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. 2015 Aug;36(2):485-92.
doi: 10.3892/ijmm.2015.2254. Epub 2015 Jun 19.

Acetaminophen induces JNK/p38 signaling and activates the caspase-9-3-dependent cell death pathway in human mesenchymal stem cells

Giou-Teng Yiang  1 , Yung-Lung Yu  2 , Ko-Ting Lin  3 , Jen-Ni Chen  3 , Wei-Jung Chang  2 , Chyou-Wei Wei  3
Affiliations

Acetaminophen induces JNK/p38 signaling and activates the caspase-9-3-dependent cell death pathway in human mesenchymal stem cells

Giou-Teng Yiang et al. Int J Mol Med. 2015 Aug.

Abstract

Acetaminophen (APAP) is a widely used analgesic and antipyretic drug. Generally, the therapeutic dose of APAP is clinically safe, however, high doses of APAP can cause acute liver and kidney injury. Therefore, the majority of previous studies have focussed on elucidating the mechanisms of APAP-induced hepatotoxicity and nephrotoxicity, in addition to examining ways to treat these conditions in clinical cases. However, few studies have reported APAP-induced intoxication in human stem cells. Stem cells are important in cell proliferation, differentiation and repair during human development, particularly during fetal and child development. At present, whether APAP causes cytotoxic effects in human stem cells remains to be elucidated, therefore, the present study aimed to investigate the cellular effects of APAP treatment in human stem cells. The results of the present study revealed that high-dose APAP induced more marked cytotoxic effects in human mesenchymal stem cells (hMSCs) than in renal tubular cells. In addition, increased levels of hydrogen peroxide (H2O2), phosphorylation of c-Jun N-terminal kinase and p38, and activation of caspase-9/-3 cascade were observed in the APAP-treated hMSCs. By contrast, antioxidants, including vitamin C reduced APAP-induced augmentations in H2O2 levels, but did not inhibit the APAP-induced cytotoxic effects in the hMSCs. These results suggested that high doses of APAP may cause serious damage towards hMSCs.

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Figures

Figure 1
Figure 1
Cell survival rates following treatment with APAP. (A) NRK-52E cells were treated with 7.94 mM (high-dose) and 0.794 mM APAP. (B) hMSCs were treated with 7.94 mM (high-dose) and 0.794 mM APAP. The survival rate was lower in the 7.94 mM APAP-treated hMSCs, compared with the 7.94 mM APAP-treated NRK-52E cells. Data was calculated from four independent experiments and is presented as the means ± standard deviation. The statistical differences between 7.94 mM-treated and 0.794 mM-treated group were analyzed by the Student’s t-test. *P<0.05. APAP, acetaminophen; hMSC, human mesenchymal stem cell.
Figure 2
Figure 2
Nuclear condensation. (A) Control group. (B) APAP-treated group. hMSCs were treated with 7.94 mM APAP for 2 days, nuclear condensation (white arrow) was observed in the APAP-treated cells under a phase-contrast microscope at magnifications of x400. APAP, acetaminophen; hMSC, human mesenchymal stem cell.
Figure 3
Figure 3
Western blot analysis to determine caspase activation. The activities of (A) caspase-3, (B) caspase-8 and (C) caspase-9 were analyzed on day 3 in the control (lane 1), 0.794 mM APAP-treated (lane 2) and 7.94 mM APAP-treated (lane 3) cells. Ceaved caspase-3 and cleaved caspase-9 were markedly increased in the 7.94 mM APAP-treated cells. APAP, acetaminophen; hMSC, human mesenchymal stem cell.
Figure 4
Figure 4
Western blot analysis to determine the expression of mitogen-activated protein kinases, (JNK, p38 and ERK) and their phosphorylation. (A) JNK, p38 and ERK, and (B) p-JNK, p-p38 and p-ERK were observed at 30 min in the control (lane 1), 0.794 mM APAP-treated (lane 2) and 7.94 mM APAP-treated (lane 3) cells. The levels of p-JNK and p-p38 were increased in the 7.94 mM APAP-treated cells. JNK, c-Jun N-terminal kinase; ERK, extracellular signal-regulated kinase; p-, phosphorylated; APAP, acetaminophen.
Figure 5
Figure 5
O2− and H2O2 levels. (A) O2− levels were determined in the control and high-dose APAP-treated cells. (B) H2O2 levels were determined in the control and high-dose APAP-treated cells. The levels of O2− and H2O2 levels were measured following 1 h treatment. Data were analyzed from four independent experiments and are presented as the means ± standard deviation. *P<0.05, compared to the control group. O2−, oxygen; H2O2 hydrogen peroxide; APAP, acetaminophen.
Figure 6
Figure 6
H2O2 levels. The levels of H2O2 levels were determined in the control, high-dose APAP-treated and high-dose APAP + 0.5 mM vit C-treated cells. The levels of H2O2 were measured after 1 h treatment. Data were analyzed from four independent experiments and are presented as the means ± standard deviation.*P<0.05, compared to the control group. H2O2 hydrogen peroxide; APAP, acetaminophen; vit C, vitamin C.
Figure 7
Figure 7
Cell survival rates. The hMSCs were treated with 0.5 mM vit C, high-dose APAP, and high-dose APAP +0.5 mM vit C-treated cells. No significant difference in survival rates were observed between the APAP-treated and APAP + vit C-treated cells. Data were calculated from four independent experiments and are presented as the means ± standard deviation.*P<0.05, compared to vitamin C alone group; both APAP group and APAP plus vitamin C group have significant difference. hMSC, human mesenchymal stem cell; APAP, acetaminophen; vit C, vitamin C.
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