FIAU-Induced Liver Toxicity Was Observed in TK-NOG Mice with Humanized Livers

Consistent with prior toxicology results, control (non-humanized) TK-NOG mice treated with a high dose of FIAU (400 mg/kg/d po) for 14 d tolerated the drug, and all treated control mice had normal plasma lactate (3.0 mmol/l±0.8) and ALT (63.7 U/l±15.7) levels (Figure 1A and 1B). However, chimeric TK-NOG mice with a high level (average of 12.5 mg/ml plasma human albumin, corresponding to >90%) of liver humanization (Table S1) could not tolerate treatment with this dose of FIAU. By the third day, all treated chimeric mice were lethargic, and several were overtly jaundiced. Since one mouse died and the other 14 remained extremely lethargic, drug treatment was terminated on day 4. Consistent with their appearance, all FIAU-treated chimeric mice had elevated serum lactate (15.9 mmol/l±3.8) and ALT (726 U/l±257.5) levels, which were significantly above their pretreatment values (p = 5×10−8 and 1×10−7, respectively). The lactate and ALT values were 5-fold and 11-fold, respectively, above those in control (non-humanized) TK-NOG mice treated with the same dose of FIAU for 4 or 14 d (Figure 1A and 1B). In contrast to the FIAU-treated chimeric mice, vehicle-treated chimeric mice had a normal appearance, and normal plasma ALT and lactate levels (Figure 1A and 1B). Consistent with FIAU causing a human-specific toxicity, the degree of lactate elevation correlated with the extent of liver humanization (Pearson's correlation: 0.86), and this correlation was highly significant (p<0.0001) (Figure 1C).

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larger image TIFF original image Download: Figure 1. FIAU-induced liver toxicity develops in TK-NOG mice with humanized livers. Control (non-humanized) (Cont, n = 12 per group) or chimeric (Hu, n = 15 per group) TK-NOG mice were treated with 400 mg/kg/d FIAU or vehicle (Veh) for 0, 4, or 14 d, and the plasma ALT (A) and lactate (B) levels were measured on the indicated days. The lactate and ALT levels were elevated in FIAU-treated chimeric mice, while control mice had normal ALT and lactate levels after 14 d of FIAU dosing. Each symbol represents the value measured in a control TK-NOG mouse or a TK-NOG mouse with a humanized liver; the dotted line across the graph indicates the upper limit of normal, and the short dashed lines show the average for each group. (C) The correlation between the human albumin and plasma lactate levels measured in 15 chimeric TK-NOG mice after 4 d of treatment with 400 mg/kg/d FIAU. The human serum albumin is a measure of the extent of liver humanization in chimeric TK-NOG mice. Each dot represents the values measured in one chimeric TK-NOG mouse. https://doi.org/10.1371/journal.pmed.1001628.g001

To investigate whether this toxicity was dose-dependent, chimeric TK-NOG mice with highly humanized livers (Table S1) were then treated with FIAU 100 mg/kg/d po. The 100-mg/kg/d dosing regimen was better tolerated than a 400-mg/kg/d dose; the chimeric mice were treated for 17 d before dosing was electively terminated because of weight loss. Chimeric TK-NOG mice lost weight over the 17-d treatment period, while FIAU-treated control TK-NOG mice did not (p = 0.028) (Figure S1). However, there were no deaths, and none of the FIAU-treated chimeric mice were overtly jaundiced. However, there was clear serological evidence of liver injury in the FIAU-treated mice with humanized livers; their serum ALT (235 U/l±72) was significantly increased (p = 0.0008, over 4-fold) relative to FIAU- (63 U/l±17) or vehicle-treated (60 U/l±7) control mice (Figure S1). Of importance, the serum ALT was not elevated in FIAU-treated relative to vehicle-treated control TK-NOG mice (Figure S1). Mice with humanized livers were next treated with FIAU 25 mg/kg/d po for 14 d, before the dosing was electively terminated. Although there were no deaths and the FIAU-treated mice with humanized livers did not lose body weight (Figure 2A), there was clear evidence of drug-induced liver toxicity. The FIAU-treated mice with humanized livers had statistically significant elevations of plasma transaminase (p = 0.0001; Figure 2B) and serum lactate (p = 0.013; Figure 2C) levels relative to vehicle-treated mice with humanized livers. Liver toxicity also developed in TK-NOG mice with humanized livers that were treated with a 10-fold lower FIAU dose (2.5 mg/kg/d) for 14 d. Although the mice with humanized livers treated with this dose of FIAU did not lose weight (p = 0.24) relative to vehicle-treated mice with humanized livers (Figure 3A), they had statistically significant elevations of plasma transaminase (p = 0.047; Figure 3B) and serum lactate (p = 0.008; Figure 3C) levels relative to vehicle-treated mice with humanized livers. These data establish that FIAU-induced liver toxicity could be detected in chimeric TK-NOG mice, and this dose-dependent toxicity could easily be detected at FIAU doses as low as 10-fold above the dose used in humans.

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larger image TIFF original image Download: Figure 2. Liver toxicity develops in TK-NOG mice with humanized livers treated with 25 mg/kg/d FIAU. Control (non-humanized) (TK or Cont, n = 6 per group) or chimeric (Hu, n = 6 per group) TK-NOG mice were treated with 25 mg/kg/d FIAU or vehicle (Veh) for 14 d, and their body weights (A) and plasma ALT (B) and lactate (C) levels were measured on the indicated days. (A) There was no difference in the weights of vehicle- or FIAU-treated control TK-NOG mice or TK-NOG mice with humanized livers. In (B and C), each symbol represents the value measured in a control TK-NOG mouse or a TK-NOG mouse with a humanized liver, the dotted line across the graph indicates the upper limit of normal, and the short dashed lines show the average for each group. https://doi.org/10.1371/journal.pmed.1001628.g002

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larger image TIFF original image Download: Figure 3. FIAU-induced liver toxicity is dose-dependent in TK-NOG mice with humanized livers. Control (non-humanized) (TK or Cont, n = 6 per group) or chimeric (Hu, n = 6 per group) TK-NOG mice were treated with 2.5 mg/kg/d FIAU or vehicle (Veh) for 14 d, and their body weights (A) and plasma ALT (B) and lactate (C) levels were measured on the indicated days. (A) There was no difference in the weights of vehicle- or FIAU-treated control TK-NOG mice or TK-NOG mice with humanized livers. In (B and C), each symbol represents the value measured in a control TK-NOG mouse or a TK-NOG mouse with a humanized liver, the dotted line across the graph indicates the upper limit of normal, and the short dashed lines show the average for each group. https://doi.org/10.1371/journal.pmed.1001628.g003

The histology of the livers obtained from chimeric TK-NOG mice treated with FIAU 400 mg/kg/d for 4 d had abnormalities that were restricted to the regions with engrafted human liver; the regions with remnant endogenous mouse liver were identical to those in untreated chimeric TK-NOG mice (Figure 4A and 4B). The livers of FIAU-treated mice with humanized livers had extensive vacuolar changes in the regions that contained human hepatocytes, and the presence of fat in these vacuoles was confirmed by staining with oil red O (Figure 3). No abnormalities were noted in the liver sections obtained from control TK-NOG mice that were treated with FIAU 400 mg/kg/d for 14 d (Figure 4C). Although they were less pronounced, fatty changes were also present in regions with human hepatocytes in livers obtained from at least two of the four chimeric mice examined after treatment with FIAU 100 mg/kg/d for 17 d (Figure S2). However, there were no apparent changes in fat vacuole content in the livers of the mice with humanized livers treated with FIAU 2.5 mg/kg/d for 14 d (Figure S3). Ultra-structural analysis by TEM indicated that the cytoplasm of human hepatocytes in the FIAU-treated chimeric mice had many enlarged fat vacuoles and mitochondria, which were characterized by their abnormal size and shape, and by a decreased number of cristae (Figure 5). The electron micrographs confirm that FIAU treatment caused mitochondrial damage and lipid accumulation selectively in human cells within the livers of chimeric TK-NOG mice.

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larger image TIFF original image Download: Figure 4. Histopathology of FIAU-induced liver toxicity in TK-NOG mice with humanized livers. Liver tissues were obtained from mice with humanized livers treated with FIAU 400/kg/d (#192) (A) or FIAU 0 mg/kg/d (#202) (B) for 4 d, or from a control TK-NOG mouse treated with FIAU 400 mg/kg/d for 4 d (C). The images (at 100× magnification) shown in the left column are of formalin-fixed paraffin-embedded tissues stained with a monoclonal anti–human cytokeratin 8 antibody and counterstained with hematoxylin. The antibody-stained dark brown regions have human hepatocytes, while the mouse hepatocytes appear in the light tan regions. The middle and right columns are higher magnification views (600×) of the boxed regions of the images shown on the left; the upper and lower images in (A) and (B) are from the boxed regions containing human and mouse hepatocytes, respectively. The middle column shows formalin-fixed paraffin-embedded tissues stained with H/E, and the images in the right column were stained with oil red O and counterstained with hematoxylin. The hepatocytes in livers obtained from the FIAU-treated control or from untreated mice with humanized livers do not contain the markedly increased number of vacuoles that are present in the H/E-strained sections of the FIAU-treated humanized mouse liver. Many of these vacuoles are stained with oil red O (arrows), which indicates that they contain fat. However, not all of the vacuoles are stained by oil red O (arrowheads). https://doi.org/10.1371/journal.pmed.1001628.g004