Comparable preventive effects of laboratory-grown spirulina and market spirulina against arsenic-induced alterations in the liver of adult rats

Arsenic (As) is a naturally occurring ubiquitous environmental toxicant widely distributed in the earth crust. Normally, As occurs in air, natural water, soil, vegetation, plants, forests and marine products and its concentrations are much higher depending on geographic locations. Arsenic occurs in both inorganic and organic forms and can exist in the environment in 4 redox states: arsenite (+3), arsenate (+5), arsine (-3) and elemental (0) forms [1]. Inorganic arsenicals have detrimental impacts on life supporting functions [2] while most of the organic arsenicals have negligible health effects and excreted unchanged [3]. Arsenite is 10 times more toxic than arsenate and 70 times more toxic than the methylated species, i.e., organic arsenicals [4].
Drinking water polluted by high level of arsenic is one of the most serious worldwide environmental problems [5]. It has been reported from at least 70 countries including 14 Asian countries that an estimated of 140 million people around the world are drinking water contaminated with arsenic exceeding 10.0 µg As/L (WHO standard; [6,7]. Although, drinking of high arsenic contaminated water is the primary pathway of arsenic exposure to humans [8,9], arsenic can also enter into the human food chain directly or indirectly due to its wide spread distribution in both the plant and animal kingdoms [10]. As a result, it has been reported that 85.0 million peoples are at risk of arsenic toxicity for consuming both arsenic rich drinking water and foodstuffs in Bangladesh [11,12].
Arsenicosis caused serious public health problems including melanosis, leukomelanosis, hyperkeratosis, black foot disease, hepatomegaly, neuropathy, cancer and gangrene [9], weakness, anemia, burning sensation of eyes, liver fibrosis, chronic lung disease, bone marrow depression [13], vascular remodeling, portal hypertension, noncirrhotic liver fibrosis [14].
Arsenic causes histopathological changes in tissues and organs of the exposed subject. The hepatotoxic action of arsenic includes cirrhotic portal fibrosis [15], hepatomegaly, non-cirrhotic portal fibrosis and portal hypertension [16,14], oxidative damage of liver [14,17], fatty accumulation, parenchymal cell degeneration [18], steatosis, hepatocyte degeneration [19], hepatic fibrosis [14], and liver proliferative lesions [18]. Fibrosis of liver may progress to cirrhosis and even to liver cancers [20]. Necrosis of hepatocytes and cytoplasmic blebbing and expanded sinusoidal spaces due to shrinkage and necrosis of hepatocytes were found on arsenite-exposure [21]. However, there were significant vascular remodeling with increased sinusoidal endothelial cell capillarization, vascularization of the peribiliary vascular plexus and constriction of hepatic arterioles [22]. Few focal areas of necrosis, Kupffer cell hyperplasia, and localized fibrosis in the periportal region were observed [23]. Ballooning of hepatocytes in the periportal and parenchymal areas, chromatin fragmentation and drop-out necrosis [24], and swollen hepatocytes near the centrilobular vein were observed in liver [25].
However, the first important step to control arsenicosis is the cessation of drinking arsenic contaminated water [17] and ingesting arsenic rich foods. Then, supplementation of potential antioxidants and feeding of arsenic burden reducing agent seem to be beneficial for remedy of arsnicosis [26,27]. Antioxidant supplement may have preventive effects in arsenicosis [28,29,26]. Intake of cysteine, methionine, niacin, vitamin B₁₂ and choline facilitates arsenic methylation by modulating its metabolism [30].
Spirulina (Spirulina platensis), a blue-green alga, has been considered as an excellent whole food ever known to mankind [31,32] having antioxidant properties [33,34]. It has the corrective properties against heavy metal toxicity, nephrotoxicity induced by heavy metals and drugs and also against cancer, tumor growth and malnutrition [32,35]. As arsenic induces the generation of ROS [36], alters DNA methylation pattern in many genes [37] and also inactivate enzymes in the cellular energy pathway [38], as a result, antioxidants could be helpful in altering the cytotoxicity of arsenic [39]. β-carotene is a very important antioxidant [40] and β-carotene from spirulina pose more antioxidant properties than synthetic one. Phycocyanin, a pigment of spirulina, stimulates the immune system and assists detoxification with the ability to inhibit oxidative damage in DNA [41]. Spirulina is rich in protein with all the essential amino acids, antioxidants, galaxy of phytonutrients and polysaccharides that trigger enzyme systems to enhance detoxification of arsenic [42]. Spirulina contains a lot of minerals including zinc, which is essential for a strong immune system and for powering antioxidant enzyme systems and acts synergistically with β-carotene. Micronutrients of Spirulina interact with toxic metals at several points in the body like absorption and excretion of toxic metals; transport of metals in the body; binding to target proteins; metabolism and sequestration of toxic metals; and in secondary mechanisms of toxicity such as oxidative stress [43]. Hence, spirulina intake can reduce the effects of arsenicosis by modulating its methylation, reducing oxidative stress, binding to target proteins, enhancing arsenic excretion from the body, and finally by reducing susceptibility to arsenicosis by combating malnutrition.
Therefore, this work has been taken with a view to evaluate the histopathological changes induced by inorganic arsenic and to clarify the comparative effects of laboratory grown agro-based spirulina (Ab-Sp) and market spirulina (M-Sp) in prevention of histopathological alterations in liver.

Animals
Apparently healthy 40 male Long Evans rats with minimum of 350.0g BW were selected and randomly divided into 10 groups consisting of 4 rats in each group. The rats of all groups, except that of T0, T2 and T3 groups, were fed with NaAsO₂ (Merck, Darmstadt, Germany: 0.58 mg iAs/mg) at 3.0 mg/kg BW/day in drinking water. Simultaneously, the rats of T4, T5 and T6 group were individually given 3 doses of the Ab-Sp at 1.0g, 1.5g and 2.0g/kg feed respectively while that of the T7, T8 and T9 group were similarly supplied M-Sp at 1.0g, 1.5g and 2.0g/kg feed respectively in feed. The rats of T2 were treated with only 2.0g Ab-Sp/kg feed while the T3 group rats were treated with the same dose of M-Sp (2.0g/kg feed) in feed as Ab-Sp and M-Sp treated control respectively. The rats of the T1 group were fed only with 3.0 mg NaAsO₂/kg BW/day in drinking water as arsenic treated control whereas; the rats of T0 group were supplied only normal feed and drinking water as non-treated control. The trial was continued for 90 days.  All experimental protocols were approved by the Animal Welfare and Ethical Committee, Faculty of Veterinary Science, Bangladesh Agricultural University. All efforts were made to minimize the number of mice used and their sufferings.

Feeding trial
A 0.2% NaAsO₂ stock solution was prepared with deionized water and preserved at 4°C to feed the trial rats for use of maximum 7 days. Dried powder of Ab-Sp obtained from the laboratory production and the dried powder of M-Sp collected from the local market. Required amounts of the respective doses (1.0g, 1.5g and 2.0g/kg feed) of both the Ab-Sp and M-Sp were individually mixed with pellet feed and dried at 50°C in an electric oven for at least 20 hours. Then, the spirulina mixed feed was taken into air tight polypropylene container for supplying to the trial rats for 5 days from the preparation. A new lot of spirulina mixed feed was prepared generally 12 hours before the 5^th day from the previous feed preparation.
In every morning, required amount of the prepared NaAsO₂ solution (0.2%) for the rats of a group per day at the rate of 3 mg As/kg BW/day was calculated and taken into a previously washed and sterilized waterer. Then, a small amount of drinking water was added to the solution so as not to exceed the half of the daily requirement of drinking water for the rats of that particular group. This was done to ensure drinking of the total amount of NaAsO₂ solution within 6 to 8 hours of supply. After drinking the total amount of NaAsO₂ solution, the rats were allowed to drink normal drinking water ad libitum. This As feeding process was done for the rats of T1, T4, T5, T6, T7, T8 and T9 groups. Concomitantly to the feeding of NaAsO₂ solution, the spirulina mixed feed with the respective doses of the individual spirulina (Ab-Sp and M-Sp) were supplied ad libitum to the rats of all six groups and the rats of T2 and T3 groups. This feeding process was continued for 90 days.

Sample collection
On Day 90, all the trial rats were euthanized with high dose of chloroform (Fisher Scientific UK Limited, UK) and samples were collected by opening the carcass immediate after euthanization. An intact piece of liver was collected from the sample rats. The collected samples were immediately washed in buffered neutral formalin and taken into individual stoppered glass vial filled with adequate amount of buffered neutral formalin (10 x sample volume) for fixation and kept at room temperature until processed for histopathological investigation.
After 48 hours, the fixed tissues were trimmed with a sharp blade into a suitable size and shape (about 0.5cmX0.5cm). Immediately after trimming, the liver was preserved in fresh buffered neutral formalin into new clean and dried stoppered glass vials.

Staining of tissue sections and photomicrograph
Routine hematoxylin and eosin (H & E) staining was used for the tissue sections. Preparation of stains and other necessary chemical solutions, and staining procedures were described previously [44]. Tissue sections were examined under compound microscope and photographs of tissue sections were taken under a Differential Interference Contrast (DIC) Microscope.

Examination of liver sections
The degree of changes, if any in the hepatocytes, was expressed by the number of hepatocytes with changes in respect to the total number following counting of hepatocytes.

Statistical analysis
The designs of the experiments were Randomized Complete Block Design (RCBD). The data were tabulated into a preliminary data sheet of a computer and compared with computer spread sheets to ensure the accuracy of the data. Then the data were analyzed with computer programs: Microsoft Excel and SPSS (Statistical Package for Social Science), and also using the analysis of variance technique by a computer using of MSTAT computer package program in according to the principles of RCBD. Least Significance Difference (LSD) was done to compare the variations between treatments where ANOVA showed significant differences.

Overall histopathological findings
With routine H&E staining, the liver showed normal in structures with mitotic phases in few nuclei, although a considerable number of hepatocytes showed hypertrophic hepatocytes with hypertrophic nucleus and hepatocytes with visible chromatin in the rats of all the trial groups in a variable degree (Table 1). In the rats of the T1 group, the liver was found with vacuolated hepatocytes, blood accumulation in some of the central veins, and erosions of lobular epithelium, and consequently hemorrhages in some hepatic lobules and lobular tissues as extravasations while no vacuolated hepatocytes, erosion and hemorrhage was found in rats of other trial groups (Figure 1A-D).

Hypertrophic hepatocytes with hypertrophic nucleus in the liver
The livers of the T8 group rats had the highest numbers of hypertrophic hepatocytes with hypertrophic nucleus while the lowest number was in the T0 group. However, the number of hypertrophic hepatocytes with hypertrophic nucleus in the liver of the rats of all of the groups differed significantly (p<0.01) among the groups. Arsenic induction significantly increased the numbers of hypertrophic hepatocytes with hypertrophic nucleus in the liver compared to the control. However, the numbers of that type of cell between T0 and T5, and among T1, T2, T3, T6 and T7 groups did not vary significantly (Table 1). The findings (based on percent values) show that none of the doses of any of the spirulina improved this histopathological condition at the control level. However, the lowest and intermediate doses of the Ab-Sp decreased (T4: 19.60% and T5: 48.57%) the number of the hypertrophic hepatocytes with hypertrophic nucleus in the liver of the As induced rats compared to the T1 group, but none of the M-Sp doses could do (Table 1).

Hepatocytes with visible chromatin in the nucleus in the liver
The rats of the T7 group showed the highest numbers of the hepatocytes with visible chromatin in the nucleus while the lowest number of that was in the T5. However, the numbers of the hepatocytes with visible chromatin in the nucleus varied significantly (p<0.01) among the trial groups. Although, the numbers of that type of hepatocytes did not differ significantly between the T1 and T2, among the T0, T4, T5 and T6 groups, and among the T3, T7, T8 and T9 groups. The data (based on percent values) show that all of the M-Sp doses increased compared to both T0 and T1 while all of the Ab-Sp doses decreased this histopathological condition at the control level. However, the average decreasing rate of the Ab-Sp was 34.50% vs. 67.41% compared to T0 vs. to T1 and the dose of 1.5g Ab-Sp/kg feed (52.57% vs. 76.40% decreased compared to T0 vs. to T1) was found best in reducing the numbers of hepatocytes with visible chromatin in the liver of the As induced rats (Table 1).

In the present study we the first to compare the effect of Ab-Sp and M-Sp on the histopathological changes in liver induced by iAs in rats. iAs induced variety of histopathological changes in liver tissues and spirulina was found effective in prevention of these histopathological changes.
In the present study the livers of the trial rats with arsenic dosing alone resulted in vacuolated hepatocytes, erosions of the lobular epithelium, and hemorrhages in some of the central veins and hepatic lobules including lobular tissues as extravasations. In accordance to the present findings, similar hemorrhages were found to be frequent throughout the liver [21] hepatocyte with hypertrophy and fatty infiltration as widespread vacuoles consistent with fatty droplets were observed after chronic arsenic exposure [18, 21]. However, the spirulina feeding with arsenic in rats resulted in full recovery from these conditions and the Spirulina treatments without arsenic in rats did not induce such histological changes in the liver. Besides these, hypertrophic hepatocytes with hypertrophic nucleus and hepatocytes with visible chromatin were found in the liver of rats of all the groups with variable degrees.
Arsenic feeding alone (T1) and treatments of both the spirulina without arsenic (T2 and T3) showed significantly higher (p<0.01) numbers of both hypertrophic hepatocytes with hypertrophic nucleus and hepatocytes with visible chromatin in the livers of the trial rats compared to the control. This finding indicates that arsenic dosing and both the spirulina treatments without arsenic in the rats induced the increased numbers of both hypertrophic hepatocytes with hypertrophic nucleus and hepatocytes with visible chromatin in the livers of rats above the control level.
The numbers of both hypertrophic hepatocytes with hypertrophic nucleus and hepatocytes with visible chromatin in the livers of the trial rats in all the As plus Ab-Sp groups were found lower compared to all of the As plus M-Sp groups as well as T1 groups. But, the numbers of the hypertrophic hepatocytes with hypertrophic nucleus were higher while the numbers of the hepatocytes with visible chromatin were lower in all the As plus Ab-Sp groups compared to the control. However, none of the M-Sp doses was found to reduce the numbers of both the types of the hepatocytes compared to the control and T1 groups. These findings reveal that all the doses of the Ab-Sp improved both these histological conditions from the intensity caused by arsenic dosing in the liver. It was evident that all the Ab-Sp doses decrease the numbers of the hepatocytes with visible chromatin below the control level, although the numbers of the hypertrophic hepatocytes with hypertrophic nucleus were decreased with all the Ab-Sp doses but could not return at the control level with any of the Ab-Sp doses. The Ab-Sp treatment with arsenic at the dose of 1.5g Ab-Sp/kg feed (T5) was found as the best dose among Ab-Sp doses in reducing the numbers of both types of the hepatocytes. On the other hand, none of the doses of the M-Sp reduce the number of any type of the cells at the control level and even at the T1 group level.
In conclusions, In the present study, we the first to clarify the comparative effects of Ab-Sp and M-Sp. Arsenic induced histopathological changes in liver were fully or partially recovered by spirulina feeding to the arsenic induced rats, and the Ab-Sp was found better in majority of the cases than the M-Sp in prevention of the changes.

The authors are thankful to the United States Department of Agriculture, USA, for financial support (Grant No. USDA/31/2006, BGARS-117) through the research project entitled “Detection of arsenic in the food chains and animal samples and study the preventive measure using the best cost-effective agricultural products-based spirulina against arsenicosis in man and livestock” under the Department of Pharmacology, Bangladesh Agricultural University, Mymensingh, Bangladesh. The authors also grateful to the Field Fertility Clinic (FFC) Laboratory, Department of Surgery and Obstetrics, BAU, Mymensingh for supporting the study providing DIC microscope.

AK, MAA and MZIK designed the experiment, AK, MRI, MRJ performed experiments, AK analyzed the data. AK drafted the manuscript. MZIK and MNI critically revised the manuscript.

The authors declare no conflict of interest.