Córdoba‑Jover et al. J Nanobiotechnol (2019) 17:112 https://doi.org/10.1186/s12951-019-0544-5 Journal of Nanobiotechnology RESEARCH Open Access Cerium oxide nanoparticles improve liver regeneration after acetaminophen‑induced liver injury and partial hepatectomy in rats Bernat Córdoba‑Jover1†, Altamira Arce‑Cerezo1†, Jordi Ribera1, Montse Pauta1, Denise Oró1, Gregori Casals1,2, Guillermo Fernández‑Varo1, Eudald Casals3,4,6, Victor Puntes3,4,5, Wladimiro Jiménez1,7 and Manuel Morales‑Ruiz1,2,7* Abstract Background and aims: Cerium oxide nanoparticles are effective scavengers of reactive oxygen species and have been proposed as a treatment for oxidative stress-related diseases. Consequently, we aimed to investigate the effect of these nanoparticles on hepatic regeneration after liver injury by partial hepatectomy and acetaminophen overdose. Methods: All the in vitro experiments were performed in HepG2 cells. For the acetaminophen and partial hepatec‑ tomy experimental models, male Wistar rats were divided into three groups: (1) nanoparticles group, which received 0.1 mg/kg cerium nanoparticles i.v. twice a week for 2 weeks before 1 g/kg acetaminophen treatment, (2) N-acetyl- cysteine group, which received 300 mg/kg of N-acetyl-cysteine i.p. 1 h after APAP treatment and (3) partial hepatec‑ tomy group, which received the same nanoparticles treatment before partial hepatectomy. Each group was matched with vehicle-controlled rats. Results: In the partial hepatectomy model, rats treated with cerium oxide nanoparticles showed a significant increase in liver regeneration, compared with control rats. In the acetaminophen experimental model, nanoparticles and N-acetyl-cysteine treatments decreased early liver damage in hepatic tissue. However, only the effect of cerium oxide nanoparticles was associated with a significant increment in hepatocellular proliferation. This treatment also reduced stress markers and increased cell cycle progression in hepatocytes and the activation of the transcription fac‑ tor NF-κB in vitro and in vivo. Conclusions: Our results demonstrate that the nanomaterial cerium oxide, besides their known antioxidant capaci‑ ties, can enhance hepatocellular proliferation in experimental models of liver regeneration and drug-induced hepatotoxicity. Keywords: Liver regeneration, Oxidative stress, Cerium oxide nanoparticles, Partial hepatectomy, Acetaminophen- induced liver injury *Correspondence: morales@clinic.cat † Bernat Córdoba-Jover and Altamira Arce-Cerezo contributed equally to this study 1 Biochemistry and Molecular Genetics Department, Hospital Clínic of Barcelona, IDIBAPS, CIBERehd, 170 Villarroel St., 08036 Barcelona, Spain Full list of author information is available at the end of the article © The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat​iveco​mmons​.org/licen​ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Córdoba‑Jover et al. J Nanobiotechnol (2019) 17:112 Page 2 of 12 Background regenerative livers. To test our hypothesis we studied The liver regenerative capability is essential in the success the hepatic regenerative process using two different rat of some treatments for chronic liver diseases, such as experimental models of liver regeneration that are com- tumor resection and donor liver transplantation, which monly associated with ROS production: partial hepatec- are conditioned by adequate liver regeneration [1, 2]. tomy (PHx) [35, 36] and DILI caused by acetaminophen However, there are some clinical situations in which the (APAP) overdose. In the last case, the therapeutic effect liver shows poor regenerative capacity, such as in the case of ­CeO2NPs was compared with the gold standard treat- of liver cirrhosis and severe drug-induced liver injury ment for APAP-induced injury, N-acetylcysteine (NAC) (DILI) [3, 4]. [4]. One pathological process that is common to these two clinical conditions is oxidative stress, which is caused by Results the excessive formation of reactive oxygen species (ROS). Characterization and biodistribution of cerium oxide The presence of oxidative stress has been described in nanoparticles in rats most of the clinical conditions associated with chronic Cerium oxide nanoparticles ­(CeO2NPs) were analyzed by liver injury (i.e.: nonalcoholic steatohepatitis, hepatitis HR-TEM. The engineered nanoparticles showed spheri- C viral infection, alcoholic liver cirrhosis) [5, 6]. In addi- cal morphology (Fig. 1a, b) and were predominantly in tion, recent reports suggest that drug-induced oxidative the size range of 4 nm. The UV–visible absorption spec- stress also significantly correlate with increased DILI risk trum of ­ CeO2NPs showed a characteristic absorption [7]. Concurrently, the association between high levels of peak of ­Ce4+ at 298 nm (Fig. 1c). The X-ray diffraction oxidative stress and a reduction of antioxidant defenses pattern of the ­CeO2NPs showed pure ­CeO2NPs with the has also been reported in most of these pathological situ- typical peak broadening characteristic of nanosize parti- ations [8, 9]. According to the results obtained by several cles (Fig. 1d). Measured zeta potentials of the C ­ eO2NPs studies, the imbalance between the production of ROS were (+) 41.2 mV (Fig. 1e). The hydrodynamic diameter and antioxidant defense in some of these liver diseases of the C ­ eO2NPs dispersed in saline solution at pH = 5.5 affects liver regeneration [10–12]. One of the reasons was 37 nm (Fig. 1f ). These optimally engineered nanoce- that explain this ROS effect is that ROS modulates the ria were used further in our animal studies. expression of a variety of regulators that play major roles Several studies have described that after systemic dis- in liver regeneration, including growth factors, transcrip- tribution small inorganic NPs accumulate in the liver tion factors and cell cycle proteins such as β-catenin [13], and spleen [31, 37]. In agreement, C ­ eO2NPs treated rats cyclin D [14], p53 [15], Nrf2 [16] and JNK/p38 mitogen showed ­CeO2NPs retention into the liver and, to a lesser activated kinases [17]. extent, in spleen as early as 90 min following i.v. injection. Cerium oxide nanoparticles ­ (CeO2NPs) have drawn In these organs and at this time point, ­CeO2NPs reached considerable attention as a potential therapeutic tool in concentrations of 160.9 μg and 36.0 μg of ­CeO2NPs per the prevention and treatment of oxidative stress related gram of tissue, respectively (Fig. 1g). Interestingly, cerium diseases. This interest relies on the multi-enzyme was still detected in liver and spleen for over 8 weeks mimetic properties of C ­ eO2NPs due to their unique elec- although in lower concentrations. ­ CeO2NPs retention tronic structure [18–20]. At the nanoscale, the oxygen was barely detected in the lungs and the kidneys of the vacancies that appear in the C ­ eO2 nanocrystals modify rats at different time points after the intravenous injec- their electronic structure enabling them to participate tion (Fig. 1g). as a catalyst in a wide range of reactions (e.g. promoting To investigate the antioxidant properties of C ­ eO2NPs, the simultaneous oxidation of Carbon Monoxide (CO) we induced oxidative stress in the hepatocyte cell line and hydrocarbons to ­CO2 and the reduction of Nitrogen HepG2 by ­ H2O2 treatment, as previously reported Oxides (NOx) to N2 in three way catalytic converters) [38, 39]. ROS were assessed in these cells by using the [21]. Similarly, in biological contexts, it has been reported dichlorofluorescein (DCF) assay [31]. When exposed to that cerium oxide nanoparticles can mimic enzymatic ­H2O2, ­CeO2NPs-treated HepG2 cells showed a signifi- antioxidants such as superoxide dismutase [22] and cat- cant reduction in the accumulation of DCF in compari- alase [23]. The beneficial effects of C ­ eO2NPs have been son to that observed in non-treated cells (Fig. 1h). reported in different clinical conditions associated with excess production of ROS such as neurology [24–27], Rats treated with ­CeO2NPs showed increased liver diabetes [28, 29], chronic inflammation [30], cirrhosis regeneration and hepatocellular proliferation after PHx [31] and cancer [32–34]. Oxidative stress mediate cell growth arrest and impairs Considering the above, we hypothesize that ­CeO2NPs hepatic regeneration in mice [13, 14]. Therefore, testing may improve liver regeneration by scavenging ROS in new anti-oxidant drugs to improve liver regeneration Córdoba‑Jover et al. J Nanobiotechnol (2019) 17:112 Page 3 of 12 Fig. 1 Characterization of cerium oxide nanoparticles. a, b Representative TEM images of C ­ eO2NPs at different magnifications showing the non-aggregate and spherical shape of the engineered nanoparticles. Inset in b is a High Resolution TEM image of single particle showing pure ­CeO2 atomic planes; c UV–Visible absorption spectrum of the as-synthesized C ­ eO2NPs; d XRD spectrum of the as-synthesized ­CeO2NPs after being dried under vacuum. e Z-Potential distribution and f Hydrodynamic diameter measured by DLS of the C ­ eO2NPs dispersed in the physiological media (saline solution at pH = 5.5); g Cerium concentration in liver, spleen, lung and and kidney from rats treated with C­ eO2NPs for 90 min, 3, 6 and 8 weeks (n = 4 for each group). h Oxidative stress was quantified in non-treated and ­CeO2NPs-treated HepG2 cells by measuring DCF fluorescence in basal condition and after inducing oxidative stress with 2 mM ­H2O2 added to the culture medium (#p < 0.0001 vs basal and *p < 0.0001 vs. non-treated, n = 10) has clinical interest. To this aim, we studied the effect treatment in rats that were fasted for 12 h. We did not of ­CeO2NPs treatment on liver regeneration after per- detect any significant change of these laboratory parame- forming PHx in rats. Rats were treated with 0.1 mg/kg ters between vehicle and ­CeO2NPs treated groups (Addi- ­CeO2NPs intravenously twice a week for 2 weeks before tional file 1: Figure S1). These results support the concept PHx. As shown in Fig. 2a, we did not observe any substan- that the ­CeO2NPs pretreatment is safe for the liver and tial change in body weight between the groups without is not associated with detectable side effects in the short treatment, vehicle treatment, and ­CeO2NPs treatment. term. Rats were sacrificed 6 days after the surgical proce- Also, we performed liver laboratory tests in rat serum to dure and the wet liver remnant weight/total body weight quantify the liver function (glucose and albumin) and the ratio was used to calculate the hepatic regenerative liver damage (ALT and AST) in response to the ­CeO2NPs index. Rats treated with ­CeO2NPs showed a significant Córdoba‑Jover et al. J Nanobiotechnol (2019) 17:112 Page 4 of 12 Fig. 2 CeO2NPs treatment increased liver regeneration and cell proliferation after PHx. a Body weights of control rats without treatment and rats that received vehicle or ­CeO2NPs before PHx (n = 8). b Hepatic regenerative index at day 6 after PHx (n = 8, p < 0.05). c Blood levels of ALT (*p < 0.01), AST (*p < 0.05) and LDH (*p < 0.05) in vehicle or C­ eO2NPs-treated rats after 3 h post-PHx (n = 8; mean ± SEM). d Representative immunostaining for the Ki-67 antigen in liver histological sections of rats treated with either vehicle or C ­ eO2NPs at different time points (t = 0 h, 24 h, 48 h, 7 days). Merged images show co-localization of Ki-67 (green) and nuclear DNA (DAPI, blue). Original magnification ×200 (n = 8 for each group and treatment). On the bottom, percentage quantification of positive Ki-67 liver cells for each time point and treatment (n = 8; mean ± SEM; *p < 0.05 compared with vehicle at the same time points) Córdoba‑Jover et al. J Nanobiotechnol (2019) 17:112 Page 5 of 12 11% increase in the hepatic regenerative index, com- (Fig. 3b first bar in the graph). Despite sharing beneficial pared with vehicle-treated rats (p < 0.05) (Fig. 2b). The antioxidant properties, NAC and C ­ eO2NPs treatments beneficial effect of ­CeO2NPs on liver regeneration was differed in their capability of stimulating hepatocellular also accompanied by lower blood levels of alanine ami- proliferation. The liver from rats treated with C ­ eO2NPs notransferase (ALT), aspartate aminotransferase (AST) showed an extensive positivity for Ki67, compared with and the enzyme lactate dehydrogenase (LDH) after 3 h vehicle and NAC treated rats at day 2 after APAP injec- post-PHx and compared with the vehicle group (Fig. 2c). tion (40.08 ± 5.04%, 10.70 ± 1.69% and 13.53 ± 3.13% To further investigate the cause through which ­Ki67+ cells, respectively; p < 0.05) (Fig. 3c). ­CeO2NPs improves liver regeneration, we assessed the We performed additional experiments to character- expression of the cell proliferation marker Ki67 in liver ize better the liver injury induced by APAP and the sections of rats treated with nanoparticles. Rats receiv- degree of recovery of the different experimental groups. ing vehicle or ­CeO2NPs showed absence of hepatocel- To this end, we quantified the serum concentration of lular proliferation in resting livers (t = 0 h) (Fig. 2d). transaminases (ALT, AST), glucose and albumin from However after PHx, the remnant liver from rats ­CeO2NPs-treated and vehicle-treated rats at t = 0 h, treated with ­ CeO2NPs showed a significant increase t = 48 h, and t = 96 h. We found that the concentration in ­Ki67+-hepatocytes at day 1 (*p < 0.05, t = 24 h) that of transaminases was significantly increased to a similar reached a maximum at t = 48 h after partial hepatectomy, extent in the vehicle and the ­CeO2NPs-treated groups compared with vehicle (44.6 ± 4.5% vs. 38.5 ± 6.7% ­Ki67+ 2 days after APAP administration (t = 48 h compared cells, *p < 0.05, t = 48 h); returning to nearly basal levels with t = 0 h). However, ­CeO2NPs treatment was associ- after 7 days (Fig. 2d). Therefore, improvement of liver ated with a significant decline in serum ALT, reaching regeneration caused by the ­CeO2NPs treatment is associ- basal levels at 96 h after the APAP injection; compared ated with enhanced hepatocyte proliferation. with the vehicle group (Additional file 1: Figure S2). This recovery phase marker, which is a more specific marker CeO2NPs treated rats showed decreased liver of liver injury than AST, points to a more efficient recov- damage and increased hepatocellular proliferation ery of the ­CeO2NPs-treated liver after drug-induced liver after acetaminophen‑induced liver injury damage. The previous results show us the role played by C­ eO2NPs on liver regeneration in the context of basal levels of CeO2NPs treatment stimulates cell cycle progression ROS. Next we assessed whether ­ CeO2NPs is equally in HepG2 cells and NFκB activation in vitro and in vivo effective after ROS induction caused by APAP toxicity. To ensure the generalization of the finding that ­CeO2NPs In this experimental model, excessive oxidative stress improves hepatocyte proliferation in vivo after PHx and plays a major role in APAP hepatotoxicity [40]. The APAP-induced toxicity in rats, we characterized the injury induced after 48 h of APAP administration was dynamics of the cell cycle in the human hepatocyte cell assessed in three experimental groups: rats previously line HepG2. As shown in Fig. 4a, ­CeO2NPs treatment sig- treated with C ­ eO2NPs, rats receiving vehicle and rats nificantly decreased the percentage of HepG2 cells that receiving a simultaneous treatment with NAC, which underwent apoptosis following 48 h of serum starva- is the accepted clinical treatment for APAP overdose. tion, as detected by propidium iodide staining and flow Hematoxylin–eosin staining of livers from rats belong- cytometry. Moreover, ­ CeO2NPs treatment was associ- ing to the vehicle experimental group showed that APAP ated with a higher percentage of HepG2 cells that were administration induced severe liver injury characterized in the G2/M phase of the cell cycle, reflecting the stimu- by massive necrosis, hepatocyte vacuolation and vascular latory role played by ­CeO2NPs on cell cycle progression. congestion. Liver injury was significantly reduced in the We further validate these observations in the HepG2 cells NAC and the ­CeO2NPs-treated groups, although vascu- by quantifying active caspase-3 and cyclin D1 (markers lar congestion was still present in both groups despite the of apoptosis and cell cycle progression, respectively). NAC and the ­CeO2NPs treatments (Fig. 3a). As expected, ­CeO2NPs treatment of HepG2 cells reduced the amount APAP treatment induced a significant increase in hepatic of activated caspase-3 and increased the levels of cyclin ROS that was quantified by measuring 4-hydroxynon- D1 following 48 h of serum starvation (Fig. 4b). enal in the liver tissue (HNE), a widely accepted marker The NF-κB/Rel family of transcription factors (NFκB) is of lipid peroxydation and oxidative damage. Interestingly, a central hub of signaling pathways that regulates apopto- both NAC (Fig. 3b second bar in the graph) and C ­ eO2NPs sis and cell proliferation. In addition, the activity NFκB (Fig. 3b third bar in the graph) are equally effective as is modulated by ROS [41–43]. These confluent evidences antioxidant treatments for treating APAP-induced oxi- let us to assess whether the activity of NF-κB is affected dative stress when compared with the vehicle condition by the ­CeO2NPs treatment in vitro and in vivo. In HepG2 Córdoba‑Jover et al. J Nanobiotechnol (2019) 17:112 Page 6 of 12 Fig. 3 CeO2NPs treatment reduces histological damage and increases cell proliferation after APAP-induced injury. a Hematoxylin-eosin stained liver sections (n = 13). After vehicle or ­CeO2NPs treatments, rats received 1 g/kg APAP and were sacrificed after 48 h (vehicle + APAP and ­CeO2NPs + APAP, respectively). Another group was treated with 300 mg/kg NAC 1 h after APAP (NAC + APAP). Also, healthy non-treated rats were included as experimental controls (upper left panel), ×100. b Quantification of HNE in liver from vehicle + APAP, NAC + APAP and C ­ eO2NPs + APAP groups (n = 13; mean ± SEM; *p < 0.05). c Immunostaining for Ki-67 in liver of rats treated with vehicle + APAP, NAC + APAP and C ­ eO2NPs + APAP. Ki-67 (green) and DAPI (blue), ×200. On the right, quantification of Ki-67 positive cells (n = 13; mean ± SEM; *p < 0.05) (See figure on next page.) Fig. 4 CeO2NPs stimulates cell cycle progression and NF-κB activation. a Flow cytometry of HepG2 showing cell cycle profiles from propidium iodide DNA staining after vehicle or ­CeO2NPs treatment (n = 5; #p < 0.01 and *p < 0.05). b Western blot for activated caspase 3 and cyclin D1 abundance from HepG2 incubated with vehicle or ­CeO2NPs. β-actin was used as loading control (mean ± SEM; n = 5; *p < 0.05 versus vehicle in the same experimental condition). O.D.: optical density. c Western blot for IκBα abundance from HepG2 (mean ± SEM; n = 5; *p < 0.05). d Transcription factor immunosorbent assay for NF-κB (p65) activity in HepG2 (n = 5, *p < 0.05). e Western blot for IκBα abundance in the liver vehicle and ­CeO2NPs-treated rats before and 3 h post-PHx (mean ± SEM; n = 5; #p < 0.01) Córdoba‑Jover et al. J Nanobiotechnol (2019) 17:112 Page 7 of 12 Córdoba‑Jover et al. J Nanobiotechnol (2019) 17:112 Page 8 of 12 cells treated with ­CeO2NPs, the abundance of IκBα (that oil with free radical scavenging capacity, protects rat liver function as an inhibitor of NFκB) significantly decreased against ischemia/reperfusion injury [46] and prevents when compared to vehicle-treated HepG2 cells (p < 0.05) liver injury in hepatectomized mice under I/R by blunt- (Fig. 4c). Next, the transcriptional activity of NF-κB ing oxidative stress, mitochondrial damage, endoplasmic was assessed by detecting specific p65 DNA binding in reticulum stress and apoptosis [47]. Exogenous admin- nuclear extracts from ­CeO2NPs treated and non-treated istration of GSH or resveratrol significantly decreased HepG2 cells. As shown in Fig. 4d, ­CeO2NPs treated cells oxidative stress and protect against APAP-induced liver showed increased p65 transcriptional activity compared injury [48, 49]. Similarly, pharmacological inhibition of with non-treated HepG2 cells (p < 0.05). We next investi- JNK activity, which enhances mitochondrial-derived gated whether the described stimulation of NF-κB activ- ROS production upon APAP toxicity, reduced liver dam- ity by ­CeO2NPs was also detected in regenerative livers age [50]. Some drugs approved for its clinical use in dif- in vivo. Before PHx, there were no differences in the ferent pathologies have also been tested pre-clinically expression of IκBα in the livers from vehicle or ­CeO2NPs for APAP hepatotoxicity. Both methylene blue and met- treated rats. However, and in agreement with the results formin reduced mitochondrial oxidant stress [51] and obtained in HepG2, the abundance of IκBα was signifi- protected against APAP liver injury [52]. cantly lower in the livers from ­CeO2NPs treated rats 3 h Our study has potential limitations regarding the after PHx, compared with the group of rats receiving APAP-induced toxicity model in rats. It has been shown vehicle (p < 0.01) (Fig. 4e). that rats are more resistant to acetaminophen damage than mice [53]. Despite this observation, the presence Discussion of oxidative stress in the APAP-induced toxicity model We previously described that ­CeO2NPs showed hepato- in rats has broadly been demonstrated [54–56], regard- protective activity in vivo against fibrosis [31]. Another less of the severity of the injury between species. There- study also demonstrated the beneficial effects of these fore, this experimental model in rats is still valid to test nanoparticles against hepatic oxidative damage caused by the antioxidant properties of C ­ eO2NPs treatment on the pyrrolizidine alkaloid monocrotaline, which causes liver regeneration after drug-induced injury. Regarding oxidative vascular damage in the liver [44]. Consider- the metabolism of APAP in the rat liver, we found in our ing this hepatoprotective role of ­CeO2NPs, we further study that the concentration of transaminases was sig- investigated whether these nanoparticles might also nificantly increased to a similar extent in the vehicle and stimulate other hepatoprotective functions, such as liver the ­CeO2NPs-treated groups 2 days after APAP admin- regeneration. In the present study we describe for the istration. This result confirmed that there is significant first time that C­ eO2NPs stimulate liver regeneration in toxicity induced by APAP overdose in our experimental the rat experimental models of partial hepatectomy and rat model and that the C ­ eO2NPs pretreatment does not DILI caused by acetaminophen overdose. Consistently modify the metabolism of APAP in the liver. Neverthe- with this observation, we demonstrated that C ­ eO2NPs less, the quantification of an intermediary metabolite of treatment decreases liver damage after PHx and APAP- APAP (i.e., N-acetyl-p-benzoquinone imine) would have induced liver injury. The treatment with ­CeO2NPs also supported more robustly this conclusion. leads to the reduction of oxidative stress markers and Our study presents an innovative treatment for stimulates the cell cycle progression in hepatocytes and diminish ROS in the context of liver regeneration using the activation of the transcription factor NF-κB in vitro nanoparticles. Some of the advantages of using ­ CeO2 and in vivo. nanomaterials over the traditional anti-oxidative drugs It is known that excess of oxidative stress inhibits effi- are: (1) minimal toxicity in cumulative doses [57, 58], cient regeneration after partial hepatectomy or acute (2) the multi-enzyme mimetic activities of ­ CeO2NPs, liver failure caused by DILI. For example, the genetic which targets several sources of ROS generation and (3) deficiency of Nrf2 (a regulator of the cellular antioxidant the continuous regeneration of the ­CeO2NPs catalytic response) and UCP2 (an uncoupling protein of the elec- activity, which avoids the exhaustion of its anti-oxidative tron transport chain) cause enhanced oxidative stress properties [58]. Among these theoretical benefits over and impaired liver regeneration in partially hepatecto- traditional drugs, we also observed superior therapeutic mized mice [43]. Moreover, oxidative cellular damage in performance compared with the gold standard treatment mitochondria and DNA is associated with deficient liver used for acetaminophen toxicity in patients, NAC. In our regeneration after acetaminophen toxicity [45]. There- study, ­CeO2NPs treatment is equally effective reducing fore, several strategies have been investigated by others to oxidative stress and tissue damage in rats receiving APAP diminish ROS in the context of liver regeneration in pre- overdose compared with NAC. However, while NAC clinical models. For example, thymoquinone, an essential did not affect hepatocyte proliferation in injured livers, Córdoba‑Jover et al. J Nanobiotechnol (2019) 17:112 Page 9 of 12 ­CeO2NPs robustly increased cell proliferation in vivo and besides their known antioxidant capacities, can enhance in vitro. In addition, we observed a significant decrease liver regeneration in experimental models that replicate in the percentage of C ­ eO2NPs-treated HepG2 cells that these two clinical scenarios. Hence, ­CeO2NPs treatment underwent apoptosis following 48 h of serum starvation, may provide avenues to overcome deficient liver regen- suggesting that the anti-apoptotic effect associated to the eration in patients. nanoceria treatment may also contribute to enhance liver regeneration. Methods NF-κB plays a major role in liver homeostasis and liver Synthesis and characterization of ­CeO2NPs regeneration. For instance, NF-κB (p65) knockout mice CeO2NPs were synthesized by the chemical precipita- show embryo lethality and display massive hepatocyte tion of cerium (III) nitrate hexahydrated (Sigma-Aldrich, apoptosis [59]. In addition, the transduction of rat livers St. Louis, MO, USA) in aqueous solution, as reported with different forms of IκB, an inhibitor of NF-κB activ- elsewhere [19]. Controlling the pH of synthesis, small- ity, before PHx was associated with impaired liver regen- sized nanoceria can be obtained. Here, we used 4 nm eration [60]. Furthermore, the inactivation of NF-κB nanoparticles at a concentration of 1 mg/mL. The surface in Kupffer cells as well as in hepatocytes impaired the charge of the NPs was characterized by the Z-potential regenerative process after PHx [61]. Here, we demon- in a Z-sizer (Malvern, Worcestershire, UK) while the strated that the treatment with C ­ eO2NPs lead to the crystal size was characterized by High Resolution Trans- activation of the transcription factor NF-κB in vitro and mission Electron Microscopy (HR-TEM) in the Tecnai in vivo. Our results are not sufficient to establish a robust G2 F20 (FEI, Oregon, USA). Nanocrystalline morphol- link between NF-κB activation and the beneficial effects ogy and composition was analyzed by HR-TEM (Tecnai of ­CeO2NPs treatment on liver regeneration yet. How- 200 kV) and XRD (Xpert Pannalytical, MA, USA), and ever, the agreement between our findings and the studies the light interaction by UV–VIS spectroscopy (Shimatzu, mentioned above make this relationship plausible. Kyoto, Japan). Size distribution was computer analyzed It has been described that oxidative stress can inhibit by ImageJ (National Institutes of Health, Bethesda, MD, the transcriptional activity of NFκB through different USA). ­CeO2NPs were stabilized with Tetramethylammo- mechanisms including direct interaction with NF-κB, nium hydroxide (TMAOH), which was used as vehicle inactivation of IKK (an IκB repressor), and alternative in all the experiments as a control condition, and kept at inhibitory phosphorylation of IκBα [41]. Our in vitro and 4 °C until use. Prior to the animal studies, 15 µL from the in vivo results showed that C ­ eO2NPs are associated with nanoparticles solution were dispersed on a copper grid a significant decrease in the intracellular IκBα content, coated with a formvar film. The samples were then let to which is translated in the activation of NF-κB. Therefore, dry for TEM observation and digital photomicrographs our findings suggest that the anti-oxidant property of were taken (BioScan Gatan, CA, USA). Free C ­ eO2NPs ­CeO2NPs is responsible for the increase in NF-κB acti- samples available for evaluation if requested. vation. However, our results are in disagreement with other studies which have shown that ­CeO2NPs treatment Animal experimentation and In vivo ­CeO2NPs treatment inhibits NF-κB in vivo mostly in the pathological context This study was performed in male Wistar rats (Charles- of sepsis [62–64]. One of the reasons that may explain River, Saint Aubin les Elseuf, France). Rats were fed with the seemingly contradictory findings is differences in the standard chow diet and housed on a 12 h light/12 h dark ­CeO2NPs stability used in these studies, what has been cycle. For each experiment of this study, treated rats were recently recognized as key in order to employ ­CeO2NPs administered 0.1 mg/kg C ­ eO2NPs dispersed in saline for medicine [22]. In addition, these studies used higher solution as a bolus (500 μL), given intravenously through in vivo doses of C ­ eO2NPs compared with ours (from the tail vain twice a week for 2 weeks. Control groups 0.5 mg/kg to 3.5 mg/kg b.w. compared with 0.1 mg/kg without treatment and treated with vehicle (TMAOH) b.w.). Our previous dosage standardization experiments were also included in the study. At the end of each exper- [31] indicate that at higher doses C ­ eO2NPs shows a hor- iment rats were euthanized by isoflurane anesthesia metic-like dose response characterized by an increasing overdose. dose inefficacy in ROS scavenging. CeO2NPs tissue quantification Conclusions For tissue quantification, at the end of the 2 weeks treat- In summary, drug-induced hepatotoxicity and impaired ment, rats were euthanized at days 1, 21, 42, and 56 liver regeneration are major concerns in medical practice (90 min, 3, 6 and 8 weeks respectively) and their livers, as they are leading causes of acute liver failure and liver spleens, lungs and kidneys dissected and kept at − 80 °C transplantation. Our results demonstrated that C ­ eO2NPs, for further analysis. For ­CeO2NPs quantification, samples Córdoba‑Jover et al. J Nanobiotechnol (2019) 17:112 Page 10 of 12 were diluted in an aqueous solution of H ­ NO3 2% w/w USA) and public libraries from The Comprehensive R (Trace Metal Basis; Sigma-Aldrich, St. Louis, MO, USA) Archive Network (CRAN; http://CRAN.R-proje​ct.org) and analyzed for cerium concentration by inductively rooted in the open source statistical computing envi- coupled plasma mass spectrometry (ICP-MS, Agilent ronment R, version 3.4 (http://www.R-proje​ct.org/). 7500; Agilent Technologies, California, USA). The quan- The statistical analysis of the results was performed titative results were obtained by interpolation in a stand- using unpaired Student’s t-tests and ANOVA models ard curve obtained from a commercial 1000 ppm Ce (with Tukey’s post hoc test) with normally distributed standard (Sigma-Aldrich, St. Louis, MO, USA). data. For other type of data, the Mann–Whitney U-test and the Kruskal–Wallis tests (with Dunn post hoc test) Reactive oxygen species quantification were used. Results are expressed as mean ± sem and Reactive oxygen species were measured by fluorescence considered significant at a p value lower than 0.05. spectrophotometry using 2′,7′-DCF-DA as a probe. Additional Materials and Methods are shown in For these experiments, HepG2 cells alone or treated Additional file 1. Free C­ eO2NPs samples available for with ­H2O2 in the presence or absence of C­ eO2NPs were evaluation if requested. washed with phosphate-buffered saline (PBS) and incu- bated with 10 μM DCF-DA (Thermo Fisher Scientific, Supplementary information Waltham, MA, USA) in Dulbecco’s modified Eagle Supplementary information accompanies this paper at https​://doi. org/10.1186/s1295​1-019-0544-5. medium (DMEM) for 40 min at 37 °C in the dark. The supernatant was collected to measure the extracellular production of ROS, and the intensity of fluorescence was Additional file 1. Additional materials and methods, additional figures and references. immediately read in a fluorescence spectrophotometer (FLUOstar Optima; BMG Labtech, Ortenberg, Germany) Abbreviations at 485 nm for excitation and at 520 nm for emission. CeO2NPs: cerium oxide nanoparticles; PHx: partial hepatectomy; APAP: acetaminophen; NAC: N-acetyl-cysteine; DILI: drug-induced liver injury; ROS: In vitro ­CeO2NPs treatment reactive oxygen species; DMEM: Dulbecco’s modified Eagle medium; PBS: phosphate-buffered saline; DCF: dichlorofluorescein; HNE: 4-hydroxynonenal; The human cell line HepG2 (a suitable in vitro model sys- PI: propidium iodide; ALT: alanine aminotransferase; AST: aspartate ami‑ tem for the study of polarized human hepatocytes) was notransferase; LDH: lactate dehydrogenase. obtained from the American Type Culture Collection Acknowledgements (ATCC; Manassas, VA, USA). HepG2 cells were cultured We thank Ministerio de Economía y Competitividad, Fundació La Marató de and maintained in DMEM containing 10% FBS, 100 U/ TV3 and CIBERehd (Instituto de Salud Carlos III) for the grants and financial mL penicillin and 100 μg/mL streptomycin at 37 °C in a support to this research. humidified atmosphere containing 5% C ­ O2. Cells were Authors’ contributions serum-starved and treated with 0.1 mg/mL ­CeO2NPs, or BC-J: experiments, procedures and writing of article; AA-C: experiments, vehicle as a control condition, for 48 h. procedures and writing of article; JR: experiments and generation of experi‑ mental models; MP: generation of experimental models; DO: experiments, GC: measurement of biochemical parameters, GF-V: measurement of biochemi‑ Measurement of NF‑κB activation cal parameters, EC: synthesis of C ­ eO2NPs and writing of article, VP: synthesis NF-κB activity in HepG2 cells was measured using a of ­CeO2NPs and writing of article, WJ: concept, design and writing of article; MM-R: concept, design, experiments and writing of article. All authors read NF-κB Transcription Factor Assay Kit (specific for p65), and approved the final manuscript. following the manufacturer’s instructions (Cayman Chemical, Ann Arbor, MI, USA). To this end, nuclear Funding This research was supported by grant from the Ministerio de Ciencia, extracts from HepG2 were incubated with double Innovación y Universidades (SAF2016-75358-R to MM-R), co-financed by stranded DNA immobilized in a 96-well plate that con- FEDER, European Union, a way of making Europe; Grant from the Ministerio tained the κB response motif site. The complex p65-DNA de Sanidad, Consumo y Bienestar Social (PI15/00777 to GC); Grant from Wuyi University Funding for Hight Talents Introduction (2018TP010 to EC); Grant was detected by enzyme-linked immunosorbent assay from Department of Education of Guangdong Province (2017KSYS010 to EC) using specific primary antibody directed against p65 and and Fundació La Marató de TV3 (Marató 120930 to WJ). CIBERehd is financed a secondary antibody conjugated to HRP. The results by Instituto de Salud Carlos III. were quantified by measuring absorbance at 450 nm in a Availability of data and materials FLUOstar Optima (BMG Labtech, Ortenberg, Germany). All data generated or analyzed during this study is available from the cor‑ responding author on reasonable request. Statistical analysis Ethics approval and consent to participate Quantitative data were analyzed using GraphPad Prism, All animals received human care and all procedures were approved by the Investigation and Ethics Committee of Animal Experimentation of the Univer‑ version 6 (GraphPad Software, Inc., San Diego, CA, sity of Barcelona. Córdoba‑Jover et al. J Nanobiotechnol (2019) 17:112 Page 11 of 12 Consent for publication 18. Esch F, Fabris S, Zhou L, et al. Electron localization determines defect Not applicable. formation on ceria substrates. Science. 2005;309:752–5. 19. Cafun J-D, Kvashnina KO, Casals E, Puntes VF, Glatzel P. Absence of Competing interests ­Ce3+ sites in chemically active colloidal ceria nanoparticles. ACS Nano. The authors declare that they have no competing interests. 2013;7:10726–32. 20. Wang Z, Shen X, Gao X, Zhao Y. Simultaneous enzyme mimicking and Author details chemical reduction mechanisms for nanoceria as a bio-antioxidant: a 1 Biochemistry and Molecular Genetics Department, Hospital Clínic of Barce‑ catalytic model bridging computations and experiments for nanozymes. lona, IDIBAPS, CIBERehd, 170 Villarroel St., 08036 Barcelona, Spain. 2 Work‑ Nanoscale. 2019;11:13289–99. ing Group for the Biochemical Assessment of Hepatic Disease-SEQC-ML, 21. Montini T, Melchionna M, Monai M, Fornasiero P. Fundamentals Barcelona, Spain. 3 Vall d’Hebron Institut of Research (VHIR), Barcelona, Spain. and catalytic applications of ­CeO2-based materials. Chem Rev. 4 Institut Català de Nanociència i Nanotecnologia (ICN2), CSIC and The Barce‑ 2016;116:5987–6041. lona Institute of Science and Technology (BIST), Barcelona, Spain. 5 Institució 22. Heckert EG, Karakoti AS, Seal S, Self WT. The role of cerium redox state in Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain. 6 School the SOD mimetic activity of nanoceria. Biomaterials. 2008;29:2705–9. of Biotechnology and Health Sciences, Wuyi University, Jiangmen 529020, 23. Pirmohamed T, Dowding JM, Singh S, et al. Nanoceria exhibit redox state- China. 7 Department of Biomedicine‑Biochemistry Unit, School of Medicine, dependent catalase mimetic activity. Chem Commun. 2010;46:2736–8. University of Barcelona, Barcelona, Spain. 24. D’Angelo B, Santucci S, Benedetti E, et al. Cerium oxide nanoparticles trigger neuronal survival in a human Alzheimer disease model by modu‑ Received: 25 March 2019 Accepted: 18 October 2019 lating BDNF pathway. Curr Nanosci. 2009;5:167–76. 25. Kim CK, Kim T, Choi I-Y, et al. Ceria nanoparticles that can protect against ischemic stroke. Angew Chemie Int Ed. 2012;51:11039–43. 26. Ranjbar A, Soleimani Asl S, Firozian F, et al. Role of cerium oxide nano‑ particles in a paraquat-induced model of oxidative stress: emergence of References neuroprotective results in the brain. J Mol Neurosci. 2018;66:420–7. 1. Fausto N, Campbell JS, Riehle KJ. Liver regeneration. Hepatology. 27. Kalashnikova I, Mazar J, Neal CJ, et al. Nanoparticle delivery of curcumin 2006;43:S45–53. induces cellular hypoxia and ROS-mediated apoptosis via modulation of 2. Michalopoulos GK, DeFrances MC. Liver regeneration. Science. Bcl-2/Bax in human neuroblastoma. Nanoscale. 2017;9:10375–87. 1997;276:60–6. 28. Pourkhalili N, Hosseini A, Nili-Ahmadabadi A, et al. Biochemical and cellu‑ 3. Cho Lam Wong T, Mau Lo C. Resection strategies for hepatocellular carci‑ lar evidence of the benefit of a combination of cerium oxide nanoparti‑ noma preoperative evaluation of liver function clinical and biochemical cles and selenium to diabetic rats. World J Diabetes. 2011;2:204–10. tests. Semin Liver Dis. 2013;33:273–81. 29. Khurana A, Tekula S, Godugu C. Nanoceria suppresses multiple low doses 4. Wendon J, Cordoba J, Dhawan A, et al. EASL Clinical Practical Guide‑ of streptozotocin-induced Type 1 diabetes by inhibition of Nrf2/NF-κB lines on the management of acute (fulminant) liver failure. J Hepatol. pathway and reduction of apoptosis. Nanomedicine. 2018;13:1905–22. 2017;66:1047–81. 30. Niu J, Azfer A, Rogers LM, Wang X, Kolattukudy PE. Cardioprotective 5. Morales-Ruiz M, Rodríguez-Vita J, Ribera J, Jiménez W. Pathophysiology effects of cerium oxide nanoparticles in a transgenic murine model of of portal hypertension. Panvascular medicine. Berlin: Springer; 2015. p. cardiomyopathy. Cardiovasc Res. 2007;73:549–59. 3631–65. 31. Oró D, Yudina T, Fernández-Varo G, et al. Cerium oxide nanoparticles 6. Ezhilarasan D. Oxidative stress is bane in chronic liver diseases: clinical reduce steatosis, portal hypertension and display anti-inflammatory and experimental perspective. Arab J Gastroenterol. 2018;19:56–64. properties in rats with liver fibrosis. J Hepatol. 2016;64:691–8. 7. Xu JJ, Henstock PV, Dunn MC, Smith AR, Chabot JR, De Graaf D. Cellular 32. Alili L, Sack M, Karakoti AS, et al. Combined cytotoxic and anti-invasive imaging predictions of clinical drug-induced liver injury. Toxicol Sci. properties of redox-active nanoparticles in tumor–stroma interactions. 2008;105:97–105. Biomaterials. 2011;32:2918–29. 8. Li S, Tan H-Y, Wang N, et al. The role of oxidative stress and antioxidants in 33. Nourmohammadi E, Khoshdel-sarkarizi H, Nedaeinia R, et al. Evaluation of liver diseases. Int J Mol Sci. 2015;16:26087–124. anticancer effects of cerium oxide nanoparticles on mouse fibrosarcoma 9. Kaffe ET, Rigopoulou EI, Koukoulis GK, Dalekos GN, Moulas AN. Oxidative cell line. J Cell Physiol. 2019;234:4987–96. stress and antioxidant status in patients with autoimmune liver diseases. 34. Li H, Liu C, Zeng Y-P, et al. Nanoceria-mediated drug delivery for targeted Redox Rep. 2015;20:33–41. photodynamic therapy on drug-resistant breast cancer. ACS Appl Mater 10. Ben Mosbah I, Duval H, Mbatchi S-F, et al. Intermittent selective clamping Interfaces. 2016;8:31510–23. improves rat liver regeneration by attenuating oxidative and endoplas‑ 35. Alizai PH, Bertram L, Fragoulis A, et al. In vivo imaging of antioxidant mic reticulum stress. Cell Death Dis. 2014;5:e1107. response element activity during liver regeneration after partial hepatec‑ 11. Dayoub R, Vogel A, Schuett J, et al. Nrf2 activates augmenter of liver tomy. J Surg Res. 2016;206:525–35. regeneration (ALR) via antioxidant response element and links oxidative 36. Guerrieri F, Vendemiale G, Grattagliano I, Cocco T, Pellecchia G, Altomare stress to liver regeneration. Mol Med. 2013;19:237–44. E. Mitochondrial oxidative alterations following partial hepatectomy. Free 12. di Bello G, Vendemiale G, Bellanti F. Redox cell signaling and hepatic Radic Biol Med. 1999;26:34–41. progenitor cells. Eur J Cell Biol. 2018;97:546–56. 37. Comenge J, Sotelo C, Romero F, et al. Detoxifying antitumoral drugs via 13. Sun S, Xie F, Xu X, et al. Advanced oxidation protein products induce nanoconjugation: the case of gold nanoparticles and cisplatin. PLoS ONE. S-phase arrest of hepatocytes via the ROS-dependent, β-catenin-CDK2- 2012;7:e47562. mediated pathway. Redox Biol. 2018;14:338–53. 38. Ling Liau L, Suzana M, Ghani Nur Azurah A, Hui Chua K. Hydrogen perox‑ 14. Barnouin K, Dubuisson ML, Child ES, et al. H ­ 2O2 induces a transient multi- ide induces acute injury and up-regulates inflammatory gene expression phase cell cycle arrest in mouse fibroblasts through modulating cyclin D in hepatocytes: an in vitro Model. Sains Malaysiana. 2016;45:451–8. and p21Cip1 expression. J Biol Chem. 2002;277:13761–70. 39. Jiang C, Yu S, Jiang Z, et al. N-acetyl-serotonin protects HepG2 cells from 15. Geng Y, Zhang C, Lei J, et al. Walsuronoid B induces mitochondrial and oxidative stress injury induced by hydrogen peroxide. Oxid Med Cell lysosomal dysfunction leading to apoptotic rather than autophagic cell Longev. 2014. https​://doi.org/10.1155/2014/31050​4. death via ROS/p53 signaling pathways in liver cancer. Biochem Pharma‑ 40. Du K, Ramachandran A, Jaeschke H. Oxidative stress during acetami‑ col. 2017;142:71–86. nophen hepatotoxicity: sources, pathophysiological role and therapeutic 16. Murakami S, Motohashi H. Roles of Nrf2 in cell proliferation and differen‑ potential. Redox Biol. 2016;10:148–56. tiation. Free Radic Biol Med. 2015;88:168–78. 41. Morgan MJ, Liu Z. Crosstalk of reactive oxygen species and NF-κB signal‑ 17. Tormos AM, Taléns-Visconti R, Nebreda AR, Sastre J. p38 MAPK: a dual role ing. Cell Res. 2011;21:103–15. in hepatocyte proliferation through reactive oxygen species. Free Radic 42. Imran M, Lim IK. Regulation of Btg2/TIS21/PC3 expression via reactive Res. 2013;47:905–16. oxygen species–protein kinase C-ΝFκΒ pathway under stress conditions. Cell Signal. 2013;25:2400–12. Córdoba‑Jover et al. J Nanobiotechnol (2019) 17:112 Page 12 of 12 43. Horimoto M, Fülöp P, Derdák Z, Wands JR, Baffy G. Uncoupling protein-2 55. Al-Brakati AY, Fouda MS, Tharwat AM, Elmahallawy EK, Kassab RB, Abdel deficiency promotes oxidant stress and delays liver regeneration in mice. Moneim AE. The protective efficacy of soursop fruit extract against Hepatology. 2004;39:386–92. hepatic injury associated with acetaminophen exposure is mediated 44. Amin KA, Hassan MS, Awad E-ST, Hashem KS. The protective effects of through antioxidant, anti-inflammatory, and anti-apoptotic activities. cerium oxide nanoparticles against hepatic oxidative damage induced Environ Sci Pollut Res. 2019;26:13539–50. by monocrotaline. Int J Nanomedicine. 2011;6:143–9. 56. Porto HKP, Grando MD, Ramalho LNZ, et al. Exposure to acetaminophen 45. Viswanathan P, Sharma Y, Gupta P, Gupta S. Replicative stress and impairs vasodilation, increases oxidative stress and changes arterial alterations in cell cycle checkpoint controls following acetaminophen morphology of rats. Arch Toxicol. 2019;93:1955–64. hepatotoxicity restrict liver regeneration. Cell Prolif. 2018;51:e12445. 57. Muhammad F, Wang A, Qi W, Zhang S, Zhu G. Intracellular antioxidants 46. Bouhlel A, Ben Mosbah I, Hadj Abdallah N, et al. Thymoquinone prevents dissolve man-made antioxidant nanoparticles: using redox vulnerability endoplasmic reticulum stress and mitochondria-induced apoptosis in a of nanoceria to develop a responsive drug delivery system. ACS Appl rat model of partial hepatic warm ischemia reperfusion. Biomed Pharma‑ Mater Interfaces. 2014;6:19424–33. cother. 2017;94:964–73. 58. Xu C, Qu X. Cerium oxide nanoparticle: a remarkably versatile rare earth 47. Bouhlel A, Bejaoui M, Ben Mosbah I, et al. Thymoquinone protects rat liver nanomaterial for biological applications. NPG Asia Mater. 2014;6:e90. after partial hepatectomy under ischaemia/reperfusion through oxidative 59. Beg AA, Sha WC, Bronson RT, Ghosh S, Baltimore D. Embryonic lethality stress and endoplasmic reticulum stress prevention. Clin Exp Pharmacol and liver degeneration in mice lacking the RelA component of NF-κB. Physiol. 2018. https​://doi.org/10.1111/1440-1681.12961​. Nature. 1995;376:167–70. 48. Saito C, Zwingmann C, Jaeschke H. Novel mechanisms of protection 60. Iimuro Y, Nishiura T, Hellerbrand C, et al. NFkappaB prevents apop‑ against acetaminophen hepatotoxicity in mice by glutathione and tosis and liver dysfunction during liver regeneration. J Clin Investig. N-acetylcysteine. Hepatology. 2010;51:246–54. 1998;101:802–11. 49. Du K, McGill MR, Xie Y, Bajt ML, Jaeschke H. Resveratrol prevents protein 61. Maeda S, Kamata H, Luo J-L, Leffert H, Karin M. IKKβ couples hepatocyte nitration and release of endonucleases from mitochondria during aceta‑ death to cytokine-driven compensatory proliferation that promotes minophen hepatotoxicity. Food Chem Toxicol. 2015;81:62–70. chemical hepatocarcinogenesis. Cell. 2005;121:977–90. 50. Han D, Dara L, Win S, et al. Regulation of drug-induced liver injury by 62. Chen G, Xu Y. Biosynthesis of cerium oxide nanoparticles and their effect signal transduction pathways: critical role of mitochondria. Trends Phar‑ on lipopolysaccharide (LPS) induced sepsis mortality and associated macol Sci. 2013;34:243–53. hepatic dysfunction in male Sprague Dawley rats. Mater Sci Eng C. 51. Lee KK, Imaizumi N, Chamberland SR, Alder NN, Boelsterli UA. Targeting 2017;83:148–53. mitochondria with methylene blue protects mice against acetami‑ 63. Selvaraj V, Manne ND, Arvapalli R, et al. Effect of cerium oxide nano‑ nophen-induced liver injury. Hepatology. 2015;61:326–36. particles on sepsis induced mortality and NF-κB signaling in cultured 52. Du K, Ramachandran A, Weemhoff JL, et al. Editor’s highlight: metformin macrophages. Nanomedicine. 2015;10:1275–88. protects against acetaminophen hepatotoxicity by attenuation of mito‑ 64. Selvaraj V, Nepal N, Rogers S, et al. Inhibition of MAP kinase/NF-kB medi‑ chondrial oxidant stress and dysfunction. Toxicol Sci. 2016;154:214–26. ated signaling and attenuation of lipopolysaccharide induced severe 53. Davis D, Potter W, Jollow D, Mitchell J. Species differences in hepatic sepsis by cerium oxide nanoparticles. Biomaterials. 2015;59:160–71. glutathione depletion, covalent binding and hepatic necrosis after aceta‑ minophen. Life Sci. 1974;14:2099–109. 54. Lakshmi T, Sri Renukadevi B, Senthilkumar S, et al. Seed and bark Publisher’s Note extracts of Acacia catechu protects liver from acetaminophen induced Springer Nature remains neutral with regard to jurisdictional claims in pub‑ hepatotoxicity by modulating oxidative stress, antioxidant enzymes lished maps and institutional affiliations. and liver function enzymes in Wistar rat model. Biomed Pharmacother. 2018;108:838–44. Ready to submit your research ? 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