Protective efficacy of Coriandrum sativum seeds against arsenic induced toxicity in Swiss albino mice

Abstract

Arsenic poisoning in ground water is one of the most sensitive environmental pollutant causing serious pollution all over the world. Chronic arsenic exposure through drinking water to humans leads to major public health related issues. There have been very meagre studies which reported that, the plant constituents proved to exhibit protective effect from arsenicosis. Therefore, the present study aims to evaluate the protective efficacy of Coriandrum sativum seeds extract against sodium arsenite induced toxicity in Swiss albino mice. In the present study twenty-four male healthy Swiss albino mice (30 ± 5 g) were divided into four groups (n = 6), where the control group received normal diet and water; group II and group III treated with sodium arsenite (2 mg per kg body weight per day) for 2 and 4 weeks respectively. The group IV mice were administered with C.sativum seeds extract at the dose of 150 mg per kg body weight per day for 4 weeks upon sodium arsenite pretreated (2 mg/kg body weight for 4 weeks per day) mice. After the complete dose duration, all the treatment group animals were sacrificed same day for haematological, biochemical and histopathological study. In the arsenic treated mice, there were significant (p < 0.0001) changes in the serum levels of ALT, AST, ALP, urea, uric acid and creatinine as well as in the haematological parameters. In contrast, after the administration with C.sativum seeds extract upon arsenic pretreated mice, there was significant (p < 0.0001) improvement observed in the hepatic and renal biomarker parameters as well as haematological variables. In the arsenic intoxicated mice,  after administration with C.sativum seeds extract there was significant (p < 0.0001) reduction in the arsenic concentration in blood, liver and kidney tissues as well as in the serum LPO levels. Furthermore, the histopathological study showed that, C.sativum seeds extract administrated group of mice significantly restored the liver and kidney at cellular level against arsenic induced toxicity. The entire study concludes that C.sativum seeds extract possesses the ameliorative effect against arsenic induced liver and kidney intoxication.

Introduction

Arsenic poisoning in groundwater has been a major health risk in recent times due to geogenic and anthropogenic activities [1]. Arsenic occurs in both inorganic and organic forms. Inorganic arsenic is predominantly present in the environment in the form of trivalent arsenite (AsIII) or pentavalent arsenate (AsV), while in the organic form, arsanilic acid, methyl arsenic acid, dimethylarsinic acid and arsenobetaine [2]. Presently, arsenic has caused severe public health problems in developing countries such as Bangladesh, India, Nepal, China, Taiwan, Thailand, Argentina, Chile, Mexico, Ghana, Vietnam and developed countries like USA, Romania and Hungary [3]. India and Bangladesh are the two most affected countries in Asia with the problem primarily in the plain area of Ganga-Meghna-Bramhaputra. In India, states such as Bihar, West Bengal, Assam, Uttar Pradesh and Uttrakhand along the Ganges basin are gravely affected by arsenic poisoning in groundwater [4]. In Bihar, 18 districts were found with elevated levels of arsenic contamination in ground water, while 10 million people are at risk of arsenic poisoning leading to serious health problems [5–8]. The toxicity of arsenic is usually attributed to both forms, either trivalent or pentavalent. Trivalent inorganic arsenic is considered more toxic compared with pentavalent inorganic arsenic [9]. The toxicity of trivalent arsenic may be mediated by its direct association with vicinal thiols or biological ligands containing sulfur group compounds and their participation in cellular redox reactions leads to an increased generation of free radicals [10, 11]. Chronic exposure to arsenic due to contamination of drinking water in humans causes carcinogenesis in vital organs, such as the liver, kidneys, bladder, gastrointestinal tract, skin, etc. [12, 13]. Epidemiological study suggests that high level of acute and chronic arsenic exposure significantly causes skin manifestations (hyper-pigmentation, hyperkeratosis, melanosis, black foot disease etc.), reproductive complications and intellectual impairment in children [8, 14–17].

Literature study emphasizes that the plant constituents proved to exhibit protective effect against arsenic induced toxicity [18]. One of the potential medicinal plant source is Coriandrum sativum L. (family Umbelliferae) which in India has the prime cultivation and production [19]. The major polyphenol isolated from Coriandrum sativum seeds (C. sativum seeds) are quercetin, kaempferol, gallic acid, ellagic acid and caffeic acid [20]. Coriander phytoconstituents has been shown to exhibit several pharmacological effects such as antioxidant activity [21, 22], anti-diabetic activity [23], antifungal activity [24], anti-mutagenic activity [25], hepatoprotective activity [26], renal protective activity [27], cholesterol lowering activity [28], anticancer activity [29], post-coital anti-fertility activity [30]. In other studies, it was found that coriander seeds extract protected the liver and kidney damaged against lead induced toxicity in mice model [31]. Coriander also plays a preventive role against lead deposition in ICR mice [32]. Therefore, the present study was designed to evaluate the protective efficacy of C.sativum seeds extract against arsenic induced liver and kidney toxicity in Swiss albino mice.

Materials and methods

Chemical

Arsenic was used as sodium arsenite, (assay 98%) manufactured by Loba Chemie, India (CAS No.7784-46-5, Lot No.#SG59751302), which was purchased locally from the Scientific store of Patna, Bihar, India.

Preparation of hydroxy-ethanolic extract of Coriandrum sativum seeds (C. sativum)

In the present study, C.sativum seeds were purchased from local market of Patna, Bihar and identified by Dr. Sushil Kumar Singh (Botanist), Associate Professor, Department of Biotechnology, A. N. College Patna, Bihar, India. The plant seeds were washed through running tap water to remove dirt and soil and finally rinsed with distilled water. The seeds were dried at 37 °C temperature, thereafter grinded to fine powder. Then fine powder was collected through fine sieve and soaked in absolute ethanol for 48 h. The ethanolic extract was separated by using Rota vapour apparatus (Buchi, R-3, Switzerland). The extracted powder was weighed 150 mg and was mixed in 10 ml of distilled water (pre-mixed in 5% alcohol) to make hydroxy-ethanolic extract and vortexed rigorously for 2 h for complete mixing up of the compounds in the solution to make it appropriate for the delivery to the treated animals.

Animals

Twenty-four healthy male Swiss albino mice (Mus musculus), weighing 30 ± 5 g of 8 weeks old, were obtained from animal house of Mahavir Cancer Sansthan and Research Centre, Patna, India (CPCSEA Regd-No. 1129/bc/07/CPCSEA). The experimental work was permitted by the Institutional Animal Ethics Committee (IAEC) with IAEC No. 2015/01 dated 16/12/2015. The mice were acclimatized in laboratory housing conditions under 12 h light and dark cycles (room temperature maintained at 22 ± 2 °C) for 7 days prior to the start of the treatment. These experimental mice were housed in conventional polypropylene cages and stainless-steel grill top and provided the diet (prepared by the laboratory itself) and water ad libitum.

Experimental design

Mice were randomly divided into four groups (n = 6).

Group I: Vehicle control. Mice were intragastrically given distilled water for 4 weeks.

Group II and III: Arsenic treated- Mice were intragastrically induced (by gavage method) sodium arsenite (2 mg/kg body weight/day) for 2 and 4 weeks respectively.

Group IV: Coriandrum sativum seeds (C.sativum seeds) extract treated- Mice were orally administration with C.sativum seeds extract at the dose of 150 mg/kg body weight/day for 4 weeks upon arsenic pretreated group of mice (2 mg/kg body weight/day for 4 weeks).

Dose selection

Sodium arsenite was selected to make the arsenic model. The dose selection of sodium arsenite was based on the LD₅₀ calculation. The final dose was selected to 2 mg/kg body weight. The sodium arsenite dose was dissolved in 10 mL distilled water and intragastrically induced by gavage method [33].

The hydroxy-ethanolic extract dose of C.sativum seeds was also calculated after the LD₅₀ estimation which was found to be 1800 mg/kg body weight and the final dose was titrated to 150 mg/kg body weight as the treatment dose, which is approximately 1/10th dose of the LD₅₀. After the completion of the dosing period, mice were sacrificed by cervical dislocation and blood samples were collected through the retro-orbital puncture from each group of mice. Blood samples were centrifuged at 3000 rpm and serum were extracted and stored at − 20 °C. The liver and kidney tissues were also dissected out for the determination of arsenic concentration on Atomic Absorption Spectrophotometer. Furthermore, the tissue samples were also fixed for the histopathological study.

Haematological assay

After sacrificing the mice, the whole blood was collected in EDTA coated vacutainer for the haematological parameter’s analysis. The red blood cells (RBC), haemoglobin (HGB), haematocrit (HCT), Mean corpuscular hemoglobin (MCH), Mean corpuscular volume (MCV), platelets (PLT) and white blood cells (WBC) were further analyzed using fully automated haematological analyzer (BC-2800, Mindray, China).

Liver and kidney biochemical assay

Biochemical parameters analysis were performed through the serum by standard kit process (Coral crest) through (UV- Vis) spectrophotometer (UV-10, Thermo Scientific, USA). The liver function test (LFT) as alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured according to method of Reitman and Frankel, [34], alkaline phosphatase (ALP) by using method Kind and King, [35]. The Kidney Function Test (KFT) were analyzed through urea by the method of Fawcett and Scott, [36]; Berthelot, [37]. Creatinine by the method of Bones and Tausky, [38] and uric acid by the method of Fossati and Prencipe, [39].

Lipid peroxidation (LPO) assay

Thiobarbituric acid reactive substances (TBARS), as a marker of LPO, were evaluated through the double heating method based on the principle of spectrophotometric measurement of color reproduced during the reaction to thiobarbituric acid (TBA) with malondialdehyde (MDA) [40]. For this study, 2.5 mL of 100 g/L solution of trichloroacetic acid (TCA) was mixed with 0.5 mL serum in a centrifuge tube and heated in the water bath at 90 °C for 15 min. After cooling the solution at room temperature, the mixture was further centrifuged at 3000 rpm for 10 min and 2 mL of the supernatant was mixed with 1 mL of 6.7 g/L TBA solution in a test tube which was further heated in water bath at 90 °C for 15 min and left for cooling at the room temperature. Thereafter, the absorbance was measured by UV–Visible spectrophotometer (Thermo Scientific UV-10 USA) at 532 nm.

Assay of arsenic concentration in blood, liver and kidney tissues

The whole blood, liver and kidney tissue samples were utilized for arsenic estimation using Atomic Absorption Spectrophotometer. The blood and tissue samples were double digested using concentrated HNO₃ on hot plate, under fume hood and estimated as per the protocol of (NIOSH, 1994) through Graphite Furnace Atomic Absorption Spectrophotometer (Pinnacle 900 T, Perkin Elmer, Singapore) at Mahavir Cancer Sansthan and Research Centre, Patna, Bihar [41].

Histopathological study

For the histopathological study, liver and kidney tissues were dissected and collected from all the group of sacrificed mice. The tissue samples were washed in normal saline, grossed into small pieces and finally fixed in 10% formalin for 24 h. Thereafter, tissues were dehydrated through process of graded series of ethanol and finally embedded into paraffin. Thin sections of 5 µm thickness were sliced through digital rotary microtome (Thermo Scientific Microm HM 340E) and stained with hematoxylin and eosin (H&E) for the histopathological study under light microscope.

Statistical analysis

Results are presented as mean ± Standard Deviation (SD) for six mice each group and total variation represented in a set of data was analyzed through one-way analysis of variance (ANOVA). Differences among mean variance has been analyzed by applying Tukey’s test at p < 0.05 was considered statistically significant. Calculations were performed with the GraphPad Prism Program (GraphPad 5 Software, Inc., San Diego, USA).

Results

Bodyweight and organ weights

In 2 weeks of arsenic treatment, significant (p < 0.001) decrease in the liver and kidney weight were observed in comparison to the control group. However, after 4 weeks of arsenic treatment the liver, kidney and body weight significantly (p < 0.0001) decreased in comparison to the control group. In contrast, after the administration with C.sativum seeds extract upon arsenic intoxicated mice, there was significant (p < 0.001) increase in the body weight, kidney weight and (p < 0.0001) liver weight as compared to the 4 weeks arsenic treated mice (Table 1).

Table 1.

Body weight and organ weights in different treatment groups of mice

Parameters	Control	2 Weeks arsenic treated	4 Weeks arsenic treated	C. sativum administration
Body weight (g)	30.83 ± 0.60	28.50 ± 0.76 ns†	26.17 ± 1.30^	32.17 ± 1.16##
Liver weight (g)	4.69 ± 0.10	4.14 ± 0.08**	3.59 ± 0.09^^^	4.38 ± 0.08###
Kidney weight (g)	1.35 ± 0.03	1.09 ± 0.06**	0.99 ± 0.03^^^	1.26 ± 0.03##

Data are given as mean ± SD (n = 6 each group). ^**(p < 0.001), ^ns†(non-significant) vs control mice; ^{^^^}(p < 0.0001), ^{^}(p < 0.05), vs control mice; ^###(p < 0.0001),^##(p < 0.001) vs 4 weeks arsenic treated mice

Relative organ weights

In 2 weeks of arsenic treated mice, a non-significant difference was observed in the relative liver and kidney weight in comparison to the control group. Also, non-significant differences were observed in 4 weeks arsenic treated mice in relative liver and kidney weights in comparison to the control mice. However, after 4 weeks of arsenic treatment, there was non-significant difference observed in the relative liver and kidney weight in comparison to the control group. In contrast, after the administration with C. sativum seeds extract upon arsenic intoxicated mice, there was also a non-significant difference observed in the relative liver and kidney weight in comparison to the 4 weeks arsenic treated mice (Table 2).

Table 2.

Relative organ weights in different treatment groups of mice

Parameters	Control	2 Weeks arsenic treated	4 Weeks arsenic treated	C.sativum administration
Relative Liver weight (g)	0.14 ± 0.01	0.15 ± 0.02 ns†	0.14 ± 0.03 ns	0.13 ± 0.01 ns††
Relative Kidney weight (g)	0.04 ± 0.00	0.03 ± 0.01 ns†	0.03 ± 0.00 ns	0.03 ± 0.00 ns††

Data are given as mean ± SD (n = 6 each group). ^ns†(non-significant) vs control mice; ^ns(non-significant), vs control mice; ^ns†† (non-significant) vs 4 weeks arsenic treated mice (Table 2)

Assay of haematological parameters

In the 2 weeks arsenic treated mice, significant (p < 0.0001) changes in HGB, HCT, RBC (p < 0.05), WBC (p < 0.001) and platelets (p < 0.001) counts, while the non-significant changes were found in MCV, MCH, MCHC in comparison to the control group. Apart from 4 weeks arsenic treated mice, there were significant (p < 0.0001) changes found in the RBC, HGB, HCT, MCHC, WBC, platelets, MCH (p < 0.05) and non-significant changes in MCV levels as compared to the control mice. In contrast, after the administration with C. sativum seeds extract upon arsenic pretreated mice there was significant (p < 0.0001) restoration in the RBC, HGB, HCT, MCHC, WBC and platelets count and non-significant changes found in the MCV and MCH level in comparison to the 4 weeks arsenic treated mice (Table 3).

Table 3.

Haematological parameters activity in different treatment groups of mice

Parameters	Control	2 Weeks arsenic treated	4 Weeks arsenic treated	C. sativum administration
RBC (× 106/mm3)	6.54 ± 1.16	4.86 ± 1.05*	2.53 ± 0.55^^^	5.70 ± 1.05###
HGB (g/dL)	13.70 ± 0.96	9.56 ± 1.40***	6.81 ± 1.12^^^	12.45 ± 1.36###
HCT (%)	39.27 ± 1.84	26.53 ± 3.26***	16.95 ± 3.23^^^	34.52 ± 4.13###
MCV (fL)	61.32 ± 9.35	55.92 ± 8.83 ns†	68.22 ± 12.77 ns	61.35 ± 7.47 ns††
MCH (pg)	21.32 ± 2.66	20.08 ± 2.93 ns†	27.58 ± 5.34^	22.12 ± 2.53 ns††
MCHC	34.87 ± 1.26	35.88 ± 0.92 ns†	40.40 ± 1.60^^^	36.13 ± 1.25###
WBC (× 103/mm3)	7.23 ± 1.28	10.60 ± 1.59**	14.75 ± 1.98^^^	8.31 ± 1.28###
Platelets (× 103/mm3)	365.3 ± 78.37	235.5 ± 33.39**	98.33 ± 20.68^^^	275.7 ± 48.49###

Data are given as mean ± SD (n = 6 each group). ^***(p < 0.0001), ^**(p < 0.001), ^*(p < 0.05), ^ns†(non-significant) vs control mice; ^{^^^}(p < 0.0001), ^{^}(p < 0.05), ^ns(non-significant) vs control mice; ^###(p < 0.0001), ^ns††(non-significant) vs 4 weeks arsenic treated mice

Assay of liver and kidney biochemical parameters

In 2 weeks of arsenic treatment, significant (p < 0.0001) elevations were observed in AST, ALP, creatinine and (p < 0.05) urea levels in comparison to the control group. However, after 4 weeks of arsenic treatment the ALT, AST, ALP, urea, and creatinine levels increased significantly (p < 0.0001) in comparison to the control group. In contrast, after the administration with C.sativum seeds extract upon arsenic intoxicated mice, there was significant (p < 0.0001) decrease in the serum level of ALT, AST, ALP, urea, uric acid, and creatinine levels as compared to 4 weeks arsenic treated mice (Table 4).

Table 4.

Liver and kidney function - biochemical parameters activity in different treatment groups of mice

Parameters	Control	2 weeks arsenic treated	4 weeks arsenic treated	C. sativum administration
ALT (U/mL)	32.25 ± 3.94	35.72 ± 7.09 ns†	173.7 ± 5.98^^^	69.55 ± 6.14###
AST (U/mL)	47.08 ± 4.25	31.30 ± 3.69***	135.1 ± 5.62^^^	52.08 ± 2.98###
ALP (KA units)	2.45 ± 0.44	5.60 ± 1.47***	9.93 ± 1.57^^^	5.38 ± 0.56###
Urea (mg/dL)	14.33 ± 2.58	18.02 ± 0.21*	22.67 ± 3.08^^^	12.53 ± 1.44###
Uric acid (mg/dL)	4.55 ± 0.73	3.40 ± 0.28 ns†	5.38 ± 1.21 ns	2.31 ± 0.23###
Creatinine (mg/dL)	0.51 ± 0.11	2.15 ± 0.10***	2.45 ± 0.67^^^	0.20 ± 0.10###

Data are given as mean ± SD (n = 6 each group). ^***(p < 0.0001), ^*(p < 0.05), ^ns†(non-significant) vs control mice; ^{^^^} (p < 0.0001), ^ns(non-significant) vs control treated mice; ^###(p < 0.0001) vs 4 weeks arsenic treated mice

Assay of arsenic concentration in blood, liver and kidney tissues

There was significant (p < 0.0001) arsenic contamination in the blood, liver and tissues in 2 and 4 weeks arsenic treated mice respectively as compared to the control mice. However, after the administration with C.sativum seeds extract upon arsenic pretreated mice, there was significant (p < 0.0001) decrease in the arsenic concentration in the blood, liver and kidney tissues as compared to the 4 weeks arsenic treated mice (Table 5).

Table 5.

Arsenic concentration in different treatment groups of mice

Group	Control	2 Weeks arsenic treated	4 Weeks arsenic treated	C.sativum administration
Blood (µg/L)	0.62 ± 0.41	33.25 ± 3.74***	85.78 ± 5.55^^^	27.08 ± 4.46###
Liver (µg/kg)	N.D	106.1 ± 10.45***	197.4 ± 11.19^^^	50.90 ± 5.37###
Kidney (µg/kg)	0.57 ± 0.40	82.58 ± 11.64***	142.3 ± 13.35^^^	54.80 ± 8.03###

Data are given as mean ± SD (n = 6 each group). ^***(p < 0.0001) vs control mice; ^{^^^}(p < 0.0001) vs control mice; ^###(p < 0.0001) vs 4 weeks arsenic treated mice (N.D- Not detected)

Assay of lipid peroxidation (MDA)

The MDA levels, in the arsenic treated mice significantly increased in the 2 weeks (p < 0.05) and 4 weeks (p < 0.0001) arsenic treated group mice respectively than the control mice. However, after the administration of C. sativum seeds extract at the dose of 150 mg/kg body weight upon arsenic pretreated mice, there was significant (p < 0.0001) reduction in the serum levels of lipid peroxidation (Fig. 1).

Fig. 1.

Effect of C. sativum against arsenic induced MDA activity. Value are expressed mean ± SD (n = 6), ^*(p < 0.05) vs control mice; ^{^^^}(p < 0.0001) vs control mice; ^###(p < 0.0001) vs 4 weeks arsenic treated mice

Histopathological observations

Histopathological study of liver

In the present histopathological study, the control mice liver section showed normal histoarchitecture of hepatocytes, well arranged around the central vein (Fig. 2A, Table 6). Arsenic treated mice liver section showed mild degeneration in the hepatocytes as well as dilatations in sinusoids (Fig. 2B, Table 6), while in 4 weeks arsenic treated mice liver showed significant degeneration in hepatocytes, with rupture in the central vein along with severe dilatation and vacuolizations in sinusoids (Fig. 2C, Table 6). But, in the C.sativum seeds extract 150 mg/kg body weight administration upon arsenic pretreated mice liver section showed significant regeneration in hepatocytes with defined nucleus and mild congestion in central vein along with dilatation in the sinusoids (Fig. 2D, Table 6).

Fig. 2.

Microphotographs section of liver stained with hematoxylin and eosin (H&E). a Control mice liver section showing defined histoarchitecture of hepatocytes (H) along with central vein (CV) X500. b 2 weeks arsenic treated mice liver section showing degeneration in hepatocytes (H) X500, c 4 weeks arsenic treated mice liver microphotograph showing significant degeneration in hepatocytes, with rupture in the endothelial cells of central vein (CV) along with dilatation in sinusoids X500. d C.sativum seeds extract administration upon arsenic pretreated mice liver section showing significant restoration in hepatocytes with well-defined nucleus X500

Table 6.

Quantitative histological damage scoring in liver tissues of different treatment group of mice (n = 6, values are expressed as mean ± SD)

Group	Control	2 Weeks arsenic treated	4 Weeks arsenic treated	C. sativum administration
Degenerated hepatocytes	1.83 ± 0.75	27.67 ± 4.08***	67.83 ± 5.77^^^	36.50 ± 4.32###
Central vein damage	0.83 ± 0.75	6.50 ± 1.87**	14.67 ± 2.80^^^	11.33 ± 2.80 ns
Portal vein damage	2.00 ± 1.26	4.16 ± 2.92 ns	16.33 ± 2.06^^^	12.00 ± 3.03#
Vacuolizations	0.66 ± 0.51	3.83 ± 3.06 ns	14.33 ± 2.16^^^	10.17 ± 1.47##
Haemorrhages	0.33 ± 0.51	4.33 ± 2.25*	11.17 ± 3.43^^^	4.50 ± 1.04###

Hepatocyte’s degeneration was assessed in each mouse by counting the degeneration among 100 hepatic cells; rest histopathological changes were examined randomly in selected 20 microscopic fields (X40; H&E). ^***(p < 0.0001), ^**(p < 0.001), ^*(p < 0.05), ^ns (non-significant) vs control group of mice; ^{^^^}(p < 0.0001), vs control mice; ^###(p < 0.0001), ^##(p < 0.001), ^#(p < 0.05), ns (non-significant) vs 4 weeks arsenic treated group of mice

Histopathological study of kidney

In the present histopathological study, the control mice kidney section showed normal histopathological architecture of nephrocytes with well defined proximal and distal convoluted tubules (Fig. 3A, Table 7). In the arsenic treated mice kidney section showed degeneration in glomerulus and vacuolization in the Bowman’s capsule basement (Fig. 3B, Table 7). However, in 4 weeks arsenic treated mice kidney section showed significant degeneration in nephrocytes, narrowing of the walls of Bowman’s capsule basement due to swollen glomerulus along with the vacuolations in distal and proximal tubules (Fig. 3C, Table 7). But, after the administration of C.sativum seeds extract 150 mg/kg body weight upon arsenic pretreated mice kidney section showed significant restoration in the nephrocytes as well as glomerulus and well defined distal and proximal tubules were observed (Fig. 3D, Table 7).

Fig. 3.

Microphotographs of kidney tissue section stained with hematoxylin and eosin (H&E). a Control mice kidney section showed normal histoarchitecture of proximal and distal convoluted tubules (PCT and DCT) X500. b 2 weeks arsenic treated mice kidney section showing degeneration in glomerulus and vacuolization in Bowman’s capsule (BC) X500 c, while in 4 weeks arsenic treated mice kidney showed significant degeneration in glomerulus along with vacuolization in proximal and distal convoluted tubule (PCT and DCT) X500. d After the administration with C. sativum seeds extract upon arsenic pretreated mice, significant restoration in nephrocytes with glomerulus was observed X500

Table 7.

Quantitative histological damage scoring in kidney tissues of different treatments group of mice (n = 6, values are expressed as mean ± SD)

Group	Control	2 Weeks arsenic treated	4 Weeks arsenic treated	C. sativum administration
Tubules damage	1.50 ± 1.04	3.66 ± 1.36 ns	29.00 ± 5.06^^^	11.67 ± 1.21###
Glomerulus degeneration	1.83 ± 0.98	2.16 ± 1.16 ns	9.16 ± 2.92^^^	4.83 ± 1.47##
Vacuolizations	0.66 ± 0.81	2.50 ± 1.87 ns	3.83 ± 1.72^^	1.16 ± 0.75#
Haemorrhages	0.33 ± 0.51	1.00 ± 1.26 ns	5.50 ± 1.04^^^	1.66 ± 1.21###

Tubules degeneration were assessed in each mouse by counting the degeneration among 100 tubules; rest histopathological changes were examined randomly in selected 20 microscopic fields (X40; H&E). ^ns(non-significant) vs control group of mice; ^{^^^}(p < 0.0001), ^{^^}(p < 0.001) vs control mice; ^###(p < 0.0001), ^##(p < 0.001), ^#(p < 0.05) vs 4 weeks arsenic treated group of mice

Discussion

Chronic arsenic exposure is known to cause various types of toxic effects involving multiple organ system with complex mechanisms [42]. Arsenic as metalloid element is highly toxic and causes degeneration at the cellular level. The liver and kidney are the vital organs, in which liver plays important role of elimination of arsenic methylated species through excretion via urine by the kidney [43]. Approximately, 70% of inorganic arsenic is excreted in the urine primarily through the kidney. Excretion through the gut has mixture of inorganic, monomethylated and demethylated forms of arsenic [44]. In the present study, there was significant elevations in the serum levels of ALT, AST and ALP in arsenic induced mice group in comparison to the control mice group, which confirmed the liver impairment. Arsenic (iAs^III) has high affinity to a potential sulfhydryl-reactive compound and binds with thiol groups of proteins or enzymes in the liver and undergoes biotransformation. It interferes with the integrity of hepatic plasma membrane leading to split out of ALT, AST and ALP in serum [45]. The ALT, AST and ALP are usually measured to indicate liver impairment and other problems with bones, gallbladder, kidney and heart. ALT and AST are known to be primarily localized inside liver cells; however, ALP is present in a wide variety of tissues including liver, bones, intestines, kidneys and other organs. In the present histopathological study, arsenic treated mice liver section showed severe degeneration in the hepatocytes with the rupturing in the endothelial cells of the central vein along with the hyperproliferation of Kupffer cells and dilatation in the sinusoidal spaces. The findings are in line with the previous studies reported by several researchers [33, 46]. However, after the administration with C.sativum seeds extract upon arsenic intoxicated mice group, there was significant reduction in the liver biomarker parameters such as ALT, AST and ALP. The C.sativum seeds extract administration stabilized the integrity of hepatocellular membrane and significant improvement in the histological alterations along with the mild degeneration in the central veins. These results suggested that C.sativum seeds ameliorated the toxic effects of arsenic induced liver injury due to the presence of antioxidant cascade mechanism. In the present study, probably the quercetin the most active polyphenol component present in C. sativum seeds, could have played an important role by providing protection against hepatotoxicity [20, 47–49]. Ghosh et al. [50] reported that quercetin significantly improved the antioxidant enzymes along with the upregulation of the cytochrome C, which potentially prevents the oxidative attacks and maintains the cellular redox against arsenic induced liver and brain oxidative damages. The polyphenol quercetin may be responsible for the potential protection against arsenic induced hepatotoxicity. In this study, we also observed the kidney damage by the arsenic intoxication. In the present study, arsenic treated mice showed significant increase in the serum levels of urea, uric acid and creatinine in comparison to the control mice group, which was consistent with previous reports [51]. The liver produces urea through the urea cycle as a waste product of the protein metabolism, terminated in kidney as urea is excreted [52]. High level of serum urea in arsenic treated mice may impair the liver and kidney functions. Uric acid is the end product of purine metabolism and it is formed from the guanine and hypoxanthine via xanthine in reactions catalyzed by guanase and xanthine oxidase. The toxic effect of arsenic alters the guanase and xanthine oxidase which elevates the serum uric acid levels [53]. The increased levels of creatinine after arsenic induced toxicity are due to the enhanced formation of metabolic waste products of skeletal muscles. Serum creatinine parameters denotes the glomerular filtration rate [54]. Arsenic damages the renal cells and releases creatine phosphokinase which is responsible for the conversion of phosphocreatine to creatinine [51]. In addition, arsenic treated mice kidney through histopathological examination showed degeneration in the renal tubules (PCT and DCT) and Bowmen’s capsules along with the high degree of degeneration and swellings in the glomerulus. There was a significant renal damage causing glomerular damage and impairment in the infiltration part. In contrast, after the administration with C.sativum seeds extract upon arsenic pretreated mice there was significant decrease in the renal biochemical parameters such as the urea, uric acid and creatinine levels. However, there was marked restoration in the glomerular and renal tubules. The membrane stabilizing properties of C.sativum could have ameliorated the damage caused by arsenic induced histopathological alterations. The efficiency of C.sativum was due to the presence of several flavonoids and polyphenol compounds such as quercetin 3- glucoronide linalool, camphor, geranyl acetate, geraniol and cumarins [55]. These active constituents played the vital role in combating the arsenic induced damage [56]. The results are in line with the previous studies reported by several researchers who suggested that C.sativum seeds protects the renal cytotoxicity on heavy metal induced intoxication [31]. Recent study states that arsenic metabolism generates the reactive oxygen species (ROS) leading to oxidative stress which results in the changes in the cell structure and functions [57, 58]. In the present study, there was significant elevation in the levels of lipid peroxidation in arsenic treated group in comparison to the control group. Lipid peroxidation (LPO) is caused by free radicals leading to oxidative degradation of polyunsaturated fatty acids constitutive of cellular membranes. Their destruction leads to the production of reactive aldehyde such as malondialdehyde (MDA) which is the relevant method to measure the peroxidation damage caused in cell membranes [59]. In the present study, oral administration of C.sativum seeds extract at the dose of 150 mg/kg body weight for 4 weeks upon arsenic pretreated mice significantly reduced the LPO levels. The antioxidant properties of coriander seeds possess large amounts of carotenoids, tocopherols (Vitamin E) and phospholipids [60, 61], which acts through different mechanisms. Carotenoids acts as a primary antioxidant by trapping free radicals and as secondary antioxidants by quenching singlet oxygen [62]. Tocopherols acts as antioxidant as it can bind with the lipid peroxyl radicals, preventing the propagation of free radical chain reaction. This natural antioxidant mechanism protects the cellular membrane injuries caused by lipid peroxides and other free radicals [63].

In the present study, it was observed that, arsenic treated mice significantly showed altered variation in the RBC, HGB, WBC and PLT counts. These findings are in consistent with the previous reports on mice [64]. This could be due to the arsenic binding with the haemaglobin, hence arsenic accumulation in blood showed significant alterations in the haematological parameters [65, 66]. In the present study, increase in the WBC counts may be due to the increased levels of thrombopoietin [67]. But, after the administration with C. sativum there were significant restoration in RBCs, HGB, WBCs and PLTs counts. The protective effect of C.sativum seeds extract against arsenic toxicity may attribute to the antioxidant potential action of its component. One of the studies reported that C.sativum significantly improves the RBC, WBC, HGB against lead nitrite induced toxicity in Swiss albino mice [68].

Moreover, in arsenic treated mice there was significant arsenic contamination in the blood, liver and kidney tissue. The elevated arsenic concentration in blood, liver and kidney tissues was also correlated with the histopathological findings. The present study findings are well in line with the previous studies which reported arsenic deposition in vital organs like the liver, kidney, lungs and blood [64, 69, 70]. Although, C. sativum seeds extract administration upon arsenic intoxicated mice significantly eliminated the arsenic contamination in the blood, liver and kidney tissues. The possible role of C. sativum seeds may be correlated with the normalisation in the functions of the liver and kidney which also enhances the biomethylation efficacy of arsenic, and thus eliminates the methylated arsenic through urine. Moreover, the quercetin present in the C.sativum seeds plays the major role in scavenging the metal induced toxicity while the metal chelation effect of it protects from the metal induced renal damage [71–73]. The gut biome is also damaged by the arsenic exposure but antioxidant activity of C.sativum seeds controls the damage at much extent [74, 75].

The present study thus, concludes that hydoxy-ethanolic seeds extract of C.sativum 150 mg/kg body weight significantly restores the hepatic and renal biochemical parameters as well as at cellular level against the deleterious effect of arsenic-induced toxicity. Due to the presence of antioxidant properties, there was also a reasonable correlation observed between the biochemical and the histopathological findings. The entire finding suggests that C.sativum seeds possesses antitoxic and antidote properties against arsenic induced toxicity.

Funding

This research work was financially supported by the institute itself (Mahavir Cancer Sansthan and Research Centre, Patna, Bihar, India).

Declarations

Conflict of interest

The authors declare that they have no conflicts of interest concerning this article.