Hepatotoxic Manifestations of Arsenic Trioxide Loaded Poly (Lactide-co-Glycolide) Nanoparticles in Wistar Rat

Present study reports on the hepatotoxic manifestations of arsenic trioxide loaded poly lactide-co-glycolide nanoparticles (As2O3-PLGA-NPs) in rats. As 2 O 3 -PLGA- NPs enhances the activity of serum transaminases. As 2 O 3 -PLGA-NPs are potential inducer of lipid peroxidation in mitochondria as well as microsomes. Mitochondrial lipid peroxidation was higher than the microsomal lipid peroxidation. CYP 450 2E1 was lower in the liver of As 2 O 3 -PLGA- NPs treated rats in comparison to arsenic trioxide treated rats. GSH showed lower values than control rats and arsenic trioxide treated rats. Glutathione-S-transferase inhibited by arsenic trioxide, non signi�cant increase was recorded in the liver of As 2 O 3 -PLGA- NPs treated rats. As 2 O 3 -PLGA- NPs readily accumulates in liver and induces peculiar histopathological changes viz. intracytoplasmic/intranuclear inclusions and apoptosis. Since As 2 O 3 -PLGA- NPs are being considered as a suitable chemo-preventive agent against different types of cancer, its toxicological properties are of prime concern from bio-safety point of view. Thus, present observations seem to be important from human health risk assessment point of view.


Introduction
Arsenic is number one substance in the most recent comprehensive, environmental, response, compensation and liability act priority list of hazardous substances published by the Agency for Toxic Substances and Disease Registry (ATSDR 2007).It has been associated with several human diseases including cancer, diabetes, skin lesions, respiratory disorders and cardiovascular effects.Best studied mechanism of action (MOA) of arsenic toxicity known today is the formation of reactive oxygen species (ROS) and nitrogen species (NS) ( In recent years, several studies have appeared suggesting e cient use of arsenic trioxide in the treatment of promyelocytic leukemia (Evens et al. 2004) and a number of solid tumors including breast cancer (Ye et al. 2005;Chow et al. 2004;Platanias 2009).Further, nanoparticles of arsenic trioxide has now been prepared and tested for their therapeutic effects on liver cancer cells (Wang et al., 2009), haematological malignancy (Ahn et al. 2013) and osteosarcoma (Li et al. 2007).Increasing experimental evidences suggest that special physiochemical properties of these nanoparticles might pose potential risk to human health (Nel et al. 2006).Therefore, considerable effort should be made to identify the potential toxicity of nanoparticles to cells and organisms.Nanoparticles of different sizes and composition do present toxicological problems.However, a comprehensive mechanism of nanoparticles cytotoxicity has not been investigated.
Recent advances in drug delivery have encouraged the development and use of nanoparticles as drug carrier.Several agents have been developed as drug carriers in the past, for example, encapsulation of drugs in poly lactide-co-glycolide (PLGA) nanoparticles is a safe system for human application (Zhao and Zhang 2009).Ahn et al. (2013) demonstrated that the antitumor e cacy of arsenic trioxide loaded nanobins was superior to free arsenic trioxide.Li et al. (2007) investigated the therapeutic e cacy of the magnetic arsenic trioxide nanoparticles against osteosarcoma in vivo tumor models.
The objective of the present investigations was to study the toxicity of arsenic trioxide loaded PLGA nanoparticles (As 2 O 3 -PLGA-NPs) in the liver of rat.As 2 O 3 -PLGA-NPs toxicity is expected to be helpful in the management of cancer therapy.Further, biosafety issue of nanoarsenic is also addressed.Toxicological evaluation of As 2 O 3 -PLGA-NPs is expected to add our knowledge on their therapeutic e cacy, if any.
Commercial kits for the determination of alanine transaminases, aspartate transaminase, and alkaline phosphatase were procured from Span Diagnostics, Surat (India).Other chemicals and reagents used in this study were of analytical grade and procured from E. Merck (India) and Glaxo (India).

Preparation of arsenic trioxide loaded PLGA nanoparticles
A polymer, PLGA was used to prepare arsenic trioxide nanoparticles with w/o/w double emulsion evaporation technique with minor modi cations (Zhao and Zhang 2009).Brie y, the rst emulsion was formed between an organic solution of the polymer (100 mg PLGA in 2ml methylene chloride) and an aqueous solution of arsenic trioxide.Then 12ml aqueous solution containing 2.25% of polyvinyl alcohol was added to this primary emulsion and sonicated (Sonics and Materials Inc. USA) to obtain the double emulsion.Afterwards the solvent evaporation was carried out by gentle magnetic stirring at room temperature.The suspension was washed with distilled water and centrifuged three times at 12,000 rpm.Subsequently, the sediments were freeze dried and sterilised.Finally arsenic trioxide PLGA nanoparticles were obtained.
Characterization of As 2 O 3 -PLGA nanoparticles, animals and treatments Physicochemical properties of As 2 O 3 -PLGA nanoparticles were veri ed through transmission electron microscope, scanning electron microscope, Zetasizer and X-Ray diffraction.Due permission from institutional ethical committee was sought before making these experiments.Male albino rats of Wistar strain weighing 250±50g were procured from the animal facility of Jamia Hamdard, New Delhi.They were housed in the animal house of Department of Zoology, Ch.Charan Singh University Meerut (India).
All the rats were offered pelleted food (Golden Feeds, New Delhi) and tap water ad libitum and maintained under standard laboratory conditions (room temperature 25±5°C and relative humidity 50±5%).After acclimatization to laboratory conditions for two weeks, the rats were divided into three groups, each containing ve rats.Rats of group A were administered a sublethal dose of As 2 O 3 -PLGA-NPs dissolved in saline (0.1 mg/100g body weight) by gavage on each alternate day for thirty days.Rats of group B were also given same dose of As 2 O 3 whereas rats of group C were offered saline only to serve as controls.

Analyses
After scheduled treatments, the rats were starved overnight and sacri ced next morning by light ether anesthesia.Liver was carefully removed and weighed using electronic balance (Sartorius, Germany) and processed for further investigations.
Arsenic accumulation in liver 1g wet liver was digested in 10ml of concentrated nitric acid (A.R. grade) at 80°C for 16 hr.It was diluted to 100ml with double distilled water.2ml aliquot of the digest was analysed for inorganic arsenic by hydride generation at pH 6.0 using sodium borohydride as the reducing agent.The analyses were performed using atomic absorption spectrophotometer (Electronic Corporation, India).Absorption was recorded at 193.7 nm, using a hollow cathode lamp for arsenic (Electronic Corporation, India).

Histopathological observations
Small pieces of liver were xed in 10% neutral formalin at room temperature for 24 hours.They were washed in running tap water overnight.After dehydration, pieces were embedded in para n, sectioned (5-6 µ thick) on a rotary microtome and stained with hematoxylin and eosin.The lesions were observed under a light research microscope (Nikon, Japan).

Lipid peroxidation
Lipid peroxidation in the liver of experimental rats was determined by measuring mitochondrial and microsomal malondialdehyde following the method of Jordan and Schenkman (1982).Microsomes were separated using an ultracentrifuge (Sorvel, USA) following the method of Schenkman and Cinti (1978).Thiobarbituric acid reactive substances were measured at 532 nm using a spectrophotometer (Systronics, India).1', 1', 3-tetramethoxypropane (Sigma, USA) was used as the standard.
The absorbance was recorded at 340 nm.CYP 450 2E1.(EC 1.14.13)Microsomes were separated using an ultracentrifuge (Sorvel,USA).Enzyme activity was estimated following the method of Koop (1986).In brief, the reaction mixture consisted of microsomal protein (0.2 mg/ml), 0.1 M potassium phosphate, pH 6.8 and 1 mM p-nitrophenol.Samples were incubated at 37ºC for 3 min prior to the addition of NADPH to start the reaction.After 10 min, the reaction was stopped with 1.5N perchloric acid.Formation of p-nitrocatechol was measured at 510 nm.

Protein measurement
Protein content in the liver samples was measured applying the method of Lowry et al (1951).Bovine serum albumin (Sigma, USA) was used as standard.

Statistical analysis
The data was analysed using SPSS software version 20.P values of less than 0.05 were accepted to be signi cant.
Biological observations i.e. change in the body weight and liver/ body weight relationship apparently showed no sign of adverse effects of As 2 O 3 -PLGA-NPs in rat.As 2 O 3 -PLGA-NPs treatment helped the rats in gaining weight ( g. 5).
Hepatosomatic index was higher in As 2 O 3 -PLGA-NPs treated rats in comparison to control rats (table 1).
Liver is a soft target for bioaccumulation of arsenic.Arsenic concentration in liver was found to increase in arsenic trioxide treated rats.Arsenic accumulated in liver of As 2 O 3 -PLGA-NPs treated rats however concentration was found to be low in comparison to arsenic trioxide treated rats (table 2).Observations on serum transaminases exhibited the state of liver function in As 2 O 3 -PLGA-NPs treated rats.Higher values were recorded for AST and ALT both in the serum of As 2 O 3 -PLGA NPs treated rats.Arsenic trioxide treatments also impaired liver function.However As 2 O 3 -PLGA-NPs were found to be more toxic.Arsenic trioxide inhibited ALP, however, As 2 O 3 -PLGA-NPs caused increased e ux of enzyme in the serum (table 1).Hepatotoxicity of arsenic trioxide has been mainly attributed to reactive oxygen species thus generated and measured in terms of a phenomenon known lipid peroxidation.Our results showed that As 2 O 3 -PLGA-NPs are potential inducer of lipid peroxidation in mitochondria as well as microsomes.Mitochondrial lipid peroxidation was higher than the microsomal lipid peroxidation (table 3).CYP 450 2E1, was also estimated to study the metabolic fate of As 2 O 3 -PLGA-NPs.Values of this enzyme were lower in the liver of As 2 O 3 -PLGA-NPs treated rats in comparison to arsenic trioxide treated rats (table 3).
Oxidative stress caused by As 2 O 3 -PLGA-NPs, if any, was also studied.GSH thus estimated showed lower values than control rats and arsenic trioxide treated rats (table 2).Status of detoxication enzyme i.e. glutathione-S-transferase was also studied in the liver of As 2 O 3 -PLGA-NPs treated rats.Though it was inhibited by arsenic trioxide, non signi cant increase was recorded in the liver of As 2 O 3 -PLGA-NPs treated rats (table 2).
Finally, toxic impairment of liver caused by As 2 O 3 -PLGA-NPs was examined by histopathological observations.These observations showed peculiar and speci c lesion in the form of nuclear and cytoplasmic changes.Several of the nuclei of hepatic parenchyma stained black and were lled with intranuclear inclusions.A few apoptotic bodies were also localized.Mild necrosis was recorded in the centrilobular region.Thus histopathological results suggested that As 2 O 3 -PLGA-NPs caused severe hepatotoxicity in rat.T.S. of liver of As 2 O 3 -PLGA-NPs treated rat shows the presence of several intracytoplasmic inclusions, apoptotic bodies and necrotic spaces (Fig. 6A).These pathological changes are wanting in arsenic trioxide treated rats.However, arsenic trioxide treated rat shows perilobular necrosis in liver ( g.6B).Fig. 6C shows the normal structure of liver of control rat with hepatocytes radiating from central vein.

Discussion
Cellular responses induced by nanomaterials or nanoparticles have been extensively studied in recent years by a number of laboratories using in vitro and /or in vivo systems.The nanomaterials have been found to be potentially cytotoxic, therefore, their safety needs to be thoroughly assessed prior to the use in nano-based consumer products and technologies including nanomedicine (Sauer et al. 2014;Hussain et al. 2015).
Recent observations from a few workers suggesting that nano-arsenic can be applied to treat haematological malignancies, liver cancer and osteosarcoma have raised concerns for its toxicological evaluation in terms of hepatotoxicity, hematotoxicty and nephrotoxicity (Ahn et al. 2013;Li et al. 2007).Further, a comparison between the toxicity of bulk arsenic and nano-arsenic needs to be made to justify its application as a therapeutic agent.
Our results on initial characterization of nano-arsenic trioxide made through TEM showed that they were round / spherical in shape, measured less than 100 nanometer and formed agglomerates with PLGA.This information has been found to be critical in determining the biological response to nanoparticles (Richman and Hutchinson 2009;Sapsford et al. 2011).Oral administration of these particles for 30 days to rats caused a non signi cant increase in their body weight.While oral administration of arsenic trioxide inhibited the growth of rats.Hepatosomatic index increased in As 2 O 3 -PLGA-NPs treated rats as compared to arsenic trioxide treated rats.This observation associates As 2 O 3 -PLGA-NPs with the synthesis of major molecules viz.proteins, lipids, and carbohydrates.Further, accumulation of nanoparticles of arsenic trioxide decreased in liver as compared to the bulk metalloid.It has been observed that excretion of nanoparticles is quicker than the bulk particles.Opsonisation, the process that prepares foreign materials to be more e ciently engulfed by macrophages occur under certain conditions for nanoparticles depending on size and surface characteristics (Moghimi et al. 2005).Nanoparticles such as As 2 O 3 -PLGA-NPs can pass through the gastrointestinal tract and are rapidly eliminated through faeces and urine (Hagens et al. 2007).However, some nanoparticles accumulate in the liver during the rst-pass metabolism (Oberdorster et al. 2005).
We observed that liver function as determined through activity of serum transaminases was also affected by As 2 O 3 -PLGA-NPs.The injury was higher in comparison to arsenic trioxide treated rats.Liver function disturbances caused by arsenic trioxide have been studied earlier in our laboratory (Singh and Rana 2009).However, present report con rms that liver is a target organ of nanoparticles.In general, nanoparticles stimulate macrophages via reactive oxygen species (ROS) and calcium signalling to make proin amatory cytokines such as TNF α (Brown et al. 2004).
Thus hepatocytes function is inhibited by oxidative stress/ pro-in ammatory cytokines induced pathological changes in liver.The pathological changes caused by As 2 O 3 -PLGA-NPs in the liver included formation of intranuclear inclusion bodies, apoptosis and mild centrilobular necrosis.Arsenic trioxide has also been found to induce in ammatory effects in the liver (Singh and Rana 2009).
We could notice that As 2 O 3 -PLGA-NPs caused signi cant mitochondrial as well as microsomal lipid peroxidation in liver.These results are most important in delineating the MOA manifested by As 2 O 3 -PLGA-NPs and are in agreement with similar reports.In general, nanotoxicity has been attributed to the generation of ROS (Fu et al. 2014).It has been hypothesized that cellular internalization of NPs activates immune cells including macrophages and neutrophils contributing to the generation of ROS/RNS (Risom et al. 2005;Knaapen et al. 2004).NPs with smaller size are reported to induce higher ROS owing to their unique characteristics such as high surface to volume ratio and high surface charge.It is the particle size that determines the reactive groups / sites on NP surface.Therefore, As 2 O 3 -PLGA-NPs induced higher generation of ROS as compared to arsenic trioxide.Nano-sized Sio 2 and Tio 2 and multiwalled carbon nanotubes have been reported to induce greater ROS as compared to their larger counterparts (Sohaebuddin et al. 2010).Arsenic trioxide induced lipid peroxidation has been reported earlier.Reactive species are formed in vitro and in vivo in the presence of arsenic and include superoxide anion, hydroxyl radical, hydrogen peroxide, RNS and arsenic centred arsenic peroxyl radicals (Kitchen 2001; Shi et al. 2004).In brief, it is concluded that arsenic with its carrier PLGA caused hepatotoxic effects through oxidative stress.Protective effects of antioxidants, yet to be made will con rm this hypothesis.

Declarations
Hughes et al 2006; Kitchen and Ahmad 2003; Kitchen and Conolly 2010).ROS formed by arsenic are involved in several of the proposed MOAs including genotoxicity, signal transduction, cell proliferation and inhibition of DNA repair (Hughes et al. 2010).
NPs-mediated toxicity is oxidative stress.Manke et al. (2013) reviewed different mechanisms of NPs induced oxidative stress and toxicity.It is known that metal based NPs induce oxidative damage to cellular macromolecules such as proteins, lipids and DNA via Fenton type and Haber-Weiss type reactions.The outcome of oxidative damage is membranous lipid peroxidation, protein denaturation and alteration of calcium homeostasis.As 2 O 3 -PLGA-NPs, as shown by present results on GSH caused hepatic parenchymal damage via oxidative stress.

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Figure 3 Size 4 X
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Figure 5 Body
Figure 5 Results are expressed as mean ± S.E.(n=5),where n is number of observations * Values are signi cant when compared with control group (p< 0.05).Results are expressed as mean ± S.E.(n=5), where n is number of observations * Values are signi cant when compared with control group (p< 0.05).
NS -Non signi cant values