Ecotoxicology and Environmental Safety 157 (2018) 255–265

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Ecotoxicology and Environmental Safety

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Spirulina platensis attenuates the associated neurobehavioral and T inflammatory response impairments in rats exposed to lead acetate ⁎ Samah R. Khalila, , Hesham A. Khalifab, Sabry M. Abdel-Motalb, Hesham H. Mohammedc, Yaser H.A. Elewad,e, Hend Atta Mahmoudb a Forensic Medicine and Toxicology Department, Faculty of Veterinary Medicine, Zagazig University, Egypt b Pharmacology Department, Faculty of Veterinary Medicine, Zagazig University, Egypt c Veterinary Public Health Department, Faculty of Veterinary Medicine, Zagazig University, Egypt d Histology and Cytology Department, Faculty of Veterinary Medicine, Zagazig University, Egypt e Laboratory of Anatomy, Department of Biomedical Sciences. Graduate school of Veterinary, Hokkaido University, Sapporo, Japan

ARTICLE INFO ABSTRACT

Keywords: Heavy metals are well known as environmental pollutants with hazardous impacts on human and animal health Caspase-3 because of their wide industrial usage. In the present study, the role of Spirulina platensis in reversing the oxi- Comet dative stress-mediated brain injury elicited by lead acetate exposure was evaluated. In order to accomplish this HSP70 aim, rats were orally administered with 300 mg/kg bw Spirulina for 15 d, before and simultaneously with an Lead intraperitoneal injection of 50 mg/kg bw lead acetate [6 injections through the two weeks]. As a result, the co- Open field test administration of Spirulina with lead acetate reversed the most impaired open field behavioral indices; however, Spirulina platensis this did not happen for swimming performance, inclined plane, and grip strength tests. In addition, it was observed that Spirulina diminished the lead content that accumulated in both the blood and the brain tissue of the exposed rats, and reduced the elevated levels of oxidative damage indices, and brain proinflammatory markers. Also, because of the Spirulina administration, the levels of the depleted biomarkers of status and interleukin–10 in the lead-exposed rats were improved. Moreover, Spirulina protected the brain tissue (cerebrum and cerebellum) against the changes elicited by lead exposure, and also decreased the reactivity of HSP70 and Caspase–3 in both cerebrum and cerebellum tissues. Collectively, our findings demonstrate that Spirulina has a potential use as a food supplement in the regions highly polluted with heavy metals.

1. Introduction consumption because of its long history of usage as a food source and its ideal safety profile in human and animal studies (Karkos et al., 2011). Microalgae have been known for centuries as food and animal feed; Spirulina contains non-toxic , but the methods of culti- they grow in freshwater and marine ecosystems as filamentous organ- vation without proper quality controls permit contamination by other isms (Vigani et al., 2015). Spirulina is a photosynthetic cyanobacterium toxin producer species prompting the presence of harmful that is used commercially as a dietary supplement and food additive. which have general health concerns. Roy-Lachapelle et al. (2017) de- Currently, the annual production of Spirulina exceeds 3000 t on a dry tected the presence of cyanotoxins in various commercially available weight basis and is acknowledged by several companies in various products containing Spirulina at levels exceeding the tolerable daily nations. The extensive production of Spirulina is owing to its original intake levels, showed the significance of better screening by the ap- chemical composition [, polyunsaturated fatty acids, and vita- propriate authorities of algal-based food supplements like spirulina to mins]. Besides, it is a source of bioactive components, for example, guarantee the safety of these products. , β-, and , which possess anti-in- The studies conducted in the past using Spirulina platensis as a flammatory and antioxidant properties (Wang et al., 2007). Also, it is supplement have proved that it has an ability to encounter the multi- able to manage numerous diseased conditions, including diabetes and organ toxicity caused by various medications and chemicals (Simsek obesity (Anitha andChandralekha, 2010), arthritis (Kumar et al., 2009), et al., 2009; Banji et al., 2013). Spirulina also reversed the modified cardiovascular diseases (Deng and Chow, 2010), and allergies (Vo et al., immune response and hepatic damage prompted by the herbicide 2012). Moreover, Spirulina is generally accepted to be safe for atrazine in the common carp (Khalil et al., 2017, 2018). It has been

⁎ Corresponding author at: Forensic Medicine and Toxicology, Faculty of Veterinary Medicine, Zagazig University, 44511 Zagazig, Egypt. E-mail address: [email protected] (S.R. Khalil). https://doi.org/10.1016/j.ecoenv.2018.03.068 Received 6 February 2018; Received in revised form 22 March 2018; Accepted 24 March 2018 Available online 04 April 2018 0147-6513/ © 2018 Elsevier Inc. All rights reserved. S.R. Khalil et al. Ecotoxicology and Environmental Safety 157 (2018) 255–265 hypothesized that the modulating potency of Spirulina, which includes considered to be safe. For experimental use, a working solution of the the central nervous system, could be considered a part of the ther- test compounds was prepared by diluting them with distilled water. apeutic regimes for neurodegenerative diseases, such as, Alzheimer's and Parkinsonism (Pabon et al., 2012), nigrostriatal dopamine system 2.2. Animals and experimental design injury (Strömberg et al., 2005), cerebrovascular accident models (Thaakur and Sravanthi, 2010), and for neurotoxicity by means of free Forty male Sprague–Dawley rats (150–200 g) obtained from the radical generation to protect the dopaminergic neurotransmission laboratory animals farm of Faculty of Veterinary Medicine (Zagazig (Tobón-Velasco et al., 2013). University, Egypt), were used in the present study. The animals were Lead is an environmental contaminant causing occupational health maintained in clean stainless steel cages under controlled environ- hazards that can be perceived in the environment and biological sys- mental conditions. The animals had free access to water and feed tems as it is widely used in industry (El-Nekeety et al., 2009). Both throughout the experimental period, and they were accommodated to environmental and occupational exposures cause significant and ser- the laboratory conditions for two weeks before being used in the ex- ious medical issues in the industries where lead and lead-based com- periments. The experiments were performed following the guidelines of ponents are used for the production of ammunition, bearing metals, the National Institutes of Health (NIH) for the Care and Use of bronze and brass, cable covering, sheet lead, and solder. Besides, lead Laboratory Animals in scientific investigations and approved by the can be used in ceramics, weights or ballast, containers or tubes, oxides, Ethics of Animal Use in Research Committee (Zagazig University, and gasoline additives (Schwartz and Levin, 1991). Although the use of Egypt). lead as an additive in household paint has ceased, the lead-containing The animals were randomly assigned into four experimental groups; paint is nonetheless found in buildings built before the 1960s therefore, each group consisted of ten animals. The control group in- (Bradberry, 2016). Lead can enter the body by inhaling, by ingesting volved rats that were intraperitoneally (IP) injected with distilled water the contaminated soil and water, and by consuming food. After ab- as a vehicle. Spirulina-administered animals received Spirulina orally at sorption, lead accumulates in several tissues, such as kidney, liver, a dose of 300 mg/kg bw for 30 days via gastric tube (Simsek et al., brain, and bones, and impacts various biological activities at the mo- 2009). Lead acetate-exposed animals were injected (IP) with lead lecular, cellular, and intracellular levels, which may result in morpho- acetate at a dose of 50 mg/kg bw (three times a week) for two weeks logical perturbations that cannot be reversed even after the levels of (Ahmed et al., 2013). The animals in the Spirulina/Lead acetate co- lead have diminished in the body (Flora et al., 2006). treated group were orally administered with Spirulina for 15 days be- Studies on both animals and humans have demonstrated lead-in- fore and 15 days simultaneously with an IP injection of lead acetate at duced deleterious effects in hepatic tissue (Khalil et al., 2018), cardi- the same course as previously described. On the day of sacrifice, the ovascular, immune, and reproductive systems (Patra and Swarup, 2004; final body weight of each rat was recorded by weighing each animal in Shah and Altindag, 2005; Teijón et al., 2006), and nerve dysfunction all the groups for estimating the body weight changes. (Ademuyiwa et al., 2007). Among the organ systems influenced by lead, the nervous system is particularly sensitive to lead exposure, which, 2.3. Behavioral response evaluation therefore, has received much research consideration for a long time. Exposure to low doses of lead causes injurious impacts on the nervous In order to evaluate the behavioral changes provoked by lead system, including hindered cognition and memory, as well as debili- acetate exposure and/or Spirulina administration, the rats were trans- tated peripheral nerve functions (White et al., 2007). Despite the var- ferred to the testing room in their home cages and kept there for about ious studies dealing with lead toxicity, the exact mechanisms through 30 min prior to testing to accommodate them to the testing-room con- which lead causes the neurotoxic effects are not yet fully understood. ditions. All behavioral tests were done in the same testing room on the Oxidative stress, membrane alterations, deregulation, and last day of the experimental period. The experimenter was blind to/ neurotransmission impairment are viewed as key perspectives involved unaware of the identity of the animal groups. Behavioral analyses, in- in lead neurotoxicity (Sanders et al., 2009). Moreover, certain in vitro cluding the open field test, swimming performance, grip strength, and and in vivo studies demonstrated that lead could give rise to neural cell inclined plane, were performed with the different experimental groups apoptosis (Baranowska-Bosiacka et al., 2013; Sharifi et al., 2010). as described ahead. Considering the involvement of oxidative stress in lead-induced neurotoxicity (Chander et al., 2014), it is reasonable that the admin- 2.3.1. Open field test istration of an antioxidant may be an imperative therapeutic approach The rats were set individually in the center of the open field arena to deal with lead toxicity. Stemmed from this notion, the present study and behavioral parameters were recorded for 5 min, beginning 2 min was undertaken to explore the putative protective potency of Spirulina after setting the animal in the test cage. The open field apparatus was against the lead-induced neurotoxicity by addressing the behavioral then thoroughly cleaned with 5% ethanol before placing the next rat, to responses, probing into oxidative stress, tissue inflammatory reactions, eliminate the possible cueing effects of the odors left by previous ani- and cellular death, and seeking a possible link with histopathological mals. The behavioral indices observed in this test were ambulation or changes in the brain tissue. locomotion frequency (the number of floor sections entered with two feet), rearing frequency (the number of times the animal stood on its 2. Material and methods hind legs), stereotype counts (the number of grooming movements), and immobility (freezing) duration (total time in second without 2.1. Test compounds and chemicals spontaneous movements). The open field contained a central square or area that was considered an unprotected area for rats; thus, the number Lead acetate (99.6% purity) was purchased from El-Nasr of entries into this area reflected the level of anxiety-like behavior Pharmaceutical Chemical Co. ( Egypt). Spirulina platensis, as a blue- (Contó et al., 2005). The observations were recorded between 10:00 green powder, was obtained from EL-Hellow for Biological Products and 12:00 a.m. supplier (Egypt). The Spirulina powder was subjected to heavy metals analysis like lead, mercury, cadmium, and arsenic by using flame 2.3.2. Swimming performance test atomic absorption spectrophotometer, these metals are the most likely Swimming performance test was carried out by putting each animal to contaminate Spirulina. Heavy metal contents in the tested Spirulina at the middle of the glass aquarium for observation for 5–10 s, where sample were all within the permissible levels (0.29, 0.048, 0.09 and the swimming performance was scored according to the position of nose 0.037 ppm, respectively). Therefore, the tested Spirulina sample was and head on the surface of water as follows: 0 F, head and nose below

256 S.R. Khalil et al. Ecotoxicology and Environmental Safety 157 (2018) 255–265 the water surface; 1, nose below the water surface; 2, nose and top of et al. (1994). The comet assay was conducted according to the method the head at or above the water surface and ears below the surface; 3, described by Singh et al. (1988). Fifty randomly selected cells per slide similar to score two except that the water line was at the mid-ear level; were investigated. Imaging was done using a fluorescence microscope and 4, similar to score three except that the water line was at the (Zeiss Axiovert L410 Inc., Germany) fitted with a CCD camera bottom of the ears (Schapiro et al., 1970). (Olympus Inc., Japan). The comets were examined using a visual scoring method and the computer image analysis was performed using 2.3.3. Inclined plane Comet Assay Software Project. Rats were put on a flat plane in a horizontal position with the head facing the side of the board to be raised (Yonemori et al., 1998). The 2.8. Evaluation of inflammatory markers in brain tissue inclined plane performance was measured with a standard protractor to the nearest 5 degrees. The trial ceased when the rat started to slip Rat ELISA kits were used for the detection of inflammatory markers backward. Hence, there was no particular trial duration. The angle at in the brain tissue–nitric oxide, NO; tumor necrosis factor-α, TNF-α; which the rat began to slip downward was recorded. The results of two interleukin–10, IL-10. The kits were obtained from MyBioSource (San trials were averaged for each rat. Both the trials were separated by a Diego, California, USA) (Catalog No.: MBS010567, MBS704859, and one-hour interval. MBS269138 for NO, TNF-α, and IL-10, respectively). The quantitative detection of such indices was done following manufacturer's instruc- 2.3.4. Grip strength test tions. The rat's forepaw strength was monitored by having it to hold a wood dowel (5 mm diameter) that was held horizontally and raised so that the animal supported its body weight, as described by Andersen 2.9. Histopathological study et al. (1991). Time taken to release the grip was recorded in seconds. All rats attempted to grip the dowel during this grip strength testing. The 2.9.1. Light results of two trials separated by a one-hour interval were averaged for Brain specimens (cerebrum and cerebellum) from the control and each rat. treated groups were processed for histopathological investigation using a light microscope (Olympus BX51 microscope, Olympus Inc., Japan) 2.4. Blood and brain tissue collection according to Bancroft and Gamble (2008). This process involved sec- tioning the tissues, staining with hematoxylin/eosin (H&E) dye, and At the time of sacrifice, whole blood samples were collected from viewing under the light microscope. the medial canthus (orbital vessels) of the experimental rats in heparin- containing tubes for evaluating the levels of lead. Brain tissue speci- mens were obtained, dissected, rinsed with sterile physiological saline 2.9.2. Immunohistochemical analysis (0.9% NaCl), weighed, and then divided into four sets. The first one was Brain tissue (cerebrum and cerebellum) sections were processed for homogenized and the homogenates were centrifuged at 664×g at 4 °C immunohistochemical analysis and incubated overnight at 4 °C with a for 15 min to obtain the supernatants, which were then used for de- rabbit polyclonal anti-caspase (Caspase–3) primary antibody (1:100) or termining the antioxidant, oxidative stress and inflammatory indices. anti-heat shock 70 (HSP70) primary antibody (1:100) in phos- The second one was used to determine the levels of lead. The third one phate-buffered saline (PBS). After three thorough washes with PBS, was used to prepare the cells for DNA-damage investigation (comet they were incubated with a goat anti-rabbit IgG biotin-conjugated assay), by setting it in 1 mL of cold Hank's balanced salt solution con- secondary antibody (1:2000) for 20 min at 32 °C. After further in- taining 20 mM ethylenediaminetetraacetic acid (EDTA) and 10% di- cubation with horseradish peroxidase-labeled streptavidin, antibody methyl sulfoxide (DMSO), and then mincing it into fine pieces to ac- binding was visualized using 3,3′-diaminobenzidine, and the sections quire cell suspensions. The final set was fixed in 10% neutral buffered were counterstained with hematoxylin. formalin for histopathological and immunohistochemical analysis. 2.10. Data analysis 2.5. Assessment of lead levels in blood and brain tissue Data were expressed as mean ± SE, one way-ANOVA followed by In order to perform the lead levels assay, whole blood (200 µL) and Tukey's multiple comparisons post hoc test using the SPSS 16.0 com- brain tissue samples (100 mg) were digested with 4 mL of nitric/per- puter program was performed to compare mean values of Lead-exposed chloric acid mixture at room temperature for 24 h. Then, the samples groups and Spirulina-administered groups versus control one. A value were heated at 80 °C for 2 h in a water bath, cooled to room tempera- of p < 0.05 was considered as statistically significant. ture, filtered, and diluted with deionized water before analysis (Julshman, 1983). The amount of lead was quantified using Buck Sci- entific 210VGP flame atomic absorption spectrophotometer; the sui- 3. Results table wavelength for the lead was 220.35 nm. 3.1. General observations, body weight changes, and brain/body weight 2.6. Evaluation of antioxidant biomarkers in brain tissue ratios of rats in response to lead acetate exposure and Spirulina administration The superoxide dismutase (SOD and catalase CAT) activities and the levels of reduced glutathione (GSH) were estimated according to During the experiments, neither deaths nor any clinical symptoms methods described previously (Misra and Fridovich, 1972; Sinha, 1972; were recorded in the groups. However, anorexia and low activity were Beutler, 1963). observed, mainly in the lead-exposed group. Lead-exposed rats showed a significant decline in the body weight changes relative to the control 2.7. Evaluation of oxidative stress biomarkers in brain tissue gain. Spirulina administration induced an improvement in rats by lowering the decrease in the body-weight gain in Spirulina/Lead co- Lipid peroxides (MDA) were measured as described by Ohkawa treated group. On the other hand, there was no significant difference in et al. (1979). Protein carbonyl was assessed using a colorimetric assay the relative brain weight (brain somatic index) among all the experi- kit (Cayman's Chemical Company, Ann Arbor, USA) according to Levine mental groups (Table 1).

257 S.R. Khalil et al. Ecotoxicology and Environmental Safety 157 (2018) 255–265

Table 1 Effect of pre and simultaneous Spirulina supplementation (300 mg/kg bw for 30 days, orally) on body weight changes, relative brain tissue weight and Lead level in rat exposed to Lead acetate (50 mg/kg bw, every other day for two weeks, IP).

Parameters Experimental groups

Control Spirulina Lead acetate Spirulina/Lead acetate

Body weight change (gm) 22.66 ± 1.76a 22.00 ± 1.80a −10.16 ± 2.61c 15.00 ± 1.46b Relative brain weight (%) 0.87 ± 0.022 0.94 ± 0.015 0.91 ± 0.015 0.95 ± 0.028 Blood lead level (ppm) 0.11 ± 0.005b 0.05 ± 0.01b 0.54 ± 0.02a 0.09 ± 0.012b Brain lead level (ppm) 0.23 ± 0.021c 0.20 ± 0.035c 0.89 ± 0.043a 0.54 ± 0.032b

Means within the same row (in each parameter) carrying different superscripts (a, b,c) are significantly different (P< 0.05) (mean ± SE, n = 6).

3.2. Behavioral responses in response to lead acetate exposure and Spirulina 3.3. Lead levels in blood and the brain tissue of rats in response to lead administration acetate exposure and Spirulina administration

3.2.1. Open field test Lead acetate exposure significantly increased the levels of lead in There was a significant increase in the locomotors activity re- blood and the brain tissue, with greater lead accumulation occurring in presented by a significant increment in ambulation, which was asso- the brain tissue when compared with the levels recorded in the control ciated with a lowering of freezing time in rats after exposure to lead group. The supplementation of Spirulina with lead significantly de- acetate, compared with control rats. Spirulina co-administration with creased its levels in blood and the brain tissue, relative to the lead- lead exhibited a significant modulation in freezing time; however, it did exposed group. In comparison with the control level, it exhibited non- not affect ambulation behavior. Rearing and grooming behaviors did significant differences in the blood lead levels; whereas, in the brain not show any significant changes as a result of lead exposure. tissue, it exhibited significantly higher lead levels than the control Meanwhile, these rats exhibited more entries and time spent in the (Table 1). central arena than the control rats. Spirulina supplementation sig- nificantly maintained the rats' behavior in the central arena in the co- treated group, compared to the lead-exposed rats (Fig. 1). 3.4. SOD, CAT activity, and GSH content in the brain tissue of rats in response to lead acetate exposure and Spirulina administration

3.2.2. Grip strength, inclined plane, and swimming performance tests Lead exposure significantly decreased the SOD, CAT activity, and The data for swimming performance, inclined plane, and grip time GSH level in the brain tissue, compared to control group. On the other in the rats exposed to lead displayed significant deficits in comparison hand, co-administration of Spirulina with lead enhanced the SOD and to the corresponding data recorded in the control animals, indicating CAT activity and increased the GSH level. Such improvement was sig- that the rats treated with lead experienced sensorimotor deficits. In nificant in Spirulina/Lead co-treated group in comparison with the Spirulina/Lead co-treated animals, Spirulina was unable to modulate values recorded in the lead-exposed group, although it did not attain these deficits, which were observed to be non-significantly different in the control value (Table 2). comparison with the lead-exposed animals (Fig. 2).

Fig. 1. Effect of pre and simultaneous Spirulina supplementation (300 mg/kg bw for 30 days, orally) on open field behavior (ambulation, freezing time, rearing frequency, grooming frequency, central arena entrance frequency and time spent) in rat exposed to Lead acetate (50 mg/kg bw, every other day for two weeks, IP). Columns carrying different superscripts (a, b,c) are significantly different (P< 0.05) (mean ± SE, n = 6).

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3.5. MDA, PC, and DNA damage levels in the brain tissue of rats in response to lead acetate exposure and Spirulina administration

MDA (a lipid peroxidation product) levels were significantly ele- vated in the brain tissue of lead-exposed rats, compared to the control group. Co-supplementation of Spirulina with lead significantly de- creased the MDA levels compared to the lead-exposed group. The modulation recorded in the Spirulina/Lead co-treated group depicted a trend toward normal value. However, it did not match the values of control. Brain protein carbonyl (a protein oxidative damage product) level was significantly increased in the lead-exposed group, compared to the control group. Nevertheless, the Spirulina/Lead group exhibited a significant reduction in the PC level, compared to the level recorded in the lead-exposed group; this level was, however, non-significantly different from the value recorded in the control rats, indicating that Spirulina co-treatment significantly prevented carbonyl formation in this group (Table 2). The findings of DNA damage revealed a significant elevation in the tail length, tail DNA %, and tail moment in the brain tissues of the lead- exposed animals, relative to controls. Spirulina supplementation alone displayed a non-significant decline in the level of potential DNA da- mage. The modulatory potency of Spirulina was confirmed by a sig- nificant reduction in all DNA damage indices in the co-treated group, relative to those exhibited in the lead-exposed rats (Fig. 3).

3.6. NO, TNF-α, and IL-10 levels in the brain tissue of rats in response to lead acetate exposure and Spirulina administration

An exposure to lead resulted in a significant increase in NO and TNF-α levels in the brain tissue of lead-exposed group. Although these indices exhibited a significant decrease in the Spirulina/Lead co-treated group compared to the lead-exposed group, the level of NO was nonetheless significantly different from the recorded in the control, and the TNF-α level was non significantly different than of the control group. Spirulina supplementation was observed to significantly elevate the levels of interleukin–10 in the brain tissue when compared with control group. Lead exposure induced a significant decrease in the IL-10 levels compared to the control values. This decrease was modulated, as it exhibited a non-significant difference compared to the control values, in the Lead/Spirulina co-administered group (Table 2).

ff Fig. 2. E ect of pre and simultaneous Spirulina supplementation (300 mg/kg 3.7. Histopathological and immunohistochemical findings bw for 30 days, orally) on grip strength time, inclined plane test and swimming performance test in rat exposed to Lead acetate (50 mg/kg bw, every other day Histopathological observations of the H&E-stained paraffin-em- for two weeks, IP). Columns carrying different superscripts (a, b,c) are sig- bedded brain sections revealed normal meninges and meningeal blood nificantly different (P< 0.05) (mean ± SE, n = 6). vessels in both control and Spirulina-administered group. In the lead- exposed group, congestion and hemorrhage were observed in the me- ningeal blood vessels. In the Spirulina/Lead co-treated group,

Table 2 Effect of pre and simultaneous Spirulina supplementation (300 mg/kg bw for 30 days, orally) on antioxidant biomarkers (SOD, CAT activities and GSH level), oxidative stress biomarkers (MDA, and PC levels) and inflammatory response indices (NO, TNF-α and IL-10 levels) in the brain of rat exposed to Lead acetate (50 mg/ kg bw, every other day for two weeks, IP).

Parameters Experimental groups

Control Spirulina Lead acetate Spirulina/Lead acetate

SOD activity (U/gm) 395.23 ± 14.27a 427.0 ± 15.71a 235.42 ± 9.30c 299.17 ± 8.58b CAT activity (U/gm) 0.80 ± 0.03a 0.77 ± 0.02a 0.46 ± 0.03c 0.62 ± 0.02b GSH level (mmol/gm) 2.51 ± 0.14a 2.66 ± 0.15a 1.41 ± 0.05c 1.80 ± 0.05b MDA (nmol/gm) 5.38 ± 0.25a 5.20 ± 0.32a 10.85 ± 0.44c 6.94 ± 0.32b PC (nmol/gm) 0.134 ± 0.02b 0.112 ± 0.02b 0.30 ± 0.03a 0.19 ± 0.01b NO (µmol/gm) 8.64 ± 0.42c 8.44 ± 0.43c 18.70 ± 0.39a 13.21 ± 0.11b TNF-α (Pg/gm) 0.19 ± 0.01bc 0.13 ± 0.02c 1.34 ± 0.10a 0.34 ± 0.02b IL-10 (Pg/gm) 0.17 ± 0.01 b 0.29 ± 0.01a 0.086 ± 0.01c 0.14 ± 0.01b

Means within the same row (in each parameter) carrying different superscripts (a, b,c) are significantly different (P< 0.05) (mean ± SE, n = 6).

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Fig. 3. A. Fluorescent microscopic images of cells derived from brain tissue of rat from the experimental groups showing, cells with intact DNA in control and Spirulina-administered groups, cells with a tail (DNA fragments) in Lead-exposed and Spirulina/Lead co-treated groups. B. Effect of pre and simultaneous Spirulina supplementation (300 mg/kg bw for 30 days, orally) on DNA damage indices (Comet assay) in the brain of rat exposed to Lead acetate (50 mg/kg bw, every other day for two weeks, IP). Columns carrying different superscripts (a, b,c) are significantly different (P< 0.05) (mean ± SE, n = 6). meningeal edema and congestion of blood vessels were observed in the observations for Caspase–3 revealed the presence of a few apoptotic region between cerebellum and cerebrum. Moreover, the choroid cells in the cerebrum and cerebellum of the Spirulina/Lead co-treated plexus exhibited normal structure in both control and Spirulina-ad- and lead-exposed animals. These cells were rarely observed in the ministered group. In the lead-exposed group, the congestion of the control and Spirulina-administered groups (Fig. 5). choroidal blood capillaries and proliferation of the choroidal epithelial lining were prominent. In the Spirulina/Lead co-treated group, only the congestion of choroidal blood capillaries was observed. The cerebellum 4. Discussion in both control and Spirulina-administered group displayed normally arranged cerebellar cortical layers, including the outer molecular, the The naturally occurring sources of have been used in inner granular, and the middle layer of pear-shaped Purkinje cells that various in vitro and in vivo studies, and have demonstrated promising ff aligned regularly in-between the former layers. In the lead-exposed outcomes with regard to their e ects on metal-induced toxicity group, the middle Purkinje-cells layers displayed a fewer number of (Moustafa et al., 2012; Khalil et al., 2013, 2018; Khalil and Hussein, ffi intact Purkinje cells that exhibited disorganized alignment, along with 2015). The present study deals with the e cacy of Spirulina on mod- the presence of a few necrotic Purkinje cells and perineural edema. In ulating the neurotoxic alterations induced by subacute exposure to lead the Spirulina/Lead co-treated group, the Purkinje cells exhibited se- acetate. The main outcome of this study was that an IP injection of lead fi verely disorganized alignment (Fig. 4). acetate resulted in a signi cant oxidative stress-mediated damage and Immunohistochemical analysis for the detection of heat shock pro- brain injury, which was inferred from the altered behavioral, bio- tein (HSP) 70 in cerebrum and cerebellum revealed numerous blood chemical, histopathological, and immunohistochemical indices under vessels with the positive reaction to HSP70 in the Spirulina/Lead co- investigation. Besides, the supplementation of Spirulina provided a ffi treated and lead-exposed groups, compared with the control and neuroprotective e cacy through the modulation of these impairments, Spirulina-administered groups. Additionally, the immunohistochemical when given prior to and simultaneously with lead acetate. In the present study, animals that were exposed to lead acetate

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Fig. 4. Photomicrograph of brain tissue of: Control (A) and Spirulina-administered (B) groups showing normal histological structured normal meninges and me- ningeal blood vessels (Bar =100 µm). Lead-exposed group (C) showing congestion and hemorrhage in the meningeal blood vessels (Arrow) (Bar = 40 µm). Spirulina/Lead co- treated group (D) showing meningeal edema, and congestion of the blood vessels in the region between cerebellum and cerebrum (Arrow) (Bar = 200 µm). Photomicrograph of choroid plexus of: Control and Spirulina-administered (A&B) groups showing normal histological structured normal choroid plexus (Bar =200, 40 µm). Lead-exposed group (C) showing congestion of the choroidal blood capillaries and proliferation of the choroidal lining epithelial (Arrows) (Bar = 40 µm). Spirulina/Lead co-treated group (D) showing congestion of the choroidal blood capillaries only (Star) (Bar = 40 µm). Photomicrograph of cerebellum: Control (A) and Spirulina-administered (B) groups showing normally arranged cerebellar cortical layers including outer molecular, inner granular and middle pear- shaped purkinjie cells that aligned regularly in-between the former layers. Lead-exposed group (C) showing less number of intact middle purkinje cell layers that showing disorganized alignment (Arrowheads) with the presence of some necrotic purkinje cells (Arrow) and perineural edema. Spirulina/Lead co- treated group (D) showing a disorganized alignment of the purkinje cells (Arrowheads) (Bar = 50 µm) (Stain: H &E). exhibited a significant loss of body weight. This might be a conceivable systems. Lead-induced consequences on the dopaminergic system in- direct effect of lead on the gastrointestinal tract, resulting in the ma- corporate changes in the synthesis, turnover, and reuptake of dopamine labsorption of supplements, or of a diminished food intake as lead in- and its metabolites, as well as in the number of dopamine receptors fluences food satiety signals, causing premature termination of food (ATSDR, 1999). intake during a meal (Minnema and Hammond, 1994). Nonetheless, Along these lines, Bressler and Goldstein (1991) demonstrated that, certain other studies have demonstrated that exposure to lead has no at the neuronal level, lead alters the release of neurotransmitters from impact on body weight (Salehi et al., 2015; Wang et al., 2015). Spir- the presynaptic nerve endings as it mimics the action of Ca++. The ulina significantly minimized the recorded weight loss, which might release of the stored neurotransmitter is achieved by Ca++-dependent have been a result of providing the body with essential nutrients pre- phosphorylation of the cytoskeleton proteins such as synapsin I (Baher sent in Spirulina, such as high-quality proteins, vitamin, and amino and Greengard, 1987), consequently lowering the levels of neuro- acids (Sharma et al., 2007), and these nutrients might have a beneficial transmitter discharged from the secretory vesicles. Additionally, role in restoring the body weight and health. Struzyńska and Rafałowska (1994) demonstrated that lead can assault Rats exposed to lead in the present study displayed enhanced lo- the synaptic neurotransmission in two ways: either by depressing the comotors activity, evidenced by higher numbers of ambulation, lower Ca–KCl-evoked release of GABA, dopamine, and histidine or by a se- freezing duration, increased number of entries and time spent in the lective stimulation of spontaneous release of GABA and dopamine, center; however, this behavior was not indicative of lowered anxiety as however, not histidine. we also observed an increase in locomotion. As mentioned in the pre- Furthermore, the lead exposure in the present study induced a sig- vious reports, only an increase in the central locomotion or an increase nificant sensorimotor impairment that was confirmed by the inclined in the time spent in the central arena without any modification in total plane performance, and the forepaw grip of the exposed animals. These locomotion can be translated as an anxiolytic effect (Prut and Belzung, neurobehavioral deficits may reflect dysfunction in multiple anatomical 2003). Likely, this trend suggests dopaminergic impairment (Nasuti areas in the CNS, peripheral nervous system, or skeletal muscles et al., 2007). (Maurissen et al., 2003). In the present study, a marked decline in the This concept is anchored to the fact that dopamine is a neuro- grip strength score was observed, which delineates motor neurotoxicity. transmitter involved in a variety of CNS processes, including cognition, On the other hand, the cerebellum is an essential structural target re- motor activity, reward, mood, attention, and learning. Consequently, lated to lead-mediated neurotoxicity (Adhami et al., 1996); therefore, any changes in its transmission may unfavorably influence a variety of in this regard, the cerebellar damage has been pointed out as a pivotal neurological processes and prompt debilitating behavioral disorders event related to lead-induced motor deficits (Bortolozzi et al., 1999). (Jones and Miller, 2008). Assuredly, lead has been demonstrated to Such behavioral alterations are in strong concurrence with our histo- alter a number of neurotransmitter systems, including the dopamine, pathological findings demonstrating the loss of neuronal cells and norepinephrine, epinephrine, serotonin, and c-aminobutyric acid Purkinje cells, neuronal degeneration, and cerebral edema; all of which

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Fig. 5. Photomicrograph of HSP70 Immunohistochemistry in the cerebrum (Bar = 200 µm) and cerebellum (Bar = 40 µm) of: Control (A) and Spirulina –ad- ministered (B) groups, showing weak of few cells. Lead–exposed group (C) showing an intense positive reaction to HSp70 in numerous blood vessels (Arrows). Spirulina/Lead co-treated group (D) showing moderate immunostaining to HSp70 (Dashed arrow). Photomicrograph of Caspase-3 Immunohistochemistry in cereberum and cerebellum (Bar = 40 µm) of: Control (A) and Spirulina– administered (B) groups, showing weak immunostaining of few apoptotic cells. Lead– exposed group (C) showing an intense presence of apoptotic cells positive to the Caspase-3 reaction (Arrows). Spirulina/Lead co-treated group (D) showing moderate immunostaining to Caspase-3(Dashed arrow) (Bar = 100 µm). have been reported previously to have occurred because of the impact such as cadmium, iron, and fluoride (Hutadilok-Towatana et al., 2008; of lead on rat brain (Moneim et al., 2011). On the contrary, Ashafaq Bermejo-Bescós et al., 2008). Indeed, Spirulina has a rapid lead ad- et al. (2016) demonstrated that lead-treated rats did not exhibit any sorption rate and high lead adsorption capacity, and it enhances the neuromuscular impairment when tested for the grip test. elimination of heavy metals from the body (Banji et al., 2013). Lead-induced aberrant behavior was partially reversed with Lead toxicity is believed to be mainly mediated by oxidative stress, Spirulina, which prevented alterations in the neural organization. Our through the collection and auto-oxidation of 5-aminolevulinic acid, findings indicated that Spirulina exerted its protective action plausibly with the production of a superoxide anion and hydrogen peroxide by decreasing the concentration of lead in the body, as evidenced by the (Zhao et al., 2007). Lead toxicity likewise includes the alteration of decline in the elevated levels of lead in the exposed rats, as well as by mitochondrial functions through the expansion of the intracellular le- inhibiting the toxicity of lead. Along these lines, Banji et al. (2013) had vels of calcium, and declining the activities of the electron transport suggested that Spirulina obstructed the cytoarchitectural changes in the chain components, changing the mitochondrial energy metabolism and brain tissues induced by fluoride exposure, and enhanced the avail- resulting in a free radical generation (Gleichmann and Mattson, 2011). ability of thyroid hormones, which in turn might be responsible for the It is well known that the disruption of the prooxidant/antioxidant improved behavior. These results are in accordance with the previous balance is the core mechanism through which lead damages various study demonstrating behavioral disturbances upon lead exposure and tissues (Chander et al., 2014). their subsequent reversal through antioxidant treatments (Sansar et al., In our findings, the damage was clearly demonstrated by the im- 2011). provement in the formation of lipid peroxidation and protein oxidation In the present study, elevated lead levels were observed in the whole products, which was accompanied by depletion in the antioxidant en- blood and the brain tissue of lead acetate-exposed rats, which dimin- zymes’ activities, and the GSH levels in the brain tissue of rats upon ished upon Spirulina supplementation, indicating that Spirulina could exposure to lead. Several in the antioxidant defense systems reduce lead levels in both blood and the brain tissue in rats. Kaushal may secure the imbalance between the prooxidants and antioxidants et al. (1996) recorded that nearly 15% of the administered lead dose induced by lead exposure, and most of them become inactive due to the remained in the body of the rat following the IP injection. Furthermore, direct binding of lead with their sulfhydryl groups (Rendón-Ramírez the decrease in the lead levels caused by Spirulina administration was et al., 2014), altering their function and suppressing their activities mostly related to the chelating capacity of Spirulina for heavy metals, (SOD, CAT, and glutathione) (Ercal et al., 2001). Furthermore,

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Spirulina administration reversed the alterations induced by lead confirmed by an elevation in the MDA levels in the lead-exposed ani- acetate in the antioxidant enzymes, and these findings were similar to mals, demonstrating membrane damage. Besides, lead readily crosses those in the previous study (Ponce-Canchihuamán et al., 2010). Such the blood-brain barrier (BBB) and directs structural alteration of the effect was achieved by improving the brain tissue antioxidant capacity BBB components, which further results in morphological and functional mechanisms because of better antioxidant supply, thus reducing the changes in the brain architecture (Prasanthi et al., 2005). Morpholo- oxidative damage represented by the reduction of MDA and PC for- gical perturbations observed in the brain tissue were supported by mation. previous neuropathological data, which revealed pathological changes Moreover, lead promotes DNA damage in the brain tissue of the following the lead exposure (Chander et al., 2014; Bazrgar et al., 2015). exposed rats. Lead has been accounted to be aneugenic, clastogenic, Besides, Spirulina's restoration potency in the brain tissue was reliable and mutagenic; it induces chromosomal aberrations, DNA damage, and even in terms of biochemical outcomes. This implies that Spirulina ef- micronuclei and nuclear alterations (Bonacker et al., 2005). In the fectively prevented the lead-induced neurotoxic damage in rat. present study, administration of Spirulina significantly lessened the Oxidative stress initiates apoptosis in various cell types. In order to DNA damage induced by lead exposure. Bhat and Madyastha (2000) investigate the relationship between oxidative stress and the expression revealed that the polysaccharides in Spirulina improved both the repair profiles of the apoptotic-mediated pathway, a pro-apoptotic gene pro- activity of the damaged DNA excision and the unscheduled DNA duct, Caspase–3, was selected for immunohistochemical analysis. The synthesis. Similarly, phycocyanin and in Spirulina apoptotic process is regulated by the expression of several proteins, possess a potent anti-cyclooxygenase–2 and antioxidant activity to including the members of the family of caspases. Our biochemical – scavenge peroxynitrite, and therefore, can lessen the OONO -induced findings suggest a crosstalk between lead-induced apoptosis and acti- oxidative damage to DNA (Abou-El Fotoh et al., 2013). Besides, the vation of oxidative stress-responsive cell signaling. In fact, DNA damage protection against genotoxicity may be explained by the fact that si- and DNA strand breaks, have been known to occur during the process of multaneous treatment with Spirulina would allow interception of the apoptosis, which was clearly detected in the current study. Moreover, free radicals generated by lead before they can reach DNA and induce the increase in the MDA levels is an indication of lipid peroxidation, genotoxicity. This is confirmed by the data obtained in the present which could cause structural damage to mitochondrial membranes and study which demonstrated that Spirulina pretreatment reduced the potentiate their dysfunction, activating the release of cytochrome c, lead-induced lipid peroxidation and prevented the reduction in anti- prompting the initiation of a variety of caspases (Nicholls and Budd, oxidant indices. 2000). Furthermore, a declined Caspase–3 expression level was ob- Spirulina ameliorated the effects of lead on the brain tissue by fa- served in the Spirulina/Lead co-treated group. These findings suggest cilitating the displacement of lead, resulting in reduced lead accumu- that Spirulina could serve as a biologically effective therapeutic agent lation in the body, through its potential antioxidant efficacy via its that can increase cell survival in the rat brains through Caspase–3 radical-scavenging ability, which was evident from a 2,2-diphenyl-1- down-expression and cell death suppression. picrylhydrazyl (DPPH) scavenging and β-carotene linoleic acid assay In response to cellular stress, the expression of HSPs elevates dra- (Banji et al., 2013). The ability of Spirulina to neutralize the oxidative matically. It is notable as a pervasive adaptation mechanism in organ- stress could be attributed to the blend of antioxidants present in it, isms that enables them to survive and adapt to different environmental including , β-carotene, vitamin E and C, and . stressors. The immunohistochemical data obtained for HSP70, whose Additionally, Spirulina contains riboflavin, α-lipoic acid, xanthophyll expression was increased in the brain tissue in response to the stress phytopigments, magnesium, selenium, and manganese, which also ex- generated by lead exposure as depicted by positive staining for HSP70 hibit antioxidant potency (Bermejo-Bescós et al., 2008). in the cerebrum and cerebellum, reflected the activation of this in- The promotion of the proinflammatory mediators (NO and TNF-α) tracellular buffer system, which responds to the oxidative stress when and the reduction in the levels of the anti-inflammatory one (IL-10) in the antioxidant exhaustion occurs (Kalmar and Greensmith, the rat brain suggest that lead enhances the inflammatory process. Our 2009). results were in line with those obtained by Liu et al. (2012), who ob- In conclusion, Spirulina has displayed a protective role against lead served that lead exposure significantly increased microglia activation, acetate-mediated brain damage, probably by scavenging the free radi- and up-regulated the release of cytokines, including TNF-α, IL-1β, and cals and chelating lead, thereby reducing the lead burden in rats. Thus, iNOS, in these cells. Spirulina supplementation evoked anti-in- it is worthwhile using Spirulina as a food supplement in the geo- flammatory effects through the reversal of NO and TNF-α generated graphical areas with high lead exposure in order to minimize the risk of and the suppressed IL-10 levels because of lead exposure in rats. Such neurological alterations. Nevertheless, further investigation is required potency may be attributed to c-phycocyanin, which may impact the for elucidating the molecular mechanism through which Spirulina ex- function of mast cells which are a critical source for the synthesis and hibits this protective effect against the subacute toxicity of lead. release of prostaglandin D2, ROS, leukotrienes, and cytokines, such as TNF-α, which trigger the release of interleukin (IL)–1β,IL–6, as well as References block the phosphorylation of p38 mitogen-activated protein kinases (MAPK), which in turn regulate the synthesis of cytokines (Shih et al., Abou-El Fotoh, M.F., Khalil, S.R., El-Sharkawy, N.I., Ahmed, M.M., 2013. Role of spirulina 2009). Furthermore, Spirulina diminishes nitrite generation, suppresses platensis on mitomycin c- induced genotoxicity in ehrlich ascites carcinoma bearing albino mice: single-cell gelelectrophoresis (comet assay) and semiquantitative RT- inducible nitric oxide synthase expression, and lessens liver microsomal PCR analysis. IJCR 5 (9), 2636–2640. lipid peroxidation (Romay et al., 2003). Also, the constituents present Ademuyiwa, O.U.R.N., Ugbaja, R.N., Rotimi, S.O., Abam, E., Okediran, B.S., Dosumu, in Spirulina, provide high nutritive value and a potent anti-in- O.A., Onunkwor, B.O., 2007. Erythrocyte acetylcholinesterase activity as a surrogate fl indicator of lead-induced neurotoxicity in occupational lead exposure in Abeokuta, ammatory activity other than the antioxidant potency (Alvarenga Nigeria. Environ. Toxicol. Pharmacol. 24 (2), 183–188. et al., 2011). As anti-inflammatory activities, phycocyanin inhibits the Adhami, V.M., Husain, R., Husain, R., Seth, P.K., 1996. Influence of iron deficiency and proinflammatory cytokines, such as TNF α, and also the cycloox- lead treatment on behavior and cerebellar and hippocampal polyamine levels in – ygenase–2 (COX–2) expression (Romay et al., 1998). neonatal rats. Neurochem. Res. 21 (8), 915 922. Ahmed, M.B., Ahmed, M.I., Meki, A.R., AbdRaboh, N., 2013. Neurotoxic effect of lead on The biochemical data together with histopathological observations rats: relationship to Apoptosis. Int. J. Health Sci. 7 (2), 192–199. (an altered normal-tissue architecture of brain) obviously showed that Alvarenga, R.R., Rodrigues, P.B., Cantarelli, V.D.S., Zangeronimo, M.G., Silva Júnior, lead acetate injured the brain; however, the structure of the brain was J.W.D., Pereira, L.J., et al., 2011. Energy values and chemical composition of spir- ffi ulina (Spirulina platensis) evaluated with broilers. 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