molecular Biomedical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27606.
  • | 2 Department of Molecular Biomedical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27606.
  • | 3 Department of Population Health and Pathobiology, College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27606.
  • | 4 Center of Comparative Medicine and Translational Research, College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27606.
  • | 5 Department of Molecular Biomedical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27606.
  • | 6 Center of Comparative Medicine and Translational Research, College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27606.

Abstract

OBJECTIVE To investigate the cytotoxic effects of azathioprine, 6-mercaptopurine, and 6-thioguanine on canine hepatocytes.

SAMPLE Commercially available cryopreserved canine primary hepatocytes.

PROCEDURES The study consisted of 2 trials. In trial 1, hepatocytes were incubated with azathioprine, 6-mercaptopurine, or 6-thioguanine at 1 of 6 concentrations (0.468, 0.937, 1.875, 3.750, 7.500, or 15.000 μmol/L) for 24, 48, or 72 hours. At each time, cell viability and lactate dehydrogenase (LDH) activity were determined for each thiopurine-concentration combination, and alanine aminotransferase (ALT) activity was determined for cells incubated with each thiopurine at a concentration of 15 μmol/L. In trial 2, hepatocytes were incubated with azathioprine, 6-mercaptopurine, or 6-thioguanine at 1 of 3 concentrations (18.75, 37.50, or 75.00 μmol/L) for 24 hours, after which the free glutathione concentration was determined for each thiopurine-concentration combination and compared with that for hepatocytes incubated without a thiopurine (control).

RESULTS Incubation of hepatocytes with each of the 3 thiopurines adversely affected cell viability in a time- and concentration-dependent manner; however, this decrease in cell viability was not accompanied by a concurrent increase in LDH or ALT activity. Likewise, free glutathione concentration for hepatocytes incubated for 24 hours with supratherapeutic thiopurine concentrations (> 18.75 μmol/L) did not differ significantly from that of control cells.

CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that thiopurines adversely affected the viability of canine hepatocytes in a time- and concentration-dependent manner but had a nonsignificant effect on the LDH and ALT activities and free glutathione depletion of those hepatocytes.

Abstract

OBJECTIVE To investigate the cytotoxic effects of azathioprine, 6-mercaptopurine, and 6-thioguanine on canine hepatocytes.

SAMPLE Commercially available cryopreserved canine primary hepatocytes.

PROCEDURES The study consisted of 2 trials. In trial 1, hepatocytes were incubated with azathioprine, 6-mercaptopurine, or 6-thioguanine at 1 of 6 concentrations (0.468, 0.937, 1.875, 3.750, 7.500, or 15.000 μmol/L) for 24, 48, or 72 hours. At each time, cell viability and lactate dehydrogenase (LDH) activity were determined for each thiopurine-concentration combination, and alanine aminotransferase (ALT) activity was determined for cells incubated with each thiopurine at a concentration of 15 μmol/L. In trial 2, hepatocytes were incubated with azathioprine, 6-mercaptopurine, or 6-thioguanine at 1 of 3 concentrations (18.75, 37.50, or 75.00 μmol/L) for 24 hours, after which the free glutathione concentration was determined for each thiopurine-concentration combination and compared with that for hepatocytes incubated without a thiopurine (control).

RESULTS Incubation of hepatocytes with each of the 3 thiopurines adversely affected cell viability in a time- and concentration-dependent manner; however, this decrease in cell viability was not accompanied by a concurrent increase in LDH or ALT activity. Likewise, free glutathione concentration for hepatocytes incubated for 24 hours with supratherapeutic thiopurine concentrations (> 18.75 μmol/L) did not differ significantly from that of control cells.

CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that thiopurines adversely affected the viability of canine hepatocytes in a time- and concentration-dependent manner but had a nonsignificant effect on the LDH and ALT activities and free glutathione depletion of those hepatocytes.

In dogs, azathioprine is widely used as an immunosuppressive drug for the treatment of immune-mediated hemolytic anemia, pemphigus, and inflammatory bowel disease. Azathioprine is metabolized to 6-mercaptopurine. Both thiopurines and 6-thioguanine are used as immunosuppressant drugs, particularly in human medicine.1 Thiopurine metabolites such as 6-TGN inhibit de novo purine synthesis and can be incorporated as fraudulent nucleotides during DNA and RNA synthesis, which eventually leads to inhibition of cell proliferation.1

Metabolism of thiopurines is complex. Most studies of thiopurine metabolism or toxicosis have involved humans1,2 and rats.2–5 The exact metabolic pathway of thiopurines in dogs has yet to be determined. The reduction of azathioprine to 6-mercaptopurine and an imidazole group is catalyzed by glutathione transferase. Results of in vitro studies3,4 conducted with rat hepatocytes indicate that free glutathione concentrations decrease during the metabolism of azathioprine to 6-mercaptopurine. 6-Mercaptopurine is metabolized by 3 competitive pathways; it can be inactivated by XOD to form thiouric acid, metabolized by TPMT to form 6-MMP, or activated by HPRT to form 6-TIMP, which is then metabolized to 6-TGN, the active and therapeutic compound, or methylated by TPMT to form methyl-6-TIMP. Similar to 6-mercaptopurine, 6-thioguanine can be metabolized by HPRT to form 6-TGN, inactivated by XOD to form thiouric acid, or methylated by TPMT to form methyl-6-thioguanine. Thus, the enzymes TPMT, HPRT, and XOD play important roles in the complex metabolism of thiopurines. Although impairment of TPMT activity is associated with severe bone marrow toxicosis in human patients,6 variations in the activities of the other thiopurine metabolic enzymes have not been investigated to the same extent.7,8

The pathogenesis of thiopurine-induced hepatotoxicosis is not well understood. In human medicine, 5 of 232 (2.1%) adult patients with inflammatory bowel disease that were treated with azathioprine developed hepatotoxicosis during 1 clinical trial,9 and 16 of 92 (17%) pediatric patients with inflammatory bowel disease that were treated with 6-mercaptopurine had aspartate aminotransferase and ALT activities that were greater than twice the respective upper reference limits in another clinical trial.10 Dogs treated with azathioprine can also develop hepatopathy.11 Thiopurineinduced hepatoxicosis appears to be associated with high concentrations of 6-MMP and methyl-6-TIMP.1,10 Results of in vitro studies3,4 suggest that depletion of free glutathione concentration leads to the generation of reactive oxygen species. In hepatocytes, the metabolism of azathioprine causes depletion of free glutathione concentration, which results in the generation of reactive oxygen species and makes the cells prone to oxidative stress. Reactive oxygen species can induce lipid peroxidation or affect DNA and protein synthesis, which in turn causes hepatotoxicosis. This method of hepatotoxic pathogenesis is similar to that for acetaminophen or ethanol intoxication.12,13

The purpose of the study reported here was to investigate the cytotoxic effects of azathioprine, 6-mercaptopurine, and 6-thioguanine on canine hepatocytes. This study was undertaken as a first step toward determining the tolerance of dogs to thiopurine administration and their susceptibility to thiopurineinduced hepatotoxicosis.

Materials and Methods

Study design

Because results of another study14 indicate that cryopreserved and freshly isolated canine hepatocytes behave similarly in several functional assays, we chose to use commercially available cryopreserved hepatocytes for the study reported here. The study consisted of 2 trials. One trial was performed to determine the cytotoxic effects for each of 6 concentrations (0.468, 0.937, 1.875, 3.750, 7.500, and 15.000 μmol/L) of 3 thiopurines (azathioprine, 6-mercaptopurine, and 6-thioguanine) on canine hepatocytes, and the other trial was preformed to investigate glutathione depletion associated with each of 3 high concentrations (18.75, 37.50, and 75.00 μmol/L) of the 3 thiopurines on canine hepatocytes.

Cytotoxic effects of thiopurines on canine hepatocytes (trial 1)

Hepatocyte culture—Four batches of commercially available plateable canine (Beagle) hepatocytesa were obtained for the trial. All batches were routinely tested for phase I and phase II enzyme activities by the manufacturer as described.15

The hepatocytes were thawed and cultured in accordance with the manufacturer's instructions. Briefly, 96-well culture plates were coated with rat-tail collagenb 24 hours before cells were plated. The vials containing the hepatocytes were transferred on dry ice from a liquid nitrogen freezer to a 37°C water bath for thawing. Once thawed, the contents of each vial were transferred to a centrifuge vial that contained 48 mL of thaw medium.c Each vial was centrifuged at 70 × g for 4 minutes at room temperature (20°C), and the medium was decanted. The remaining pellet was resuspended in thaw medium, and the cells were counted.

Each well of a prepared 96-well collagen-coated culture plate was inoculated with 1.0 × 105 hepatocytes in 100 μL of thaw medium. The plates were incubated at 37°C with 5% CO2 for 5 hours to allow the cells time to attach to the wells. Then, the thaw medium in each well was replaced with 100 μL of maintenance mediumc that contained an additional nutrition overlay.d The plates were incubated at 37°C with 5% CO2 for an additional 24 hours.

Thiopurine treatment—Treatments consisted of 3 thiopurines (azathioprine, 6-mercaptopurine, and 6-thioguanine) at each of 6 concentrations (0.468, 0.937, 1.875, 3.750, 7.500, and 15.000 μmol/L). The concentrations were selected to be consistent with the thiopurine concentrations used in another in vitro study2 of human and rat hepatocytes. Each thiopurinee was dissolved in dimethyl sulfoxidee solution to achieve a concentration of 10 mg/mL. Sterile filtered maintenance medium was used to further dilute each thiopurine solution to each of the 6 treatment concentrations.

Twenty-four hours after the hepatocytes were plated on 96-well culture plates, 100 μL of each treatment was added to each designated well. Control wells were treated with 100 μL of vehicle solution (0.1% dimethylsulfoxide in maintenance medium) without any thiopurine. The plates were then incubated at 37°C with 5% CO2.

Cell viability assay—For each treatment, cell viability was determined in triplicate for incubation periods of 24 and 72 hours and determined in each of 6 wells for the 48-hour incubation period. At each time, each of 3 control wells that were designated for analysis were treated with 10 μL of LDH lysis buffer solutionf and incubated at 37°C for 5 minutes. Then, the supernatant from the wells being analyzed was removed and replaced with 100 μL of a 10% viability assay solution.g The viability assay solution contained a tetrazolium compound (3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]-2H-tetrazolium) that is bioreduced by viable cells into a colored formazan product that is soluble in tissue culture medium and indicative of dehydrogenase activity in metabolically active cells. The plates were incubated at 37°C for 90 minutes. Following incubation, 80 μL of the supernatant from each well was transferred to a well on a separate 96-well plate, and the light absorbance of each well was read at 490 nm. Cells that were treated with the vehicle solution served as the controls for 100% viability (positive control), and cells that were treated with the LDH lysis buffer solution served as the controls for 0% viability (ie, cells incubated with the LDH lysis buffer solution did not produce a colored formazan product; negative control). Cell viability results for the cells in each of the treated wells were calculated as the percentage of light absorbance compared with that for the positive control. For each treatment at each time, the mean cell viability was calculated for the 3 or 6 wells analyzed.

Determination of LDH activity—The LDH activity was also determined for each treatment after 24, 48, and 72 hours of incubation. The LDH activity was determined in the cell supernatant that was removed from the wells prior the addition of the cell viability assay solution by use of an LDH cytotoxicity assay.f The assay consisted of an enzymatic reaction that, in the presence of LDH, produces a red formazan product that can be measured spectrophotometrically. Briefly, for each well analyzed, 50 μL of the supernatant was combined with 50 μL of the reaction mixture solution and incubated at room temperature (20°C) for 30 minutes. The reaction was stopped by the addition of 50 μL of the stop solution. The light absorbance was measured for each sample at 490 and 680 nm. The supernatant obtained from the cells treated with the LDH lysis buffer solution served as the positive control (ie, maximum LDH activity), and the supernatant obtained from the cells treated with only the vehicle solution served as the negative control (minimum LDH activity).

Determination of ALT activity—For each thiopurine administered at a concentration of 15 μmol/L, the ALT activity was determined in duplicate after 24, 48, and 72 hours of incubation by use of an automated biochemical analyzerh at the Clinical Pathology Laboratory at the College of Veterinary Medicine at North Carolina State University. The ALT activity was measured without pyridoxal phosphate activation in accordance with the International Federation of Clinical Chemistry and Laboratory Medicine protocol.

Glutatione depletion in canine hepatocytes following treatment with high thiopurine concentrations (trial 2)

Hepatocyte culture—Trial 2 consisted of 2 experiments. In experiment 1, commercially available cryopreserved plateable canine (Beagle) hepatocytesa were plated into each well of a 24-well culture platei that was precoated with collagen by the manufacturer in a manner similar to that described for trial 1 except that each well was inoculated with 5.0 × 105 cells/well in 0.5 mL of maintenance medium. Commercially available fresh canine (Beagle) hepatocytesj that were plated on a 24-well culture plate at a concentration of 5.0 × 105 hepatocytes/well by the manufacturer were used for experiment 2.

Thiopurine treatment—For each experiment, treatments consisted of azathioprine, 6-mercaptopurine, or 6-thioguanine at each of 3 concentrations (18.75, 37.50, and 75.00 μmol/L). The concentrations were selected to be consistent with the thiopurine concentrations used in another in vitro study.3 Thiopurinese were dissolved in dimethyl sulfoxidee solution and diluted with sterile filtered maintenance medium to achieve each of the desired treatment concentrations in the same manner as that described for trial 1.

For each experiment, 24 hours after the hepatocytes were plated, 500 μL of each treatment was added to each of 6 wells. Negative control wells (n = 2) were treated with 500 μL of vehicle solution (0.1% dimethyl sulfoxide in maintenance medium) without any thiopurines. Each of the positive control wells (n = 2) were incubated with 500 μL of 0.05% H2O2 at 37°C for 10 minutes, then the H2O2 was replaced with 500 μL of maintenance medium. The plates were then incubated at 37°C with 5% CO2 for 24 hours.

Determination of glutathione concentration—For each experiment, glutathione concentration was determined in duplicate for each treatment by use of a commercially available detection kit,k and the mean was calculated and used for analysis. Briefly, 24 hours after thiopurine treatment, the medium was removed from all of the wells and the plate was frozen at −80°C. The cells were lysed during 3 vigorous freeze-thaw cycles; for each cycle, the culture plate was placed in a −80°C freezer for 15 minutes and a 37°C water bath for 10 minutes. Then, the lysed cells in each well were resuspended in 0.3 mL of 5% sulfosalicylic acid dyhydrate solutione and placed in a centrifuge tube. The cell suspension was vortexed and centrifuged at 8,000 × g for 10 minutes, and the supernatant was decanted and analyzed to determine the glutathione concentration. For each supernatant sample, assay buffer solution provided in the kit was used to create serial dilutions from 1:5 to 1:15. Standards, free thiol group detection reagent, and reaction mixture solutions were prepared in accordance with the manufacturer's recommended protocol. A 0 standard solution was created with 5% sulfosalicyclic acid dihydrate solution diluted 1:5 with assay buffer solution. For each supernatant dilution and standard, 50 μL was transferred into a well of a 96-well black plate. To determine free (reduced) glutathione concentration, 25 μL of the thiol group detection reagent solution was added to each well, and the plate was incubated in the dark for 15 minutes at room temperature. Binding of the reagent to the free thiol group of glutathione yields a fluorescent product. The fluorescent emission produced in each well was read at 510 nm with excitation at 390 nm. For determination of total glutathione concentration, 25 μL of the reaction mixture solution was added to each well, and the plate was incubated in the dark at room temperature for another 15 minutes. Then, the fluorescent emission of each well was read at 510 nm with excitation at 390 nm.

Statistical analysis—All results for cell viability, LDH activity, and glutathione concentration were reported as the percentage of the respective results for controls. Cell viability and LDH activity at 24, 48, and 72 hours after incubation with a thiopurine were compared among treatments by means of ANOVA followed by a Dunnett multicomparison post hoc test. As only 2 independent settings were performed for trial 2 (calculation of free glutathione concentration), no statistical analysis was done. All analyses were performed with statistical software,l and values of P < 0.05 were considered significant.

Results

Trial 1

Cell viability (expressed as a percentage of the cell viability for cells not treated with a thiopurine [control]) for cells treated with azathioprine, 6-mercaptopurine, or 6-thioguanine at each of the 6 concentrations (0.469, 0.937, 1.875, 3.750, 7.500, and 15.000 μmol/L) evaluated was summarized (Figure 1). Following incubation for 24 hours, the cell viability for the cells treated with azathioprine did not differ significantly from that for the control cells at any concentration evaluated; however, the cell viability for cells treated with 6-mercaptopurine at 7.5 and 15 μmol/L was significantly lower than that for the control cells, and the cell viability for cells treated with 6-thioguanine was significantly lower than that for the control cells at most concentrations evaluated. Cell viability tended to decrease as thiopurine concentration increased and was particularly noticeable after 48 and 72 hours of incubation. The exception was cell viability after 24 hours of incubation with 6-thioguanine, which remained fairly constant across all concentrations evaluated.

Figure 1—
Figure 1—

Mean ± SD cell viability for canine primary hepatocytes that were incubated with azathioprine (A), 6-mercaptopurine (B), or 6-thioguanine (C) at each of 6 concentrations (0.469, 0.937, 1.875, 3.750, 7.500, and 15.000 μmol/L) for 24 (black bars), 48 (gray bars), or 72 (white bars) hours. Each thiopurine-concentration combination was replicated in triplicate for incubation periods of 24 and 72 hours and in each of 6 wells for the 48-hour incubation period, and results are reported as the percentage of cell viability for the thiopurine-treated cells, compared with the cell viability for control cells that were incubated with a 0.1% dimethyl sulfoxide solution without a thiopurine. *Value differs significantly (P < 0.05) from the corresponding value for the control cells.

Citation: American Journal of Veterinary Research 76, 7; 10.2460/ajvr.76.7.649

The LDH activity for cells treated with each thiopurine at concentrations of 0.937, 3.750, and 15.000 μmol/L and positive control cells (cells treated with the lysis buffer solution) as a percentage of the LDH activity for cells not treated with a thiopurine or lysis buffer (negative control) was summarized (Figure 2). As expected, the LDH activity for the positive control cells was significantly greater than the LDH activity for the negative control cells following 24, 48, and 72 hours of incubation (except for 6-mercaptopurine incubation for 24 hours). However, the LDH activity for the cells treated with the thiopurines did not differ significantly from that for the negative control cells at any concentration or time.

Figure 2—
Figure 2—

Mean ± SD LDH activity for canine primary hepatocytes that were incubated with azathioprine (A), 6-mercaptopurine (B), or 6-thioguanine (C) at each of 3 concentrations (0.937, 3.750, and 15.000 μmol/L) and positive control cells (non–thiopurine-treated hepatocytes that were treated with LDH lysis buffer solution to induce maximum cell lysis) for 24 (black bars), 48 (gray bars), or 72 (white bars) hours. Each thiopurine-concentration combination was replicated in triplicate for incubation periods of 24 and 72 hours and in each of 6 wells for the 48-hour incubation period, and results are reported as the percentage of LDH activity for the thiopurine-treated or positive control cells, compared with the LDH activity for negative control cells that were incubated with a 0.1% dimethyl sulfoxide solution without a thiopurine. *Value differs significantly (P < 0.05) from the corresponding value for the negative control cells.

Citation: American Journal of Veterinary Research 76, 7; 10.2460/ajvr.76.7.649

For all cells treated with 1 of the 3 thiopurines at a concentration of 15 μmol/L, the ALT activities in the supernatants were within the range of ALT activities (53 to 249 U/L) observed in the supernatants of the control cells that were not treated with any thiopurines at all observation times. Thus, ALT activity appeared to be unaffected by the concurrent 50% to 80% decrease in cell viability.

Trial 2

When data from both experiments of trial 2 were combined, the free glutathione concentration did not differ from that for the negative control cells (cells not treated with a thiopurine) for any of the cells treated with a thiopurine at any high concentration (18.75, 37.50, and 75.00 μmol/L) evaluated (Table 1). Conversely, the free glutathione concentration in the positive control cells (hepatocytes incubated with 0.05% H2O2 for 10 minutes) was considerably less than the free glutathione concentration of the negative control cells (mean ± SD percentage of the negative control, 16.1 ± 0.9%). The free glutathione concentration for cells treated with azathioprine at 75 μmol/L was substantially lower than that for the negative control cells in one of the experiments, which resulted in the fairly large SD for that particular treatment.

Table 1—

Free (reduced) glutathione concentration for canine primary hepatocytes that were incubated with azathioprine, 6-mercaptopurine, or 6-thioguanine at each of 3 concentrations (18.75, 37.50, and 75.00 μmol/L) for 24 hours expressed as the mean ± SD percentage of the free glutathione concentration for cells that were not treated with a thiopurine (negative control).

ThiopurineConcentration (μmol/L)Mean ± SD % of negative control
Azathioprine18.75100.8 ± 18.9
 37.50102.5 ± 20.9
 75.00 
6-Mercaptopurine18.75104.8 ± 9.3
 37.50116.1 ± 29.1
 75.00112.0 ± 25.2
6-Thioguanine18.75101.0 ± 19.1
 37.50106.9 ± 17.4
 75.0097.1 ± 25.4

For each thiopurine-concentration combination, the results represent mean ± SD for duplicate samples within 2 experiments (ie, n = 4 samples). Commercially available cryopreserved plateable canine (Beagle) hepatocytes were used for experiment 1, and commercially available freshly plated canine (Beagle) hepatocytes were used for experiment 2.

Discussion

Results of the present study indicated that the viability of canine hepatocytes was inversely proportional to both the concentration and duration of time that the cells were exposed to thiopurines. However, there was no apparent concurrent effect on the LDH activity of canine hepatocytes or on glutathione depletion or ALT activity when cells were treated with thiopurine concentrations above the therapeutic range (≥ 15 μmol/L). These findings suggested that canine hepatocytes are susceptible to thiopurine-induced cytotoxicosis, although the pathogenesis of that cytotoxicosis remains unknown.

The range of concentrations (0.469 to 15.000 μmol/L) we chose to evaluate for each thiopurine in trial 1 was chosen to be consistent with the plasma thiopurine concentrations likely to be achieved in dogs after administration of therapeutic (1 to 2 mg/kg) or supratherapeutic (> 2 mg/kg) doses of azathioprine. Interestingly, for all 3 thiopurines evaluated in the present study, concentrations considered therapeutically relevant for human patients (eg, 6.9 μmol of 6-mercaptopurine/L after IV infusion or 0.7 to 3.7 μmol of 6-mercaptopurine/L after PO administration16,17) were associated with a significant decrease in the viability of canine hepatocytes after 48 and 72 hours of incubation.

Results of another study2 indicate that the in vitro susceptibility of rat hepatocytes to thiopurine-induced cytotoxicosis is substantially greater than that of human hepatocytes. Incubation of human hepatocytes with azathioprine, 6-mercaptopurine, or 6-thioguanine at a concentration of 5 or 25 μmol/L for 48 and 72 hours resulted in only a moderate decrease in cell viability, whereas incubation of rat hepatocytes with the same thiopurine concentrations for the same duration resulted in a marked decrease in cell viability.2 On the basis of the findings of the present study, it appears that the susceptibility of canine hepatocytes to thiopurine-induced cytotoxicosis falls somewhere between that of human and rat hepatocytes.

The time- and concentration-dependent effects of thiopurine treatment on canine hepatocyte viability did not substantially differ among the 3 thiopurines evaluated. This was somewhat unexpected because 6-thioguanine is a poor substrate for TPMT, which limits production of the hepatotoxin methyl-6-TIMP.1 6-Thioguanine is less cytotoxic to human hepatocytes than is azathioprine.1 We expected that 6-thioguanine would likewise be less cytotoxic than azathioprine to canine hepatocytes, but our findings did not support that supposition.

Interestingly, the thiopurine-induced decrease in cell viability in the present study was not accompanied by an increase in either the LDH or ALT activity in the cell supernatant. In in vitro studies3,4 of rat hepatocytes, a decrease in metabolic activity, or cell viability, was associated with an concurrent increase in LDH activity. In an in vivo study18 that involved laboratory Beagles, administration of a high dose of azathioprine (4 mg/kg) was associated with an increase in the activities of ALT and aspartate aminotransferase from basal levels after approximately 5 days. It is possible that LDH release and induction of ALT production might also be delayed in vitro. In the present study, hepatocyte viability was markedly decreased after 72 hours of incubation with the thiopurines, even at the lowest concentrations evaluated, and we chose to not incubate the hepatocytes for a longer duration. Therefore, we might have missed any increases in LDH and AST activities that were delayed > 72 hours.

Investigators of another study3 concluded that apoptosis was not involved in the pathogenesis of thiopurine-induced cytotoxicosis in rat hepatocytes. Because the LDH activity in the supernatant of cells incubated with thiopurines did not differ significantly from that in the supernatant of control cells that were not treated with a thiopurine in the present study, we cannot definitively conclude that apoptosis was not associated with the thiopurine-induced decrease in the viability of canine hepatocytes.

During trial 2 of the present study, we treated canine hepatocytes with higher thiopurine concentrations, compared with those used in an in vitro study3 of rat hepatocytes to determine whether acute depletion of free glutathione concentration might contribute to the cytotoxicity of thiopurines. Our findings indicated that the free glutathione concentration in the supernatants of cells after 24 hours of incubation with azathioprine, 6-mercaptopurine, or 6-thioguanine at the highest concentration (75 μmol/L) evaluated did not differ markedly from that in the supernatant of cells not treated with a thiopurine. This finding is similar to results of another study4 in which abnormal depletion of the free glutathione concentration was not observed for a human hepatocyte cell line that was incubated with azathioprine (500 μmol/L) for 16 hours. Conversely, in that same study,4 the free glutathione concentration was significantly depleted for rat hepatocytes following incubation with azathioprine (250 μmol/L) for 16 hours. Incubation of rat hepatocytes with azathioprine (150 μmol/L) for 24 hours resulted in acute depletion of the free glutathione concentration by approximately 80%.3 Thus, it appears that canine hepatocytes are less prone than are rat hepatocytes to acute glutathione depletion following thiopurine treatment.

In a recent retrospective study,19 5 of 34 (15%) dogs treated with azathioprine develop hepatotoxicosis. In that study,19 hepatotoxicosis was defined as a serum ALT concentration greater than twice the upper reference limit.

It is possible that an individual dog may have a metabolic profile that favors the accumulation of hepatotoxic metabolites such as 6-MMP or methyl-6-TIMP, which makes them particularly susceptible for thiopurine-induced hepatoxicosis in vivo. Results of a study20 of a large population of dogs indicate that the activity of TMPT, the enzyme that produces inactive azathioprine metabolites, varies greatly among individual dogs.

Findings of the present study suggested that canine hepatocytes are similar to human hepatocytes in that they are not as susceptible to thiopurine-induced cytotoxicosis as are rat hepatocytes. Incubation of canine hepatocytes with any of 3 thiopurines (azathioprine, 6-mercaptopurine, or 6-thioguanine) resulted in a decrease in cell viability in a concentration- and time-dependent manner, but did not affect LDH or ALT activities or depletion of free glutathione concentrations at even the highest concentrations evaluated (75 μmol/L). Consequently, the pathogenesis of thiopurine-induced cytotoxicosis remains unknown.

Acknowledgments

The authors declare no conflicts of interest.

Supported by a grant from the Center of Comparative Medicine and Translational Research, College of Veterinary Medicine, North Carolina State University.

ABBREVIATIONS

6-MMP

6-methylmercaptopurine

6-TGN

6-thioguanine nucleotide

6-TIMP

6-thioinosine monophosphate

ALT

Alanine aminotransferase

HPRT

Hypoxanthine phosphoribosyl transferase

LDH

Lactate dehydrogenase

TPMT

Thiopurine methyltransferase

XOD

Xanthine oxidase

Footnotes

a.

Canine primary hepatocytes, lot Nos. DB268 (male; n = 1 vial), DB274 (female, 3 vials), and DB297 (male; 1 vial), Gibco, Frederick, Md.

b.

Collagen type I, Roche Diagnostics, Mannheim, Germany.

c.

Gibco, Frederick, Md.

d.

Matrigel (end concentration, 250 μg/mL), BD Biosciences, Bedford, Mass.

e.

Sigma-Aldrich, St Louis, Mo.

f.

Pierce Biotechnology, Rockford, Ill.

g.

CellTiter 96 AQueous One Solution cell proliferation assay, Promega, Mannheim, Germany.

h.

COBAS INTEGRA 400 plus, Roche Diagnostics, Indianapolis, Ind.

i.

Collagen I plated wells, BD Biosciences, Bedford, Mass.

j.

Freshly isolated canine primary hepatocytes, lot No. DGB141 (male; n = 1 24-well plate), Triangle Research Lab, Charlottesville, Va.

k.

Glutathione fluorescent detection kit, Arbor Assays, Ann Arbor, Mich.

l.

Prism, version 6.01, GraphPad Software, La Jolla, Calif.

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Contributor Notes

Address correspondence to Dr. Bäumer (wbaeumer@ncsu.edu).