Aminotriazole Alleviates Acetaminophen Poisoning via Downregulating P450 2E1 and Suppressing Inflammation

Aminotriazole (ATZ) is commonly used as a catalase (CAT) inhibitor. We previously found ATZ attenuated oxidative liver injury, but the underlying mechanisms remain unknown. Acetaminophen (APAP) overdose frequently induces life-threatening oxidative hepatitis. In the present study, the potential hepatoprotective effects of ATZ on oxidative liver injury and the underlying mechanisms were further investigated in a mouse model with APAP poisoning. The experimental data indicated that pretreatment with ATZ dose- and time-dependently suppressed the elevation of plasma aminotransferases in APAP exposed mice, these effects were accompanied with alleviated histological abnormality and improved survival rate of APAP-challenged mice. In mice exposed to APAP, ATZ pretreatment decreased the CAT activities, hydrogen peroxide (H2O2) levels, malondialdehyde (MDA) contents, myeloperoxidase (MPO) levels in liver and reduced TNF-α levels in plasma. Pretreatment with ATZ also downregulated APAP-induced cytochrome P450 2E1 (CYP2E1) expression and JNK phosphorylation. In addition, posttreatment with ATZ after APAP challenge decreased the levels of plasma aminotransferases and increased the survival rate of experimental animals. Posttreatment with ATZ had no effects on CYP2E1 expression or JNK phosphorylation, but it significantly decreased the levels of plasma TNF-α. Our data indicated that the LD50 of ATZ in mice was 5367.4 mg/kg body weight, which is much higher than the therapeutic dose of ATZ in the present study. These data suggested that ATZ might be effective and safe in protect mice against APAP-induced hepatotoxicity, the beneficial effects might resulted from downregulation of CYP2E1 and inhibiton of inflammation.


Experimental protocol
Fresh suspensions of APAP were prepared before each experiment by dissolving the compound in warm phosphate buffered saline (PBS, pH 7.2). Acute liver injury was induced in mice by intraperitoneal (i.p.) injection of APAP (350 mg/kg). To evaluate the prophylactic effects of ATZ, vehicle or various dose of ATZ (125 mg/kg, 250 mg/kg or 500 mg/kg, dissolved in normal saline, i.p., the volume of ATZ solutions in each of experimental groups was 5 ml/kg) was administered 0.5 h prior to APAP challenge (n = 8 per group). The mice were then returned to their cages and provided food and water ad libitum. The animals were sacrificed by decapitation at 8 h after APAP exposure, blood samples were harvested for detecting TNF-α and measuring aminotransferases. The right lobe of the liver was fixed in formalin for morphological analysis. Remaining liver tissues were thoroughly washed in cold physiological saline and stored at -80°C until required. To evaluate the therapeutic effects, ATZ (500 mg/kg, i.p.) was administered at 1 h, 2 h or 4 h after APAP challenge, blood and liver samples were harvested at 8 h after APAP insult (n = 8 per group).

Survival analysis
To determine the prophylactic effect of ATZ on mortality of APAP-challenged mice, vehicle or various dose of ATZ (125 mg/kg, 250 mg/kg or 500 mg/kg) was administered 0.5 h prior to APAP challenge (n = 20 per group); to determine the therapeutic effects of post-treatment with ATZ, ATZ (500 mg/kg) was administered at 1 h, 2 h or 4 h after APAP challenge (n = 20 per group). The survival rate after APAP administration was assessed four times a day for at least 7 days and the cumulative survival curve was depicted using the Kaplan-Meier method.

Determination of the median lethal dose (LD 50 ) of ATZ
To assess the safety of ATZ dose, the LD 50 of ATZ was determined. Based on the data from our pretests, the mice were randomly assigned into four groups with 20 animals in each group and they were injected intraperitoneally with a series of doses of ATZ (5200 mg/kg, 5460 mg/kg, 5733 mg/kg, and 6019 mg/kg). The percentage survival was recorded in each group for at least 7 days. The LD 50 and 95% confidence limit were calculated by the probit analysis method.

Aminotransferases determination
Liver injury was assessed at 8 h after APAP administration by measuring plasma enzyme activities of ALT and AST using the detection kits according to the manufacturer's instructions.

Histological analysis
Formalin-fixed specimens were embedded in paraffin and stained with hematoxylin & eosin routinely for conventional morphological evaluation under light microscope (Olympus, Tokyo, Japan). The degree of hepatocellular necrosis and hemorrhage were semi-quantified using a 0 (no lesion) to 4 (severe change) scoring system in 20 random fields at 100× magnification per animal (n = 3 per group) by a blinded pathologist as previously described [22].

TNF-α determination
Plasma samples were harvested and the concentrations of TNF-α were determined using ELISA kits according to the manufacturer's instructions.

MPO activity assay
The activity of MPO was determined with MPO assay kit according to the manufacturer's instructions. Briefly, the frozen liver tissues were thawed and homogenized in phosphate buffer containing 0.5% hexadecyltrimethylammonium bromide. The enzyme activity was determined by the MPO detection kit according to the manufacturer's instructions. The MPO activity was assessed according to the absorbance measured at 450 nm. All values were normalized by the total protein concentration of each sample.

CAT activity assay
The activity of CAT was determined with CAT assay kit according to the manufacturer's instructions. Briefly, samples were treated with excess H 2 O 2 for decomposition by CAT for 5 min, and the remaining H 2 O 2 coupled with a substrate was treated with peroxidase to generate a red product absorbs maximally at 520 nm. CAT activity was thus determined by measuring the decomposed H 2 O 2 . All values were normalized by the total protein concentration of each sample.

H 2 O 2 determination
The content of H 2 O 2 in liver tissue was determined with H 2 O 2 assay kit according to the manufacturer's instructions. H 2 O 2 could oxidize Fe 2+ to Fe 3+ , and then Fe 3+ reacted with xylenol orange leading to colorimetric reaction that could be further detected by a spectrometer at a wavelength of 560 nm. The concentration of H 2 O 2 was calculated according to standard concentration curve and normalized by the total protein concentration of each sample.

GSH measurement
The the content of reduced glutathione in liver tissues was determined with glutathione assay kit according to the manufacturer's instructions. The concentration of GSH was assessed according to the absorbance measured at 412 nm and calculated according to standard concentration curve. All values were normalized by the total protein concentration of each sample.

MDA measurement
Liver tissues were prepared to make 1:10 (w/v) homogenates, and the homogenates were then centrifuged at 12,000 g under 4°C for 20 min to collect supernatants for determination of MDA. The concentrations MDA was evaluated by the thiobarbituric acid-reactive substances method (TBARS) using a MDA detection kit according to the manufacturer's instructions. All values were normalized by the total protein concentration of each sample.

Western blot analysis
Total proteins from frozen liver samples were prepared according to the method described by the protein extract kit (Beyotime, Shanghai, China). The total protein concentration was determined using the BCA protein assay kit (Pierce, USA). Protein extracts were fractionated on 12% polyacrylamide-sodium dodecyl sulfate (SDS) gel and then transferred to nitrocellulose membrane. The membrane was blocked with 5%(w/v) nonfat milk in Tris-buffered saline (TBS) containing 0.05% tween-20, and then the membrane was incubated with primary antibody overnight at 4°C, followed by incubation with secondary antibody. Antibody binding was visualized with an ECL chemiluminescence system (Pierce, USA) and short exposure of the membrane to X-ray films (Kodak, Japan).

Statistical analysis
All data except the survival rate were expressed as a mean ± SD. Multigroup comparisons were performed using one-way ANOVA with the Turkey's post hoc test. The statistical significance between two groups was determined by the Student's t test. The statistical significance of the semi-quantified scores was determined by the Wilcoxon rank sum test. The survival statistics were compared with a Kaplan-Meier curve and log-rank test. Results were considered statistically significant when P<0.05.

Pretreatment with ATZ alleviated APAP-induced hepatotoxicity
The levels of ALT and AST, widely used as quantitative biochemical markers of hepatocellular damage [23], increased significantly after APAP challenge, but the elevation of these aminotransferases was suppressed in mice pretreated with ATZ in a dose and time-dependent manner (Fig 1). The histological examination found that intraperitoneal injection of APAP resulted in obvious histological abnormalities, including massive cell necrosis and extensive hemorrhage in liver tissues. Consistent with the decreased elevation of aminotransferases, pretreatment with ATZ markedly attenuated APAP-induced histological lesions (Fig 2). Our data also showed that pretreatment with ATZ dose-dependently improved the survival rate of APAPchallenged mice (Fig 3). In addition, the plasma aminotransferases level (Fig 1), the hepatic histological architecture (Fig 2) and the survival rate (data not shown) in mice treated with ATZ alone were not different from those of the control mice.

Pretreatment with ATZ suppressed APAP-induced oxidative stress
ATZ is a widely used CAT inhibitor [9][10], the intraperitoneal administration of ATZ significantly suppressed the activities of CAT in mice with or without APAP exposure ( Fig 4A). CAT is the major antioxidant enzyme that catalyzes the decomposition of H 2 O 2 [8]. Although ATZ significantly suppressed hepatic CAT activity, we unexpectedly found that ATZ reduced H 2 O 2 levels in liver tissue of APAP-challenged mice ( Fig 4B). In APAP exposed mice, hepatic contents of reduced glutathione decreased significantly, while ATZ partially reversed this abnormality ( Fig 4C). The level of MDA, an end product of lipid peroxidation in liver damage [24], was determined to evaluate the degree of oxidative injury. As shown in Fig 4D, APAP exposure induced significant increase of MDA, but ATZ markedly decreased the MDA contents in APAP exposed mice.

Pretreatment with ATZ suppressed APAP-induced inflammatory response
TNF-α is an important proinflammatory cytokine involved in progression of APAP-induced hepatotoxicity [25][26]. In APAP exposed mice, the plasma levels of TNF-α increased significantly whereas the levels of TNF-α in APAP-challenged mice were suppressed by ATZ ( Fig  5A). The infiltration of neutrophils was quantified by the MPO activity in liver tissues [27]. As expected, MPO activities increased significantly in APAP-challenged mice. In mice treated with ATZ, the upregulation of MPO by APAP was obviously lower (Fig 5B).  The degree of hepatocellular necrosis and hemorrhage were semi-quantified using a 0 (no lesion) to 4 (severe change) scoring system in 20 random fields at 100× magnification per animal (n = 3 per group). Data were expressed as mean ± SD, ## P<0.01, as compared with the APAP group.

Pretreatment with ATZ decreased JNK activation
The phosphorylation and activation of JNK is a crucial molecular event in the pathogenesis of APAP hepatotoxicity [21]. Western blot analysis indicated that APAP challenge markedly increased the phosphorylation level of JNK in the liver, while ATZ suppressed the level of phosphorylated JNK (Fig 6).

Pretreatment with ATZ downregulated CYP2E1 expression
APAP is metabolically activated mainly by CYP2E1 [28]. The present study showed that APAP-challenge markedly increased the contents of hepatic CYP2E1 protein. Treatment with ATZ significantly suppressed the induction of CYP2E1 (Fig 7).

Posttreatment with ATZ also attenuated APAP induced liver injury
Since the pre-insult treatment protocol is different from clinic settings and seems unrealistic in clinical treatment, the pharmacological efficiency of ATZ treated with clinically relevant post- insult administration strategy was also tested. The experimental data indicated that ATZ administered at 1 h or 2 h after APAP challenged could also decrease the elevation of ALT and AST. However, treatment with ATZ at 4 h after APAP exposure had no obvious effect on the levels of ALT and AST (Fig 8A-8F). In addition, the administration of ATZ at 1 h or 2 h after APAP exposure significantly improved the survival rate of mice (Fig 9). The data also indicated that posttreatment with ATZ significantly suppressed APAP-induced production of TNF-α but it had no effects on the levels of phosphorylated JNK or CYP2E1 (Fig 10).

The safety of ATZ in mice
Finally, the LD 50 and the 95% confidence limits of ATZ were determined to evaluate the safety of ATZ treatment. Our data indicated that the LD 50 of ATZ in mice was 5367.4 mg/kg body weight and the 95% confidence limits of ATZ were 5260.4-5476.6 mg/kg (Table 1), which is much higher then the dose of ATZ in the present study.

Discussion
APAP overdose is the most common cause of drug-induced liver injury in developed countries [2]. The severe oxidative stress induced by the highly reactive metabolite NAPQI plays central roles in the pathogenesis of APAP-induced hepatotoxicity [20]. The endogenous antioxidant enzymes CAT usually provides protective benefits in oxidative stress [8]. However, the present study found that the widely used CAT inhibitor ATZ significantly protected mice from APAPinduced acute liver injury. The protective benefits were evidenced by suppressed elevation of plasma aminotransferases, improved histopathological abnormality and increased survival rate.
It is well known that the CAT plays defensive roles in oxidative stress via catalyzing the decomposition of H 2 O 2 [8]. Inhibition of CAT by ATZ resulted in enhanced the cytotoxicity of Alzheimer's amyloid-beta peptide in both neuronal and non-neuronal cells [13], ATZ treatment also resulted in significantly higher elevations in intracellular free radicals and significantly lowered cell viability of fibroblasts exposed to UV radiation [14]. But in the present study, as well as in our previous study, suppression of CAT by ATZ decreased the levels of H 2 O 2 , reduced the generation of MDA and attenuated APAP or CCl 4 -induced hepatotoxicity [16]. The decreased levels of H 2 O 2 and MDA in these studies could not be explained by the suppressed activity of CAT. Although there was no direct evidence indicating that CAT could provide beneficial effects in APAP poisoning, the crucial protective function of CAT in CCl 4induced hepatotoxicity was well proven in acatalasemic mice and transgenic mice [17,29]. Therefore, the hepatoprotective effects of ATZ in APAP or CCl 4 -induced hepatotoxicity might not be attributed to its inhibitory effects on CAT. The level of H 2 O 2 is determined by both the biochemical generation and its degradative clearance. In the present study, inhibition of CAT by ATZ might block the degradation of H 2 O 2 , but these were not accompanied with increase in H 2 O 2 . Therefore, we questioned whether ATZ could modulate other target that involved in the metabolism of APAP and the generation of H 2 O 2 . The CYP2E1 is the major enzyme catalyzing the metabolism of APAP to hepatotoxic reactive metabolite [30]. The expression of CYP2E1 was induced by APAP and other hepatotoxic substance such as alcohol [28,[31][32]. The induction of CYP2E1 was implicated in alcohol-mediated increases in APAP hepatotoxicity [33], whereas suppression of CYP2E1 expression by tea polyphenols and other compounds attenuated APAP hepatotoxicity [34][35]. In the present study, the induction of CYP2E1 in APAP exposed mice was inhibited by pretreatment with ATZ, which might lead to suppressed generation of reactive metabolite and alleviated liver damage. Interestingly, the hepatotoxicity of CCl 4 also largely depends on CYP2E1, CCl 4 is mainly metabolized by CYP2E1 in the hepatocytes to produce the highly reactive free radicals, which lead to cellular damage [36]. Therefore, downregulation of CYP2E1 also seems to be a plausible explanation for the protective effects of ATZ on CCl 4 hepatotoxicity.
There is increasing evidence that the JNK plays a pivotal role in the induction of APAP hepatotoxicity [20]. The JNK is sustained phosphorylated and activated in human and murine liver with APAP-induced hepatotoxicity [37]. The activated JNK translocates to mitochondria, inducing mitochondria permeability transition and triggering hepatocyte death [38]. Inhibition of JNK by specific JNK inhibitors effectively rescued mice from APAP-induced liver injury doi:10.1371/journal.pone.0122781.g008 [37]. It was reported that JNK was sensitive to APAP-induced generation of reactive oxygen species such as H 2 O 2 [21,39]. In the present study, the induced phosphorylation of JNK in APAP-exposed mice could be suppressed by pretreatment with ATZ, which was correlated well with the downregulated CYP2E1 and the alleviated liver injury.  Posttreatment with ATZ suppressed the levels of plasma TNF-α but had no effects on JNK phosphorylation or CYP2E1 expression in APAP exposed mice. Mice were treated with vehicle or ATZ (500 mg/kg) at 1 h after APAP exposure. Hepatic levels of phosphorylated JNK, total JNK, CYP2E1, β-actin (A) and plasma levels of TNF-α (B) were determined at 8 h after APAP exposure. Data were expressed as mean ± SD, n = 8. ## P<0.01, as compared with the APAP group. More importantly, ATZ treated with clinically relevant post-insult administration strategy at the early stage of APAP exposure also decreased the levels of plasma aminotransferases and increased the survival rate APAP-challenged mice. However, the alleviated liver injury by ATZ posttreatment was not associated with CYP2E1 downregulation and JNK inhibition. Therefore, other CYP2E1-independent mechanisms might also be involved in the hepatoprotective effects of ATZ. In addition to oxidative stress induced tissue injury, the recruitment of neutrophils and the production of proinflammatory cytokines are critical for the progression of APAP hepatotoxicity [40][41]. In the present study, pretreatment with ATZ significantly suppressed APAP-induced elevation of hepatic MPO and the production of TNF-α. The plasma level of TNF-α in APAP-exposed mice also decreased in ATZ-posttreated group. The anti-inflammatory effects of ATZ have been confirmed in LPS-stimulated neutrophils in vitro and LPS-induced acute lung injury in vivo [15]. Moreover, TNF-α is a representative proinflammatory cytokine acting as a detrimental factor in various liver disorders and the presence of TNF-α significantly enhanced the toxicity of APAP to hepatocytes [25,[42][43]. These data suggested that the sterile inflammation in APAP challenged mice was inhibited after ATZ treatment. The anti-inflammatory action of ATZ might be another beneficial mechanism underlying the hepatoprotective effects of ATZ in APAP poisoning.
Taken together, the present study has shown that ATZ effectively attenuated liver damage and decreased mortality induced by APAP poisoning. The downregulation of CYP2E1 induction and the suppression of inflammation might be the important mechanisms underlying these protective benefits of ATZ. Importantly, treatment with ATZ with clinically relevant post-insult administration strategy could also provide therapeutic benefits and the therapeutic dose of ATZ is much lower than its LD 50 . Therefore, ATZ seems effective and safe in protecting mice against APAP-induced hepatotoxicity, but the underlying mechanisms remain to be further investigated.