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Cerium oxide nanoparticles: In pursuit of liver protection against doxorubicin-induced injury in rats
Biomedicine & Pharmacotherapy 103 (2018) 773–781
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Cerium oxide nanoparticles: In pursuit of liver protection against doxorubicin-induced injury in rats
T
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Heba G. Ibrahima, Noha Attiaa,b, , Fatma El Zahraa A. Hashema, Moushira A.R. El Heneidya a
Department of Medical Histology and Cell Biology, Faculty of Medicine, University of Alexandria, Alexandria, Egypt Department of Basic Sciences, The American University of Antigua-College Of Medicine, University Park, Jabberwock Beach Road, P.O. Box 1451, Coolidge, Antigua and Barbuda b
A R T I C LE I N FO
A B S T R A C T
Keywords: Cerium oxide nanoparticles Oxidative stress Doxorubicin Antioxidant Hepatic inflammation
Doxorubicin (DOX) is considered as a backbone in several chemotherapeutic regimens. Nevertheless, the reported systemic toxicity usually hampers its broad application. Interestingly, Cerium oxide nanoparticles (CeONPs) depicted promising regenerative antioxidant and hepatoprotective potentials against multiple oxidative stress-induced pathologies. Thus, the aim of the present study was to determine either CeONPs would display hepatoprotective properties once concomitantly administered with DOX or not. Male Sprague Dawley rats were divided into four groups (n = 10) in a two weeks study: Control (received saline, IP injection thrice a week), CeO (0.5 mg/kg, IP injection once a week), DOX (2.5 mg/kg, IP injections thrice a week) and DOX + CeO (received both treatments). Hepatic toxicity was assessed by histological and ultrastructural studies. In addition, serum transaminases (ALT, AST) and malondialdehyde (MDA), an oxidative stress marker, were evaluated. CeONPs were not only proved to be safe at the proposed dose, but also their concomitant administration with DOX managed to mitigate DOX-induced hepatic insult on both histological and biochemical aspects. Such hepatoprotective behavior was referred to the noticed antioxidant action CeONPs as highlighted by the significant difference in MDA levels.
1. Introduction Doxorubicin (DOX), one of the anthracyclines, is among the most widely used chemotherapeutic agents. It plays an essential role in the treatment of many malignancies such as lymphoma, leukemia and sarcomas. However, using DOX faces restrictions owing to its systemic side effects: cardiac, hepatic and renal toxicity [1]. In the current study, hepatic insult was chosen as it affects not only liver function in 40% of patients receiving DOX (as one of the potentially affected organs) [2], but also it may affect DOX metabolism and clearance as the liver is the main organ concerned with DOX detoxification. Such hepatic toxicity could be due to oxidative stress, apoptosis and interference with the electron transport chain [3]. Over the years, many studies discussed different strategies to overcome DOX-mediated toxicity, however no ideal agent is yet recommended. Although dexrazoxane was the only FDA approved agent against DOX toxicity, it is rarely used clinically due to its reported side effects including decreased DOX efficacy and increasing the incidence of acute myeloid leukemia and myelodysplastic syndrome [4,5]. Antioxidans as vitamins A, E, C as well berberine, resveratrol and wheat germ oil were widely investigated to assess their potential to mitigate DOX-imposed hepatotoxicity [6,7]. ⁎
Different DOX formulations as pegylated and non-pegylated liposomes were reported to increase the efficacy and selectivity of DOX with decreased liver toxicity [8,9]. Nowadays, nanomedicine affords innovative solutions in different scientific areas through unique physical, chemical, and biological features of the materials in their nanoscale form (< 100 nm) [10]. Cerium, the first member in the lanthanide group, is known to be the most abundant among the rare earth metals. Cerium oxide nanoparticles (CeONPs) enter in the synthesis of various industrial products such as: oxygen sensors, ultraviolet filters and polishing materials in glass and optics industry. They also show importance as a diesel fuel additive to increase combustion efficiency and decrease diesel soot emissions [11]. It is interesting how cerium oxide (CeO) can exist in both the trivalent state (Ce3+) and the tetravalent state (Ce4+) oxidation states permitting redox reactions [12]. CeONPs show numerous promising biomedical applications due to their regenerative ability to scavenge free radicals in addition to their anti-angiogenic, anti-inflammatory and anti-apoptotic properties [13]. So far, several studies had suggested the use of ceria nanoparticles for treatment of many reactive oxygen species-induced pathologies [14] such as neuronal diseases [15] and retinopathies, either inherited or acquired [16]. Similarly, CeO proved to
Corresponding author at: Department of Medical Histology and Cell Biology, Almowasah Campus, Faculty of Medicine, University of Alexandria, Alexandria, Egypt. E-mail address: [email protected] (N. Attia).
https://doi.org/10.1016/j.biopha.2018.04.075 Received 24 February 2018; Received in revised form 8 April 2018; Accepted 9 April 2018 0753-3322/ © 2018 Elsevier Masson SAS. All rights reserved.
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by a Humalyzer junior photometer (Human Diagnostics, Germany) [22].
have a protective role against endometriosis [17], microvascular dysfunction in hypertension [18] and acute kidney injury [19]. On the other hand, opposite, to the obvious protective role of CeONPs in healthy tissues, CeONPs could prevent tumor progression and sensitize tumor cells to radiation [20]. Recently, an inspiring in vitro study [21] has reported that the co-administration of CeONPs with DOX could boost its anti-neoplastic activity in melanoma cells without hampering the viability of normal stromal cells. Thus, the aim of the present study is to verify the in vivo efficacy of CeONPs to mitigate doxorubicin-induced hepatotoxicity in rats. To the best of our knowledge, this would be the first in vivo study to determine the protective impact of CeONPs on the liver of DOX-treated animals.
2.4.2. Liver function assays Serum alanine aminotransferase (ALT) and Aspartate aminotransferase (AST) were measured using calorimetric kits obtained from Spectrum Diagnostics (Cairo, Egypt). Absorbance of specimens was measured against reagent blank at 546 nm with the aid of Humalyzer junior photometer (Human Diagnostics, Germany) [23]. 2.5. Histology study 2.5.1. Light microscopy Liver specimens (0.5 cm3) were fixed in 10% formol saline. All samples were dehydrated in a graded series of ethanol, cleared in xylol and embedded in paraffin. 6 μm thick paraffin sections were stained by hematoxylin and eosin (H&E) [24]. Photomicrographs were acquired using an Olympus microscope (equipped with a digital camera) at the center of excellence for research in regenerative medicine and its applications (CERRMA), Faculty of Medicine, University of Alexandria.
2. Materials and methods 2.1. Materials CeONPs of size < 25 nm (product number 544841) were purchased from Sigma–Aldrich (St Louis, MO, USA) and suspended in distilled water at a concentration of 0.4 mg/ml. Immediately before in vivo administration, the suspension was sonicated at 230 V for 2 min using USR3/2 907 sonicator (Julabo Labortechnik, Seelbach, Germany). Doxorubicin hydrochloride solution (2 mg/ml), Adricin®, was purchased from EIMC United pharmaceutical (Cairo, Egypt).
2.5.2. Transmission electron microscopy Liver specimens (1 mm3) were fixed in 3% glutaraldehyde in PBS (pH 7.4) for 24 h at 4 °C, washed and post-fixed with 1% OsO4 for 2 h. All samples were dehydrated in a graded series of ethanol and embedded in epoxy resin. Ultrathin sections (90 nm) were obtained with a diamond knife on an LKB microtome. Subsequently, they were mounted on copper grids and double-stained with uranyl acetate and lead citrate [25]. Electron micrographs were obtained by TEM (Jeol 100 CX, Tokyo, Japan) equipped with a digital camera in the electron microscopy unit, Faculty of Science, University of Alexandria.
2.2. Animals Forty adult male Sprague Dawley rats (6–8 weeks old, 200 ± 20 g) were used. Rats were maintained under standard laboratory conditions of temperature and humidity and 12 h light/dark cycles and were fed standard laboratory diet and tap water. Guidelines for care and use of animals, approved by the animal house center, Faculty of Medicine, University of Alexandria, were followed.
2.6. Morphometric study
2.3. Experimental design
For control and CeO groups, the surface area of hepatic sinusoids was measured in the captured images according to the adjusted threshold using NIH Fiji© program. For each group, five randomly selected H&E stained sections were analyzed. For the studied groups, the intercellular distance between the lateral domains of adjacent hepatocyte was measured in 20 randomly selected electron micrographs using the NIH Fiji© program.
In a study that extended for two weeks, rats were randomly divided into 4 groups (10 rats each):
• Group I (control) where rats received 250 μl of phosphate buffered saline (PBS) intraperitoneally every other day. • Group II (CeO) that received IP injections of CeONPs suspension at a dose of 0.5 mg/kg once per week. • Group III (DOX) in which rats were injected IP with DOX solution at a dose of 2.5 mg/kg every other day. • Group IV (DOX + CeO) that received the same treatment of both
2.7. Statistical analysis All numerical data were shown as mean ± SD. Differences were considered statistically significant at p < 0.05 calculated by the ANOVA and the Student’s t-test.
group II and III.
3. Results and discussion
Animals were weighed on days 1, 8 and 14 to determine body weight changes during the course of treatment. After 24 h of the last injection (day 15 of the study), animals were euthanized. Blood samples (5 ml) were drawn from the aorta then centrifuged at 1000×g for 10 min within 1 h after collection to obtain serum. Samples were stored at −20 °C till further use. Samples of liver tissue were obtained for histopathological assessment.
3.1. Characterization of cerium oxide nanoparticles CeONPs 3.1.1. Transmission electron microscopy (TEM) The electron micrograph (Fig. 1-A) showed multifaceted particles that depicted various shapes (triangular, quadrangular and polyhedral geometric shapes). CeONPs were mainly with the average size range of 24.06 ± 8.27 nm showing a tendency to agglomerate. Nanoparticles’ size is an important factor determining the rate of activity, cellular interaction, internalization and subcellular distribution. The fraction of redox active Ce3+ ions in the particles is inversely proportionate to the particle size [26]. Thereafter, it was recommended to use a particle size less than 30 nm to increase the percentage of Ce3+ valence status [12].
2.4. Serum biochemical study 2.4.1. Oxidative marker assay Serum Malondialdehyde (MDA) levels were measured as markers of oxidative stress. The MDA calorimetric assay was made using kits obtained from Biodiagnostic (Giza, Egypt). MDA level- an indicator of lipid peroxidation- was determined as Thiobarbituric Acid Reactive Substances (TBARS). Peroxidation of the tissue lipids results in the formation of peroxide compounds that binds with thiobarbituric acid by heating leading to chromogen formation that was measured at 530 nm
3.1.2. Ultraviolet–visible absorption spectroscopy (UV–vis) Optical responses of CeONPs provide information about the oxidation state of CeONPs used in the present work. The absorbance 774
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Fig. 1. Characterization of cerium oxide nanoparticles CeONPs. (A) TEM micrograph (scale bar = 50 nm) (B) UV absorbance in nm (C) Hydrodynamic size distribution (in nm) by the intensity. (D) Zeta potential in mV.
Fig. 2. Body weight and Biochemical studies. (A) Animals’ body weight (g) followed-up for two weeks (the duration of study). (B) Serum MDA levels (nM/ml). (C) Serum AST levels (U/L). (D) Serum ALT levels (U/L). Values represent mean ± SD (n = 10). Statistical significance was determined using ANOVA and t tests. Similar letters indicate no statistical difference (p ˃ 0.05), while different letters would indicate a true statistical difference (p ˂ 0.05).
region of the spectrum [27]. CeONPs undergo reduction reaction (Ce4+ → Ce3+) by losing oxygen from surface atoms with an increase in the number of oxygen vacancies (defect sites) on the CeONPs surface. The ratio of Ce3+/Ce4+ sites on the surface is directly proportionate
spectrum recorded was below 400 nm (in UV range) with a peak at around 308 nm (Fig. 1-B). This indicates fluorite cubic nature of CeONPs and the presence of active Ce3+ status that shows an absorbance peak in the UV range, while that of Ce4+ is located in the visible 775
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Fig. 3. Histological impact of CeO on liver tissue. (A, B) Control group and (C, D) CeO group. Cords of hepatocytes radiate from the central veins (CV). Portal tract (Pt) is present at the angle of hepatic lobule. Sinusoids (S) appear dilated in CeO group. (K) Kupffer cells and (E) endothelial cells. Some hepatocytes show mild vacuolation (arrow). Binucleated hepatocytes are present (double arrow). Scale bars (A,C) 200 μm, (B,D) 50 μm. (E) Influence of CeONPs on the sinusoidal surface area (μm²). Values represent mean ± SD (n = 5). Statistical significance was determined using t tests. Different letters indicate significant statistical differences at (p ˂ 0.05).
route to induce hepatotoxicity model of doxorubicin [34–36]. Both IP and IV administration of CeONPs has resulted in similar levels in our target organ in the current study, the liver [37]. Therefore, we were encouraged to introduce CeONPs through the same IP route as that for the DOX.
with the antioxidant/enzyme-mimetic activity of CeONPs [12]. 3.1.3. Particle size and zeta potential CeONPs suspended in distilled water had a positive zeta potential (ZP) of 24.5 ± 7.79 mV and average size of 99.82 ± 0.15 nm (Fig. 1-C and D). Higher values (˃30 mV) of (ZP) ensure better stability of the suspension with no aggregates [28]. The relatively low ZP value led to the aggregations seen in electron micrographs. As a result, the hydrodynamic diameter assessed by Zetasizer was larger than that measured by TEM due to the known tendency of CeONPs to agglomerate when once measured in suspension [29–31]. Therefore, CeONPs suspensions were sonicated immediately before injection into experimental animals to ensure uniform distribution of nanoparticles [32]. In vivo, aggregation of CeONPs is much evaded by acquiring the biomolecular corona from plasma proteins that enhances their stability and decrease the state of aggregation [33]. Several experimental studies had adopted IP
3.2. Animals’ body weight and survival In the present study, no mortalities were recorded in any of the experimental groups. As shown in Fig. 2-A, experimental animals managed to gain weight normally throughout the duration of the experiment in both control and CeO groups (p ˃ 0.05). Marked decrease in food consumption was observed in both DOX treated groups (DOX, DOX + CeO). By the end of the second week of the study, group III (DOX) showed significant weight loss compared to both control and CeO groups (p ˂ 0.05). Group IV (DOX + CeO) depicted no marked 776
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Fig. 4. Histopathological assessment of DOX versus DOX + CeO on liver tissue. (A–D) DOX-treated rats and (E, F) DOX + CeO group. (A) Marked disorganization of hepatic tissue, periportal leucocytic infiltrations (Pt) are seen. (B) Congestion of central vein (CV) and blood sinusoids(S), ballooned hepatocytes with dark eccentric nuclei (arrow) are depicted. (C) Hypereosinophillic cells (double arrows) that surround degenerated and inflammatory cells (*). Note apoptotic bodies (arrow head). (D) Portal tract is surrounded by leucocytic infiltration (L) and shows bile duct hyperplasia (b). Note Hypereosinophillic cells around the portal tract (double arrow), highly vacuolated hepatocytes with dark, eccentric nuclei (arrow) and Kupffer cells (K). (E, F) depict preserved hepatic architecture. Cords of hepatocytes radiate from central vein (CV). Normal constituents of portal tract with less sinusoidal congestion (S) and cellular infiltration are noticed. Hepatocytes depict vesicular nuclei and mild cytoplasmic vacuolation (arrow), while some are binucleated (*).Scale bars (A, E) 200 μm, (B–D and F) 50 μm.
difference from the DOX group (p ˃ 0.05). Chemotherapy is usually associated with a state of cachexia (unintentional loss of adipose tissue and muscular tissue). As a result, animals’ body weight is considered an indicator of general health in models of chemotherapy-induced fatigue [38]. The weight loss in DOX group could be attributed to the diminished food intake [data not shown] that might be the result of central (induction of chemotherapy trigger zone or vomiting center) and/or peripheral (stomatitis and gastroenteritis) mechanisms [39]. Interestingly, animals’ body weights in group IV (DOX + CeO) did not differ markedly from DOX group. As reported previously, CeONPs showed poor penetration to the blood-brain barrier (BBB) [40]. Others stated that ceria NPs could not be detected in the brain after systemic injection [37]. This could explain the inability of CeONPs to suppress central mechanisms of DOX-induced weight loss.
3.3. Biochemical study 3.3.1. Oxidative marker assay Malondialdehyde (MDA) is recognized as a major product of oxidation of polyunsaturated fatty acids. Therefore, high MDA levels are considered as an indicator of lipid peroxidation [41]. As shown in Fig. 2-B, the difference in mean serum MDA levels in groups I (control) and II (CeO) did not reach significance (p > 0.05). However, serum level of (MDA) showed more than three fold increase in group III (DOX) (24.23 ± 2 nM/ml) compared to control group in the current study (p ˂ 0.05). In group IV (DOX + CeO), MDA level (12.8.1 ± 1.3 nM/ml) was markedly lower than that of DOX group (p ˂ 0.05), which provides an evidence for the potent antioxidative action of CeONPs. 3.3.2. Liver function assay Serum ALT and AST are useful indicators of hepatocyte integrity as both are normally located intracellular. In the present study, the mean 777
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Fig. 5. Ultrastructural effects of CeONPs on rat liver. (A, B)Control group and (C, D) CeO group. (A, B) Electron micrograph showing binucleated hepatocyte. Both nuclei (N) are euchromatic with smooth outline and prominent nucleoli (nu). The cytoplasm shows sER, rER and mitochondria (m). Note bile canaliculus (b) with microvilli protruding in its lumen and junctional complexes on both sides (arrow heads). (C) Electron micrograph showing hepatocyte with euchromatic nucleus (N) and prominent nucleolus (nu), abundant mitochondria (m), rER and lipid droplets (L). Blood sinusoid (S) retained some RBCs. Kupffer cell (K). Inset: higher magnification showing mitochondria (m) containing electron dense particles (< 25 nm). (D) Electron micrograph of CeO group represents Kupffer cell depicting lysosomes(Ly), endocytotic vacuoles (V) and irregular nucleus (N). Microvilli (Mv) project into the space of Disse (arrow). Sinusoid-lining endothelial cell (e). Inset: at a higher magnification, lysosomes (Ly) with internalized electron dense particles (< 25 nm).
3.4. Histology and morphometric studies
serum levels of ALT and AST in the control group were 8 ± 2.8 U/L and 43.5 ± 2.12 U/L respectively (Fig. 2-C, -D). ALT levels in group II (CeO) were approximately similar to that of the control group (p ˃ 0.05) while AST levels showed higher values (p ˂ 0.05). This elevation could merely denote non-specific tissue injury as AST is widely distributed in the body organs. It is worth noting that ALT is considered the most liver-specific of the transaminases [24]. DOX injection in group III caused significant elevation in liver transaminases (more than three folds in ALT and two folds in AST) which designate compromised cellular integrity. However, the values of ALT and AST (7.86 ± 2.64 U/L and 59.8 ± 9 U/L, respectively) in group IV (DOX + CeO) were significantly lower than DOX group (p < 0.05). The lower levels of liver enzymes in DOX + CeO group suggest the beneficial anti-inflammatory/anti-oxidant impact of CeONPs when concomitantly administered with DOX [13].
3.4.1. Light microscopy As depicted in Fig. 3, injection of CeONPs (group II) maintained near normal hepatic architecture where hepatocytes showed granular acidophilic cytoplasm and central vesicular nuclei, some binucleated hepatocytes were frequently noticed denoting the safety of CeONPs at the given dose. Though, blood sinusoids were markedly dilated (Fig. 3C, D and E) (as indicated by the increased gap between the hepatic cords in a hepatic lobule). Similar dilatation was reported after intratracheal instillation of CeONPs at doses of 1–7 mg/kg [42]. Marzano and his colleagues reviewed different mechanisms for sinusoidal dilation; portal outflow obstruction (veno-occlusive lesion), non-specific inflammatory response secondary to various drugs or even oral contraceptives. Activation of the interleukin-6 and vascular endothelial growth factor-related pathways are thought to play a role in sinusoidal dilatation [43]. On the other hand, in DOX-treated group (III), the histological 778
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Fig. 6. Ultrastructural effects of DOX with and without CeO on liver tissue. (A–C) DOX group and (D–F) DOX + CeO group. (A–C) Electron micrograph showing some areas of cytoplasmic rarefaction (*), dilated rER, dense pleomorphic mitochondria (m) and euchromatic nuclei (N) with prominent nucleolus (nu). Sinusoid (S) retained some RBCs. Kupffer cells (K). Note the presence of apoptotic body (AP) showing dense nucleus and marked thickening of intercellular spaces (arrow). (D–E) Electron micrograph showing preserved hepatocytes’ structure with mild rarefaction (*), lipid droplets (L), dilated rER, euchromatic nucleus (N) and prominent nucleolus (nu). RBCs and a neutrophil are seen in a sinusoid (S). Note mild thickening in inter-cellular space (arrows). Inset: a higher magnification of (E) revealing pleomorphic mitochondria (m) with electron dense particles of the size range (< 25 nm). (F): Bar graph demonstrating the width of intercellular space (nm). Values represent mean ± SD. Statistical significance was determined using ANOVA and t tests. Different letters indicate significant statistical differences at (p ˂ 0.05). (n = 5).
epithelial cells as those lining the bile ducts resulting in the observed biliary hyperplasia (Fig. 4-D) and the associated inflammatory cellular infiltration [47]. Ballooning degeneration and vacuolar appearance of the hepatocytes (Fig. 4-B and D) was attributed to the hydropic changes, where ATP depletion leads to cessation of energy-dependent ion pumps of plasma membranes with subsequent accumulation of cations. This will be followed by water accumulation in the cytoplasm and membranous compartments [52]. Different apoptotic figures were noticed among the hepatocytes (Fig. 4-C) in the form of; densely compacted nuclear chromatin, nuclear fragments, hypereosinophillic cytoplasm and apoptotic bodies. Mitochondria are the most extensively injured organelles by doxorubicin, which is retained in the mitochondrial inner membrane interfering with oxidative phosphorylation, electron transport, and leading to ATP depletion, as well as an irreversible bonding with cardiolipin. Furthermore, DOX decreases mitochondrial membrane potential and leads to increased mitochondrial content of Ca++ ions [53]. As a consequence, accumulated Ca++ leads to mitochondrial swelling, followed by rupture of the outer membrane, resulting in activation of the intrinsic apoptotic pathway [54]. Ghadially [55] connected such findings to ROS production, resulting in failure of mitochondrial cation pumps. Furthermore, DOX promotes p53 expression and causes DNA damage which initiates the intrinsic pathway through the pro-apoptotic proteins as Bax and caspase–3 proteins with simultaneous inhibition of antiapoptotic Bcl-2 protein [56].
sections showed marked disorganization of hepatic tissue as well as parenchymal lesions (Fig. 4-A). At a cumulative dose equivalent to that clinically administered (450–550 mg/m2 body surface area) [44], DOX induced histological and biological effects similar to the long term experiments (6–9 weeks) [45]. Doxorubicin-induced hepatotoxicity was associated with an oxidative stress status (imbalance in the normal metabolism of oxygen either due to the excessive production of reactive oxygen species (ROS) and/or a reduction in the antioxidants and sulfhydryl groups). [46] The addition of one electron to the quinone moiety of DOX leads to the formation of semiquinone form that regenerates the quinone form by reducing molecular oxygen to ROS. In turn, such ROS can generate both hydrogen peroxide (H2O2) and hydroxyl radicals (OH) [47]. Fig. 4-B depicted congestion of central veins and sinusoids which was related to veno-occlusive lesions associated with DOX administration [48]. Vascular congestion was explained to be due to cytokinesinduced vascular endothelial cells swelling and malfunctioning that might result in retention of blood elements in the space of Disse [49]. In addition, inflammatory foci (Fig. 4-C) seen after DOX administration were attributed to the recruitment, activation and subsequent accumulation of leucocytes in response to tissue damage [50]. Periportal leucocytic infiltration seen in Fig. 4-D was previously referred to ROS formation through promoting cytokines IL-6 and IL-8 (the main modulators of hepatic inflammatory response) [51]. Moreover, the DOX-induced oxidative stress could increase the expression of endothelial growth factors and therefore, enhancing the mitotic activity of 779
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Author's contributions
Interestingly, the concomitant administration of both CeO and DOX (group IV) revealed preserved hepatic architecture. Hepatocytes demonstrated less vacuolated cytoplasm and vesicular nuclei. The portal tract showed preserved histological features with minimal cellular infiltration (Fig. 4-E and F). CeONPs act as an inhibitor of ROS generation and the scavenger of already present ones. Besides, they potentiate several antioxidant enzymes [57]. The results found in the present work supported previously published data where CeONPs succeeded to counteract the hepatotoxicity caused by monocrotaline injection in rats [13] and played an anti-hyperlipidemic role in CCl4-induced hepatotoxicity [58]. Several binucleated hepatocytes were noticed which could be considered as a sign of the preserved regenerative power of hepatocytes compared to the mononuclear shift discerned in DOX group (Fig. 4-F). Binucleated hepatocytes are more likely to undergo mitosis in case of liver injury by being more responsive to hepatocytes growth factors [59].
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3.4.2. Electron microscopy An important aspect of the current study was the emphasis on the hepatic ultrastructural features in the studied groups. Electron microscopic examination of group II (CeO) demonstrated well-preserved ultrastructure of hepatocytes (Fig. 5-C). Electron micrographs illustrated electron dense particles within the same size range of the used CeONPs (˂25 nm) in the organelles of hepatocytes and lysosomes of Kupffer cells (Figs. 5-C and D). On the other hand, group III (DOX) revealed severely affected hepatocytes where the cytoplasm depicted marked rarefaction as the hallmark of hydropic degeneration. In addition, Fig. 6-A, B and C showed dense pleomorphic mitochondria and dilated rER. Thickened intercellular space and apoptotic bodies were also seen. These findings were previously reported in mice and rats that received DOX as well as other chemotherapeutic agents as cisplatin and 5-flurouracil [48,50]. Drug-induced increase in portal blood pressure in addition to cell membrane lesion were proposed as probable causes for such dilatation of the intercellular spaces [60,61]. In group IV (DOX + CeO) hepatocytes were less rarefied/ vacuolated compared to DOX group. They had euchromatic regular nuclei, prominent nucleoli and organelles with normal morphology (Fig. 6-D and E). Electron dense particles of 14–24 nm size range seen in mitochondria of hepatocytes are believed to be the injected CeONPs (Fig. 6-E). As reported by Singh and his colleagues [62], nanoceria could be localized in mitochondria, lysosomes, endoplasmic reticulum and even the nucleus. Such wide range of intracellular distribution allows for protection against variable mechanisms of oxidative stresses. The localization of CeONPs in hepatocytic mitochondria, as seen in the current study was shown to significantly boost their antioxidative effects [63]. Furthermore, the use of CeONPs had markedly attenuated the DOX-induced dilatation of inter-hepatocyte spaces (p ˂ 0.05) (Fig. 6-F). To conclude, CeONPs -at the tested dose- did not cause any manifest harm to hepatic tissue. Moreover, thanks to their anti-oxidant effect, they managed to ameliorate the DOX-related hepatotoxic effects both at the structural and the functional levels. Such findings would pave the way for further in vivo studies conducted in various animal models of cancer rather than normal rats.
Disclosure The authors report no conflicts of interest in this work.
Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. 780
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Cerium oxide nanoparticles: In pursuit of liver protection against doxorubicin-induced injury in rats
T
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Heba G. Ibrahima, Noha Attiaa,b, , Fatma El Zahraa A. Hashema, Moushira A.R. El Heneidya a
Department of Medical Histology and Cell Biology, Faculty of Medicine, University of Alexandria, Alexandria, Egypt Department of Basic Sciences, The American University of Antigua-College Of Medicine, University Park, Jabberwock Beach Road, P.O. Box 1451, Coolidge, Antigua and Barbuda b
A R T I C LE I N FO
A B S T R A C T
Keywords: Cerium oxide nanoparticles Oxidative stress Doxorubicin Antioxidant Hepatic inflammation
Doxorubicin (DOX) is considered as a backbone in several chemotherapeutic regimens. Nevertheless, the reported systemic toxicity usually hampers its broad application. Interestingly, Cerium oxide nanoparticles (CeONPs) depicted promising regenerative antioxidant and hepatoprotective potentials against multiple oxidative stress-induced pathologies. Thus, the aim of the present study was to determine either CeONPs would display hepatoprotective properties once concomitantly administered with DOX or not. Male Sprague Dawley rats were divided into four groups (n = 10) in a two weeks study: Control (received saline, IP injection thrice a week), CeO (0.5 mg/kg, IP injection once a week), DOX (2.5 mg/kg, IP injections thrice a week) and DOX + CeO (received both treatments). Hepatic toxicity was assessed by histological and ultrastructural studies. In addition, serum transaminases (ALT, AST) and malondialdehyde (MDA), an oxidative stress marker, were evaluated. CeONPs were not only proved to be safe at the proposed dose, but also their concomitant administration with DOX managed to mitigate DOX-induced hepatic insult on both histological and biochemical aspects. Such hepatoprotective behavior was referred to the noticed antioxidant action CeONPs as highlighted by the significant difference in MDA levels.
1. Introduction Doxorubicin (DOX), one of the anthracyclines, is among the most widely used chemotherapeutic agents. It plays an essential role in the treatment of many malignancies such as lymphoma, leukemia and sarcomas. However, using DOX faces restrictions owing to its systemic side effects: cardiac, hepatic and renal toxicity [1]. In the current study, hepatic insult was chosen as it affects not only liver function in 40% of patients receiving DOX (as one of the potentially affected organs) [2], but also it may affect DOX metabolism and clearance as the liver is the main organ concerned with DOX detoxification. Such hepatic toxicity could be due to oxidative stress, apoptosis and interference with the electron transport chain [3]. Over the years, many studies discussed different strategies to overcome DOX-mediated toxicity, however no ideal agent is yet recommended. Although dexrazoxane was the only FDA approved agent against DOX toxicity, it is rarely used clinically due to its reported side effects including decreased DOX efficacy and increasing the incidence of acute myeloid leukemia and myelodysplastic syndrome [4,5]. Antioxidans as vitamins A, E, C as well berberine, resveratrol and wheat germ oil were widely investigated to assess their potential to mitigate DOX-imposed hepatotoxicity [6,7]. ⁎
Different DOX formulations as pegylated and non-pegylated liposomes were reported to increase the efficacy and selectivity of DOX with decreased liver toxicity [8,9]. Nowadays, nanomedicine affords innovative solutions in different scientific areas through unique physical, chemical, and biological features of the materials in their nanoscale form (< 100 nm) [10]. Cerium, the first member in the lanthanide group, is known to be the most abundant among the rare earth metals. Cerium oxide nanoparticles (CeONPs) enter in the synthesis of various industrial products such as: oxygen sensors, ultraviolet filters and polishing materials in glass and optics industry. They also show importance as a diesel fuel additive to increase combustion efficiency and decrease diesel soot emissions [11]. It is interesting how cerium oxide (CeO) can exist in both the trivalent state (Ce3+) and the tetravalent state (Ce4+) oxidation states permitting redox reactions [12]. CeONPs show numerous promising biomedical applications due to their regenerative ability to scavenge free radicals in addition to their anti-angiogenic, anti-inflammatory and anti-apoptotic properties [13]. So far, several studies had suggested the use of ceria nanoparticles for treatment of many reactive oxygen species-induced pathologies [14] such as neuronal diseases [15] and retinopathies, either inherited or acquired [16]. Similarly, CeO proved to
Corresponding author at: Department of Medical Histology and Cell Biology, Almowasah Campus, Faculty of Medicine, University of Alexandria, Alexandria, Egypt. E-mail address: [email protected] (N. Attia).
https://doi.org/10.1016/j.biopha.2018.04.075 Received 24 February 2018; Received in revised form 8 April 2018; Accepted 9 April 2018 0753-3322/ © 2018 Elsevier Masson SAS. All rights reserved.
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by a Humalyzer junior photometer (Human Diagnostics, Germany) [22].
have a protective role against endometriosis [17], microvascular dysfunction in hypertension [18] and acute kidney injury [19]. On the other hand, opposite, to the obvious protective role of CeONPs in healthy tissues, CeONPs could prevent tumor progression and sensitize tumor cells to radiation [20]. Recently, an inspiring in vitro study [21] has reported that the co-administration of CeONPs with DOX could boost its anti-neoplastic activity in melanoma cells without hampering the viability of normal stromal cells. Thus, the aim of the present study is to verify the in vivo efficacy of CeONPs to mitigate doxorubicin-induced hepatotoxicity in rats. To the best of our knowledge, this would be the first in vivo study to determine the protective impact of CeONPs on the liver of DOX-treated animals.
2.4.2. Liver function assays Serum alanine aminotransferase (ALT) and Aspartate aminotransferase (AST) were measured using calorimetric kits obtained from Spectrum Diagnostics (Cairo, Egypt). Absorbance of specimens was measured against reagent blank at 546 nm with the aid of Humalyzer junior photometer (Human Diagnostics, Germany) [23]. 2.5. Histology study 2.5.1. Light microscopy Liver specimens (0.5 cm3) were fixed in 10% formol saline. All samples were dehydrated in a graded series of ethanol, cleared in xylol and embedded in paraffin. 6 μm thick paraffin sections were stained by hematoxylin and eosin (H&E) [24]. Photomicrographs were acquired using an Olympus microscope (equipped with a digital camera) at the center of excellence for research in regenerative medicine and its applications (CERRMA), Faculty of Medicine, University of Alexandria.
2. Materials and methods 2.1. Materials CeONPs of size < 25 nm (product number 544841) were purchased from Sigma–Aldrich (St Louis, MO, USA) and suspended in distilled water at a concentration of 0.4 mg/ml. Immediately before in vivo administration, the suspension was sonicated at 230 V for 2 min using USR3/2 907 sonicator (Julabo Labortechnik, Seelbach, Germany). Doxorubicin hydrochloride solution (2 mg/ml), Adricin®, was purchased from EIMC United pharmaceutical (Cairo, Egypt).
2.5.2. Transmission electron microscopy Liver specimens (1 mm3) were fixed in 3% glutaraldehyde in PBS (pH 7.4) for 24 h at 4 °C, washed and post-fixed with 1% OsO4 for 2 h. All samples were dehydrated in a graded series of ethanol and embedded in epoxy resin. Ultrathin sections (90 nm) were obtained with a diamond knife on an LKB microtome. Subsequently, they were mounted on copper grids and double-stained with uranyl acetate and lead citrate [25]. Electron micrographs were obtained by TEM (Jeol 100 CX, Tokyo, Japan) equipped with a digital camera in the electron microscopy unit, Faculty of Science, University of Alexandria.
2.2. Animals Forty adult male Sprague Dawley rats (6–8 weeks old, 200 ± 20 g) were used. Rats were maintained under standard laboratory conditions of temperature and humidity and 12 h light/dark cycles and were fed standard laboratory diet and tap water. Guidelines for care and use of animals, approved by the animal house center, Faculty of Medicine, University of Alexandria, were followed.
2.6. Morphometric study
2.3. Experimental design
For control and CeO groups, the surface area of hepatic sinusoids was measured in the captured images according to the adjusted threshold using NIH Fiji© program. For each group, five randomly selected H&E stained sections were analyzed. For the studied groups, the intercellular distance between the lateral domains of adjacent hepatocyte was measured in 20 randomly selected electron micrographs using the NIH Fiji© program.
In a study that extended for two weeks, rats were randomly divided into 4 groups (10 rats each):
• Group I (control) where rats received 250 μl of phosphate buffered saline (PBS) intraperitoneally every other day. • Group II (CeO) that received IP injections of CeONPs suspension at a dose of 0.5 mg/kg once per week. • Group III (DOX) in which rats were injected IP with DOX solution at a dose of 2.5 mg/kg every other day. • Group IV (DOX + CeO) that received the same treatment of both
2.7. Statistical analysis All numerical data were shown as mean ± SD. Differences were considered statistically significant at p < 0.05 calculated by the ANOVA and the Student’s t-test.
group II and III.
3. Results and discussion
Animals were weighed on days 1, 8 and 14 to determine body weight changes during the course of treatment. After 24 h of the last injection (day 15 of the study), animals were euthanized. Blood samples (5 ml) were drawn from the aorta then centrifuged at 1000×g for 10 min within 1 h after collection to obtain serum. Samples were stored at −20 °C till further use. Samples of liver tissue were obtained for histopathological assessment.
3.1. Characterization of cerium oxide nanoparticles CeONPs 3.1.1. Transmission electron microscopy (TEM) The electron micrograph (Fig. 1-A) showed multifaceted particles that depicted various shapes (triangular, quadrangular and polyhedral geometric shapes). CeONPs were mainly with the average size range of 24.06 ± 8.27 nm showing a tendency to agglomerate. Nanoparticles’ size is an important factor determining the rate of activity, cellular interaction, internalization and subcellular distribution. The fraction of redox active Ce3+ ions in the particles is inversely proportionate to the particle size [26]. Thereafter, it was recommended to use a particle size less than 30 nm to increase the percentage of Ce3+ valence status [12].
2.4. Serum biochemical study 2.4.1. Oxidative marker assay Serum Malondialdehyde (MDA) levels were measured as markers of oxidative stress. The MDA calorimetric assay was made using kits obtained from Biodiagnostic (Giza, Egypt). MDA level- an indicator of lipid peroxidation- was determined as Thiobarbituric Acid Reactive Substances (TBARS). Peroxidation of the tissue lipids results in the formation of peroxide compounds that binds with thiobarbituric acid by heating leading to chromogen formation that was measured at 530 nm
3.1.2. Ultraviolet–visible absorption spectroscopy (UV–vis) Optical responses of CeONPs provide information about the oxidation state of CeONPs used in the present work. The absorbance 774
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Fig. 1. Characterization of cerium oxide nanoparticles CeONPs. (A) TEM micrograph (scale bar = 50 nm) (B) UV absorbance in nm (C) Hydrodynamic size distribution (in nm) by the intensity. (D) Zeta potential in mV.
Fig. 2. Body weight and Biochemical studies. (A) Animals’ body weight (g) followed-up for two weeks (the duration of study). (B) Serum MDA levels (nM/ml). (C) Serum AST levels (U/L). (D) Serum ALT levels (U/L). Values represent mean ± SD (n = 10). Statistical significance was determined using ANOVA and t tests. Similar letters indicate no statistical difference (p ˃ 0.05), while different letters would indicate a true statistical difference (p ˂ 0.05).
region of the spectrum [27]. CeONPs undergo reduction reaction (Ce4+ → Ce3+) by losing oxygen from surface atoms with an increase in the number of oxygen vacancies (defect sites) on the CeONPs surface. The ratio of Ce3+/Ce4+ sites on the surface is directly proportionate
spectrum recorded was below 400 nm (in UV range) with a peak at around 308 nm (Fig. 1-B). This indicates fluorite cubic nature of CeONPs and the presence of active Ce3+ status that shows an absorbance peak in the UV range, while that of Ce4+ is located in the visible 775
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Fig. 3. Histological impact of CeO on liver tissue. (A, B) Control group and (C, D) CeO group. Cords of hepatocytes radiate from the central veins (CV). Portal tract (Pt) is present at the angle of hepatic lobule. Sinusoids (S) appear dilated in CeO group. (K) Kupffer cells and (E) endothelial cells. Some hepatocytes show mild vacuolation (arrow). Binucleated hepatocytes are present (double arrow). Scale bars (A,C) 200 μm, (B,D) 50 μm. (E) Influence of CeONPs on the sinusoidal surface area (μm²). Values represent mean ± SD (n = 5). Statistical significance was determined using t tests. Different letters indicate significant statistical differences at (p ˂ 0.05).
route to induce hepatotoxicity model of doxorubicin [34–36]. Both IP and IV administration of CeONPs has resulted in similar levels in our target organ in the current study, the liver [37]. Therefore, we were encouraged to introduce CeONPs through the same IP route as that for the DOX.
with the antioxidant/enzyme-mimetic activity of CeONPs [12]. 3.1.3. Particle size and zeta potential CeONPs suspended in distilled water had a positive zeta potential (ZP) of 24.5 ± 7.79 mV and average size of 99.82 ± 0.15 nm (Fig. 1-C and D). Higher values (˃30 mV) of (ZP) ensure better stability of the suspension with no aggregates [28]. The relatively low ZP value led to the aggregations seen in electron micrographs. As a result, the hydrodynamic diameter assessed by Zetasizer was larger than that measured by TEM due to the known tendency of CeONPs to agglomerate when once measured in suspension [29–31]. Therefore, CeONPs suspensions were sonicated immediately before injection into experimental animals to ensure uniform distribution of nanoparticles [32]. In vivo, aggregation of CeONPs is much evaded by acquiring the biomolecular corona from plasma proteins that enhances their stability and decrease the state of aggregation [33]. Several experimental studies had adopted IP
3.2. Animals’ body weight and survival In the present study, no mortalities were recorded in any of the experimental groups. As shown in Fig. 2-A, experimental animals managed to gain weight normally throughout the duration of the experiment in both control and CeO groups (p ˃ 0.05). Marked decrease in food consumption was observed in both DOX treated groups (DOX, DOX + CeO). By the end of the second week of the study, group III (DOX) showed significant weight loss compared to both control and CeO groups (p ˂ 0.05). Group IV (DOX + CeO) depicted no marked 776
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Fig. 4. Histopathological assessment of DOX versus DOX + CeO on liver tissue. (A–D) DOX-treated rats and (E, F) DOX + CeO group. (A) Marked disorganization of hepatic tissue, periportal leucocytic infiltrations (Pt) are seen. (B) Congestion of central vein (CV) and blood sinusoids(S), ballooned hepatocytes with dark eccentric nuclei (arrow) are depicted. (C) Hypereosinophillic cells (double arrows) that surround degenerated and inflammatory cells (*). Note apoptotic bodies (arrow head). (D) Portal tract is surrounded by leucocytic infiltration (L) and shows bile duct hyperplasia (b). Note Hypereosinophillic cells around the portal tract (double arrow), highly vacuolated hepatocytes with dark, eccentric nuclei (arrow) and Kupffer cells (K). (E, F) depict preserved hepatic architecture. Cords of hepatocytes radiate from central vein (CV). Normal constituents of portal tract with less sinusoidal congestion (S) and cellular infiltration are noticed. Hepatocytes depict vesicular nuclei and mild cytoplasmic vacuolation (arrow), while some are binucleated (*).Scale bars (A, E) 200 μm, (B–D and F) 50 μm.
difference from the DOX group (p ˃ 0.05). Chemotherapy is usually associated with a state of cachexia (unintentional loss of adipose tissue and muscular tissue). As a result, animals’ body weight is considered an indicator of general health in models of chemotherapy-induced fatigue [38]. The weight loss in DOX group could be attributed to the diminished food intake [data not shown] that might be the result of central (induction of chemotherapy trigger zone or vomiting center) and/or peripheral (stomatitis and gastroenteritis) mechanisms [39]. Interestingly, animals’ body weights in group IV (DOX + CeO) did not differ markedly from DOX group. As reported previously, CeONPs showed poor penetration to the blood-brain barrier (BBB) [40]. Others stated that ceria NPs could not be detected in the brain after systemic injection [37]. This could explain the inability of CeONPs to suppress central mechanisms of DOX-induced weight loss.
3.3. Biochemical study 3.3.1. Oxidative marker assay Malondialdehyde (MDA) is recognized as a major product of oxidation of polyunsaturated fatty acids. Therefore, high MDA levels are considered as an indicator of lipid peroxidation [41]. As shown in Fig. 2-B, the difference in mean serum MDA levels in groups I (control) and II (CeO) did not reach significance (p > 0.05). However, serum level of (MDA) showed more than three fold increase in group III (DOX) (24.23 ± 2 nM/ml) compared to control group in the current study (p ˂ 0.05). In group IV (DOX + CeO), MDA level (12.8.1 ± 1.3 nM/ml) was markedly lower than that of DOX group (p ˂ 0.05), which provides an evidence for the potent antioxidative action of CeONPs. 3.3.2. Liver function assay Serum ALT and AST are useful indicators of hepatocyte integrity as both are normally located intracellular. In the present study, the mean 777
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Fig. 5. Ultrastructural effects of CeONPs on rat liver. (A, B)Control group and (C, D) CeO group. (A, B) Electron micrograph showing binucleated hepatocyte. Both nuclei (N) are euchromatic with smooth outline and prominent nucleoli (nu). The cytoplasm shows sER, rER and mitochondria (m). Note bile canaliculus (b) with microvilli protruding in its lumen and junctional complexes on both sides (arrow heads). (C) Electron micrograph showing hepatocyte with euchromatic nucleus (N) and prominent nucleolus (nu), abundant mitochondria (m), rER and lipid droplets (L). Blood sinusoid (S) retained some RBCs. Kupffer cell (K). Inset: higher magnification showing mitochondria (m) containing electron dense particles (< 25 nm). (D) Electron micrograph of CeO group represents Kupffer cell depicting lysosomes(Ly), endocytotic vacuoles (V) and irregular nucleus (N). Microvilli (Mv) project into the space of Disse (arrow). Sinusoid-lining endothelial cell (e). Inset: at a higher magnification, lysosomes (Ly) with internalized electron dense particles (< 25 nm).
3.4. Histology and morphometric studies
serum levels of ALT and AST in the control group were 8 ± 2.8 U/L and 43.5 ± 2.12 U/L respectively (Fig. 2-C, -D). ALT levels in group II (CeO) were approximately similar to that of the control group (p ˃ 0.05) while AST levels showed higher values (p ˂ 0.05). This elevation could merely denote non-specific tissue injury as AST is widely distributed in the body organs. It is worth noting that ALT is considered the most liver-specific of the transaminases [24]. DOX injection in group III caused significant elevation in liver transaminases (more than three folds in ALT and two folds in AST) which designate compromised cellular integrity. However, the values of ALT and AST (7.86 ± 2.64 U/L and 59.8 ± 9 U/L, respectively) in group IV (DOX + CeO) were significantly lower than DOX group (p < 0.05). The lower levels of liver enzymes in DOX + CeO group suggest the beneficial anti-inflammatory/anti-oxidant impact of CeONPs when concomitantly administered with DOX [13].
3.4.1. Light microscopy As depicted in Fig. 3, injection of CeONPs (group II) maintained near normal hepatic architecture where hepatocytes showed granular acidophilic cytoplasm and central vesicular nuclei, some binucleated hepatocytes were frequently noticed denoting the safety of CeONPs at the given dose. Though, blood sinusoids were markedly dilated (Fig. 3C, D and E) (as indicated by the increased gap between the hepatic cords in a hepatic lobule). Similar dilatation was reported after intratracheal instillation of CeONPs at doses of 1–7 mg/kg [42]. Marzano and his colleagues reviewed different mechanisms for sinusoidal dilation; portal outflow obstruction (veno-occlusive lesion), non-specific inflammatory response secondary to various drugs or even oral contraceptives. Activation of the interleukin-6 and vascular endothelial growth factor-related pathways are thought to play a role in sinusoidal dilatation [43]. On the other hand, in DOX-treated group (III), the histological 778
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Fig. 6. Ultrastructural effects of DOX with and without CeO on liver tissue. (A–C) DOX group and (D–F) DOX + CeO group. (A–C) Electron micrograph showing some areas of cytoplasmic rarefaction (*), dilated rER, dense pleomorphic mitochondria (m) and euchromatic nuclei (N) with prominent nucleolus (nu). Sinusoid (S) retained some RBCs. Kupffer cells (K). Note the presence of apoptotic body (AP) showing dense nucleus and marked thickening of intercellular spaces (arrow). (D–E) Electron micrograph showing preserved hepatocytes’ structure with mild rarefaction (*), lipid droplets (L), dilated rER, euchromatic nucleus (N) and prominent nucleolus (nu). RBCs and a neutrophil are seen in a sinusoid (S). Note mild thickening in inter-cellular space (arrows). Inset: a higher magnification of (E) revealing pleomorphic mitochondria (m) with electron dense particles of the size range (< 25 nm). (F): Bar graph demonstrating the width of intercellular space (nm). Values represent mean ± SD. Statistical significance was determined using ANOVA and t tests. Different letters indicate significant statistical differences at (p ˂ 0.05). (n = 5).
epithelial cells as those lining the bile ducts resulting in the observed biliary hyperplasia (Fig. 4-D) and the associated inflammatory cellular infiltration [47]. Ballooning degeneration and vacuolar appearance of the hepatocytes (Fig. 4-B and D) was attributed to the hydropic changes, where ATP depletion leads to cessation of energy-dependent ion pumps of plasma membranes with subsequent accumulation of cations. This will be followed by water accumulation in the cytoplasm and membranous compartments [52]. Different apoptotic figures were noticed among the hepatocytes (Fig. 4-C) in the form of; densely compacted nuclear chromatin, nuclear fragments, hypereosinophillic cytoplasm and apoptotic bodies. Mitochondria are the most extensively injured organelles by doxorubicin, which is retained in the mitochondrial inner membrane interfering with oxidative phosphorylation, electron transport, and leading to ATP depletion, as well as an irreversible bonding with cardiolipin. Furthermore, DOX decreases mitochondrial membrane potential and leads to increased mitochondrial content of Ca++ ions [53]. As a consequence, accumulated Ca++ leads to mitochondrial swelling, followed by rupture of the outer membrane, resulting in activation of the intrinsic apoptotic pathway [54]. Ghadially [55] connected such findings to ROS production, resulting in failure of mitochondrial cation pumps. Furthermore, DOX promotes p53 expression and causes DNA damage which initiates the intrinsic pathway through the pro-apoptotic proteins as Bax and caspase–3 proteins with simultaneous inhibition of antiapoptotic Bcl-2 protein [56].
sections showed marked disorganization of hepatic tissue as well as parenchymal lesions (Fig. 4-A). At a cumulative dose equivalent to that clinically administered (450–550 mg/m2 body surface area) [44], DOX induced histological and biological effects similar to the long term experiments (6–9 weeks) [45]. Doxorubicin-induced hepatotoxicity was associated with an oxidative stress status (imbalance in the normal metabolism of oxygen either due to the excessive production of reactive oxygen species (ROS) and/or a reduction in the antioxidants and sulfhydryl groups). [46] The addition of one electron to the quinone moiety of DOX leads to the formation of semiquinone form that regenerates the quinone form by reducing molecular oxygen to ROS. In turn, such ROS can generate both hydrogen peroxide (H2O2) and hydroxyl radicals (OH) [47]. Fig. 4-B depicted congestion of central veins and sinusoids which was related to veno-occlusive lesions associated with DOX administration [48]. Vascular congestion was explained to be due to cytokinesinduced vascular endothelial cells swelling and malfunctioning that might result in retention of blood elements in the space of Disse [49]. In addition, inflammatory foci (Fig. 4-C) seen after DOX administration were attributed to the recruitment, activation and subsequent accumulation of leucocytes in response to tissue damage [50]. Periportal leucocytic infiltration seen in Fig. 4-D was previously referred to ROS formation through promoting cytokines IL-6 and IL-8 (the main modulators of hepatic inflammatory response) [51]. Moreover, the DOX-induced oxidative stress could increase the expression of endothelial growth factors and therefore, enhancing the mitotic activity of 779
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Author's contributions
Interestingly, the concomitant administration of both CeO and DOX (group IV) revealed preserved hepatic architecture. Hepatocytes demonstrated less vacuolated cytoplasm and vesicular nuclei. The portal tract showed preserved histological features with minimal cellular infiltration (Fig. 4-E and F). CeONPs act as an inhibitor of ROS generation and the scavenger of already present ones. Besides, they potentiate several antioxidant enzymes [57]. The results found in the present work supported previously published data where CeONPs succeeded to counteract the hepatotoxicity caused by monocrotaline injection in rats [13] and played an anti-hyperlipidemic role in CCl4-induced hepatotoxicity [58]. Several binucleated hepatocytes were noticed which could be considered as a sign of the preserved regenerative power of hepatocytes compared to the mononuclear shift discerned in DOX group (Fig. 4-F). Binucleated hepatocytes are more likely to undergo mitosis in case of liver injury by being more responsive to hepatocytes growth factors [59].
H.G. Ibrahim carried out the experimental work, assisted in designing the experiments and wrote the manuscript. N. Attia shared in experimental design, performed the analytical part of the study and cowrote the manuscript. F.A. Hashem supervised the research and revised the manuscript. M.A.R. El Heneidy optimized the design of experiments, supervised the research, gave conceptual advice and revised the manuscript References [1] Y. Wang, X. Mei, J. Yuan, W. Lu, B. Li, D. Xu, Taurine zinc solid dispersions attenuate doxorubicin-induced hepatotoxicity and cardiotoxicity in rats, Toxicol. Appl. Pharmacol. 289 (1) (2015) 1–11. [2] G. Damodar, T. Smitha, S. Gopinath, S. Vijayakumar, Y. Rao, An evaluation of hepatotoxicity in breast cancer patients receiving injection doxorubicin, Ann. Med. Health Sci. Res. 4 (1) (2014) 74–79. [3] H.A. Jung, J.I. Kim, S.Y. Choung, J.S. 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3.4.2. Electron microscopy An important aspect of the current study was the emphasis on the hepatic ultrastructural features in the studied groups. Electron microscopic examination of group II (CeO) demonstrated well-preserved ultrastructure of hepatocytes (Fig. 5-C). Electron micrographs illustrated electron dense particles within the same size range of the used CeONPs (˂25 nm) in the organelles of hepatocytes and lysosomes of Kupffer cells (Figs. 5-C and D). On the other hand, group III (DOX) revealed severely affected hepatocytes where the cytoplasm depicted marked rarefaction as the hallmark of hydropic degeneration. In addition, Fig. 6-A, B and C showed dense pleomorphic mitochondria and dilated rER. Thickened intercellular space and apoptotic bodies were also seen. These findings were previously reported in mice and rats that received DOX as well as other chemotherapeutic agents as cisplatin and 5-flurouracil [48,50]. Drug-induced increase in portal blood pressure in addition to cell membrane lesion were proposed as probable causes for such dilatation of the intercellular spaces [60,61]. In group IV (DOX + CeO) hepatocytes were less rarefied/ vacuolated compared to DOX group. They had euchromatic regular nuclei, prominent nucleoli and organelles with normal morphology (Fig. 6-D and E). Electron dense particles of 14–24 nm size range seen in mitochondria of hepatocytes are believed to be the injected CeONPs (Fig. 6-E). As reported by Singh and his colleagues [62], nanoceria could be localized in mitochondria, lysosomes, endoplasmic reticulum and even the nucleus. Such wide range of intracellular distribution allows for protection against variable mechanisms of oxidative stresses. The localization of CeONPs in hepatocytic mitochondria, as seen in the current study was shown to significantly boost their antioxidative effects [63]. Furthermore, the use of CeONPs had markedly attenuated the DOX-induced dilatation of inter-hepatocyte spaces (p ˂ 0.05) (Fig. 6-F). To conclude, CeONPs -at the tested dose- did not cause any manifest harm to hepatic tissue. Moreover, thanks to their anti-oxidant effect, they managed to ameliorate the DOX-related hepatotoxic effects both at the structural and the functional levels. Such findings would pave the way for further in vivo studies conducted in various animal models of cancer rather than normal rats.
Disclosure The authors report no conflicts of interest in this work.
Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. 780
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