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Dual role of acetaminophen in promoting hepatoma cell apoptosis and kidney fibroblast proliferation

Acetaminophen (APAP), is a safe analgesic and antipyretic drug at therapeutic dose, and is widely used in the clinic. However, high doses of APAP can induce hepatotoxicity and nephrotoxicity. Most studies have focused on high-dose APAP-induced acute liver and kidney injury. So far, few studies have investigated the effects of the therapeutic dose (1/10 of the high dose) or of the low dose (1/100 of the high dose) of APAP on the cells. The aim of this study was to investigate the cellular effects of therapeutic- or low-dose APAP treatment on hepatoma cells and kidney fibroblasts. As expected, high-dose APAP treatment inhibited while therapeutic and low-dose treatment did not inhibit cell survival of kidney tubular epithelial cells. In addition, therapeutic-dose treatment induced an increase in the H2O2 level, activated the caspase-9/-3 cascade, and induced cell apoptosis of hepatoma cells. Notably, APAP promoted fibroblast proliferation, even at low doses. This study demonstrates that different cellular effects are exerted upon treatment with different APAP concentrations. Our results indicate that treatment with the therapeutic dose of APAP may exert an antitumor activity on hepatoma, while low-dose treatment may be harmful for patients with fibrosis, since it may cause proliferation of fibroblasts.

cells), hepatoma (HuH7 cells) and breast cancer (FM3A cells) (29)(30)(31)(32)(33). These studies also demonstrated, in different tumor cell types, that APAP-induced cell death is related to the proteins NF-κB, members of the Bcl-2 family, and the glycogen synthase kinase-3. In addition, APAP can enhance the chemotherapeutic anticancer effects of drugs used to treat neuroblastoma, leukemia and ovarian carcinoma (30,34,35). According to the above studies, APAP can activate different cytotoxic mechanisms in liver, kidney and tumor cells (14,19,21,31,36). To date, most studies have focused on the mechanisms of APAP-induced cytotoxicity and on how to prevent high-dose APAP-related poisoning of the liver and the kidneys. However, whether APAP can enhance cell proliferation remains unclear.
Kidney tubular epithelial cell damage can induce renal failure (37)(38)(39)(40). Kidney fibrosis, via fibroblast proliferation, can also cause renal failure (41)(42)(43). Therefore, both kidney tubular cell damage and fibroblast proliferation can cause kidney dysfunction. Recently, high-dose APAP-induced nephrotoxicity was reported and investigated (13,22,(44)(45)(46)(47). These studies found that high-dose APAP treatment can induce kidney tubular cell death in animal and cell models. In addition, numerous studies have demonstrated that high-dose APAP treatment can induce an increase in oxidative stress, causing tubular cell death through necrosis or the apoptotic pathway (13,22,44,47,48). However, there is no evidence that APAP can cause kidney dysfunction by inducing fibroblast proliferation. The present study is the first to demonstrate, to the best of our knowledge, that high doses of APAP (7.94 mM) can inhibit cell survival in kidney tubular cells (NRK-52E), while promoting cell proliferation in kidney interstitial fibroblasts (NRK-49F).
In addition, APAP can induce different cytotoxic mechanisms on different hepatoma cell lines. APAP can induce caspase-dependent apoptosis on hepatoma HuH7 and SK-Hep1 cells (31,49) and induces apoptosis and necrosis on hepatoma HepG2 cells (50). Additionally, a study demonstrated that high-dose APAP treatment can inhibit DOX-induced cell death in hepatoma HepG2 cells (36). Although APAP-induced apoptosis of hepatoma Hep3B cells was reported (51), the underlying mechanisms are still unclear.
Cell lines and cultures. The rat kidney cell lines NRK-52E (tubular epithelial cells) and NRK-49F (fibroblasts) and Hep3B cells were purchased from the Bioresource Collection and Research Center (Hsinchu, Taiwan). These cell lines were cultured in DMEM medium supplemented with 10% FBS, 2 mM L-glutamine, 100 IU/ml penicillin/streptomycin and 0.1 mM non-essential amino acids, and were maintained at 37˚C in a humidified atmosphere containing 5% CO 2 , as in (52,53).
Cell survival assay. Survival rates of NRK-52E, NRK-49F and Hep3B cells were determined with the MTT assay as previously described (54,55). Briefly, cells were cultured in 96-well plates. On the second day, cells were divided into the control and experimental groups. After cells were treated with 7.94 nM APAP, 0.794 nM APAP, 0.0794 nM APAP and 1nM TGF-B, respectively, cell survival rates were measured every day. The MTT assay was conducted daily according to the manufacturer's instructions. Absorbance was measured at 570 nm using a multi-well ELISA reader (Molecular Devices, Sunnyvale, CA, USA).

Quantification of H 2 O 2 and O 2
levels. H 2 O 2 and O 2 levels were measured using a lucigenin-amplified chemiluminescence method, as in (56,57). Briefly, 200 µl of cell lysate was mixed with 0.2 mmol/l of luminol solution (100 µl) for the quantification of the H 2 O 2 level, or with 0.1 mmol/l of lucigenin solution (500 µl) for the quantification of the O 2 level. Measurements were then performed on the CLA-FSI chemiluminescence analyzing system (Tohoku Electronic Industrial Co., Ltd., Sendal, Japan). Each assay was performed four times and results were expressed as the chemiluminescence count per 10 sec.
Data analysis. Data were obtained from four independent triplicate experiments and are presented as mean values of all data, with related standard deviations (SD).

APAP treatment reduces the survival rate of kidney tubular epithelial cells, while inducing proliferation of kidney fibroblasts.
Previous studies showed that a high dose of APAP (>5 mM) can cause cell cytotoxicity in vitro (15)(16)(17)(18)(19)(20). In accordance with these studies, we also found that high-dose (7.94 mM) APAP treatment reduces the survival rate of kidney tubular epithelial cells (NRK-52E line), in a time-dependent manner (Fig. 1A). The survival rate of NRK-52E cells did not decrease upon treatment with 1/10 of the high dose of APAP compared to high-dose treatment (Fig. 1A). These results suggest that APAP-induced cell cytotoxicity is dependent on APAP concentration and incubation time. However, to our surprise, although high-dose APAP treatment decreased the survival rate of NRK-52E cells, it promoted cell proliferation of kidney fibroblasts (NRK-49F line) (Fig. 1B). This was also observed upon treatment with 1/10 of the high dose of APAP (Fig. 1B). It is well established in the clinic that both tubular epithelial cell damage and kidney fibrosis can induce renal fail ure (13,22,41,43,47,48,63,64). Therefore, our findings indicate that APAP-induced renal failure may not only relate to the inhibition of tubular epithelial cell survival, but also to the promotion of renal fibroblast proliferation.

Low-dose APAP treatment induces proliferation of kidney fibroblasts.
Previous studies have demonstrated that high-dose APAP treatment can inhibit tubular epithelial cell survival to induce renal failure (13,22,43,47,48). In the present study, as shown in Fig. 1, high-dose APAP treatment inhibited growth of tubular epithelial cells, and induced proliferation of kidney fibroblasts. In patients with kidney fibrosis, it is important to prevent fibroblast proliferation, which further aggravates their condition. In order to enhance our understanding on the effects of APAP treatment on patients with fibrosis, it is therefore valuable to investigate whether low doses of APAP (below the therapeutic dose) can induce fibroblast proliferation. In this study, low-dose APAP treatment was applied on kidney fibroblasts to study its effects on cell growth. It is notable that low-dose APAP treatment did not inhibit cell survival of NRK-52E cells, while low-dose treatment induced cell proliferation in the fibroblast cell line NRK-49F (Fig. 2A). In addition, APAP induced fibroblast proliferation similarly to the treatment with the positive control TGF-β, and in a dose-dependent manner (Fig. 2B). APAP has not been reported to be toxic to liver and kidney cells at doses below the therapeutic dose in the clinic. However in our experiments, a low dose of APAP induced fibroblast proliferation, which may have harmful effects in patients with fibrosis. Thus, our  study suggests that these patients may be sensitive to even low doses of APAP.
The cytotoxic effects of APAP are more prominent in Hep3B compared to NRK52 cells. High-dose APAP treatment induced cytotoxic effects not only in the tubular cell line NRK-52E, but also in the hepatoma cell line Hep3B (Fig. 3A). Cell survival rates of treated Hep3B cells were lower compared to those observed in NRK-52E cells. However, at an APAP concentration that was 1/10 of the high dose (therapeutic dose), no obvious cytotoxic effects were observed in NRK-52E cells, while the survival rate of Hep3B cells was markedly reduced (Fig. 3B). Therefore, APAP exerts more prominent cytotoxic effects on Hep3B compared to NRK-52E cells. These results indicate that APAP, at a non-toxic concentration for healthy tubular cells, may exert an antitumor effect on hepatoma cells.

APAP treatment increases apoptosis of Hep3B cells via an increase in the H 2 O 2 level.
APAP-induced cytotoxic effects that relate to an increase in the generation of reactive oxygen species (ROS) were previously reported (65,66 (Fig. 4A), but not in the O 2 -, level in Hep3B cells (Fig. 4B). Therefore, APAP-induced cytotoxicity is possibly related to H 2 O 2 but not to O 2 -. In addition, microscopic observations of the nuclear morphology revealed nuclear condensation and DNA fragmentation in the APAP-treated Hep3B cells (Fig. 5). These results overall suggest that APAP can induce cell cytotoxicity via an increase in the H 2 O 2 level.

APAP activates the caspase-9/-3 cascade in Hep3B cells.
Caspase activation can induce cell apoptosis (60,61). In our study, APAP treatment also induced apoptosis of Hep3B cells, as indicated by results presented in Figs. 4 and 5. Therefore, caspase activities were next measured in Hep3B cells, focusing on the two major caspase cascades, the caspase-9/-3 and the caspase-8/-3, and using a substrate cleavage assay as previously described (60,61). The caspase-9 and -3 activities were found induced by treatment with 1/10 of the high dose of APAP ( Fig. 6A and C) However, the caspase-8 activity did not notably change upon APAP treatment (Fig. 6B). This result suggests that APAP can activate the caspase-9/-3 cascade to induce cell cytotoxicity in Hep3B cells.

Discussion
Both tubular epithelial cell damage and fibroblast proliferation can induce renal dysfunction (13,22,41,43,47,48,63,64). Numerous studies have demonstrated that an APAP overdose can reduce tubular epithelial cell survival, resulting in nephrotoxicity (13,22,(43)(44)(45)(46). Most of the studies to date have focused on high-dose APAP-induced acute intoxication of kidney tubular cells. These studies have highlighted the need to further investigate the effects of APAP and take these effects into consideration in order to prevent APAP-induced acute damage. However, it is still unclear whether low doses of APAP may cause chronic kidney damage. Our present study demonstrated that high-dose APAP treatment not only reduces survival of tubular epithelial cells, but it can also induce proliferation of fibroblasts, even at low doses. This implies that APAP-induced renal damage may occur through epithelial cell damage or fibroblast proliferation. In general, acute damage is easier to detect and diagnose compared to chronic damage; therefore, APAP overdose-induced acute intoxication is commonly observed, whereas low-dose APAP-induced damage is more likely to be ignored in the clinic. Here, we demonstrated that low-dose APAP treatment can promote fibroblast proliferation. Thus, we consider the therapeutic dose of APAP to be a safe analgesic and antipyretic agent for patients who do not show fibrosis, but potentially harmful to patients with kidney fibrosis.
The TGF-β signaling pathway was shown to be involved in renal damage (67)(68)(69). TGF-β-induced renal damage  has been associated with: i) tubular cell death (68,70,71); ii) epithelial mesenchymal transition (72,73); and iii) fibroblast proliferation (74,75). Up to now, no study has provided evidence that APAP can induce kidney fibroblast proliferation via TGF-β-related signals. In this study, NRK-49F cells (fibroblasts) treated with APAP showed a similar induction in proliferation to the one observed in the group treated with TGF-β. In addition, a previous study showed that TGF-β is significantly elevated in APAP-treated liver tissue (71). Based on these observations, we hypothesize that APAP induces kidney fibroblast proliferation via the TGF-β signaling pathway. Whether APAP also exerts effects on epithelial mesenchymal transition in kidney tubular cells warrants future investigation.  level. This is possibly the reason why N-acetyl cysteine, a substrate for glutathione synthesis, is applied on patients with APAP-induced poisoning in emergency clinical cases (77,78).
APAP-induced cell death has been extensively studied (13,22,44,47,48). These studies demonstrated that APAP induces cell death either via the apoptotic or the necrotic death pathways in different cells. In our study, features of apoptosis were observed in APAP-treated Hep3B cells, similar to previous studies (51,75). Moreover, our study further demonstrated that the caspase-9/-3 cascade is activated upon APAP treatment, while the caspase-8/-3 cascade is not. Caspase-9/-3 signaling related to mitochondrial damage and caspase-8/-3 signaling related to death receptor signals have been previously reported (60,61). Thus, our data suggest that APAP-induced cell cytotoxicity might be associated with mitochondrial damage in Hep3B cells. Finally, previous studies have shown cytotoxicity upon high-dose (>5 mM) APAP treatment in vitro (15)(16)(17)(18)(19)(20). In this study, high-dose APAP treatment induced cytotoxicity in both healthy kidney tubular cells and hepatoma cells. However, 1/10 of this dose was only cytotoxic to hepatoma cells. This suggests that non-toxic (to healthy cells) doses of APAP may be applied in the future as antitumor agents targeting cancer cells.
In summary, the present study shows that: i) APAP treatment can induce cell proliferation of kidney fibroblasts even at low doses, and thus we suggest that APAP treatment needs to be carefully monitored in patients with fibrosis; ii) APAP treatment can increase the H 2 O 2 level and activate the caspase-9/-3 cascade to cause cytotoxicity; and iii) the cytotoxic effects of APAP depend on the cell type, with hepatoma cells being more severely affected compared to healthy kidney tubular cells.