Protective effects of synbiotic diets of Bacillus coagulans, Lactobacillus plantarum and inulin against acute cadmium toxicity in rats

Background Cadmium is a heavy metal that causes oxidative stress and has toxic effects in humans. The aim of this study was to investigate the influence of two probiotics along with a prebiotic in preventing the toxic effects of cadmium in rats. Methods Twenty-four male Wistar rats were randomly divided into four groups namely control, cadmium only, cadmium along with Lactobacillus plantarum (1 × 109 CFU/day) and inulin (5% of feedstuff) and cadmium along with Bacillus coagulans (1 × 109 spore/day) and inulin (5% of feedstuff). Cadmium treated groups received 200 μg/rat/day CdCl2 administered by gavage. During the 42-day experimental period, they were weighed weekly. For evaluation of changes in oxidative stress, the levels of some biochemicals and enzymes of serum including SOD, GPX, MDA, AST, ALT, total bilirubin, BUN and creatinine, and also SOD level of livers were measured at day 21 and 42 of treatment. The cadmium content of kidney and liver was determined by using atomic absorption mass spectrophotometry. Data were analyzed using analysis of variance (ANOVA) followed by Duncan’s post hoc test. Results Treatment of cadmium induced rats with synbiotic diets significantly improved the liver enzymes and biochemical parameters that decreased AST, ALT, total bilirubin, BUN and metal accumulation in the liver and kidney and increased body weight, serum and liver SOD values in comparison with the cadmium-treated group. No significant differences were observed with MDA and GPX values between all groups (p > 0.05). Conclusions This study showed that synbiotic diets containing probiotics (L. plantarum and B. coagulans) in combination with the prebiotic (inulin) can reduce the level of cadmium in the liver and kidney, preventing their damage and recover antioxidant enzymes in acute cadmium poisoning in rat. Electronic supplementary material The online version of this article (doi:10.1186/s12906-017-1803-3) contains supplementary material, which is available to authorized users.


Background
Cadmium is a trace element and is one of the nonessential metals that has toxic effects in man [1]. It is a major concern for public health and is present at low concentration in soil, rock and drinking water. Cadmium can mainly be found in the earth's crust. It always occurs in combination with zinc. Cadmium also consists in the industries as an inevitable by-product of zinc, lead and coppe extraction. Because of its highly soluble nature compared to other metals, cadmium is taken up by plants and is stored in food and feed products and human receives it mainly through food [2]. Foodstuffs such as mushrooms, shellfish, mussels, liver, cocoa powder and dried seaweed that are rich in cadmium. They can greatly increase the cadmium concentration in the human body. Cigarette smoke transports high levels of cadmium into the lungs. Blood then transports the cadmium through the rest of the body where it can increase its toxic effects by potentiating with the cadmium that is already present from cadmium-rich food.
Cadmium is first transported to the liver through the blood stream where, it binds to proteins to form complexes that are transported to the kidneys. Cadmium accumulates in the kidneys where it damages the kidney's filtering mechanisms. This causes the excretion of essential proteins and sugars from the body and further kidney damage. The World Health Organization (WHO) specifies the tolerable intake for cadmium at 7 μg/kg of bodyweight/week. Dietary exposure to large cadmium doses has been reported to result in adverse health effects in the kidneys, liver, bone, mammary gland, breast, pancreas, colon [3][4][5] and also neurological alterations in humans such as lower attention, hypernociception, olfactory dysfunction and memory deficits [6].
Numerous studies have revealed that the mentioned metal caused oxidative stress by inducing the generation of reactive oxygen species (ROS). It has been assumed that cadmium reduced antioxidant defence system of cells via glutathione depletion, affecting some enzymes such as glutathione peroxidase (GP X ), superoxide dismutase (SOD) and increasing lipid, protein and DNA oxidation [7,8].
Chelation therapy has been applied for heavy metals poisoning excretion in therapeutic strategy. Evidence has shown that these chemical chelators (like CaNa 2 EDTA) have some side effects such as nausea, vomiting, anorexia, appetite loss, and diarrhoea [9]. Therefore, in recent years, there has been an increasing number of studies to find safe and efficient dietary compounds against heavy metals toxicity.
Synbiotic refers to a nutritional supplement which combines probiotic and prebiotic in a form of synergism [10]. Prebiotics are typically non-digestible fibre compounds that pass undigested through the gastrointestinal tract and stimulate the growth and activity of advantageous bacteria like probiotics [10]. Probiotics are live bacteria which are intended to colonize the large intestine, avoid the adherence of pathogens and confer physiological health benefits to the host [11]. Lactic acid bacteria (LAB) such as Lactobacillus acidophilus, L. plantarum and Bifidobacterium are known to be the most common probiotics. These probiotics are very sensitive to normal physiological conditions such as the very low pH of the stomach and bile salts [12]. Hence, a novel beneficial probiotic is introduced that can survive under extreme conditions. Some strains of Bacillus coagulans are able to withstand the gastrointestinal tract and continue their metabolic activities via spore production [13,14].
Probiotic bacteria may have the tendency for toxin protection due to their abilities in heavy metals binding and antioxidant effect. It is reported that L. rhamnosus LC105 and L. plantarum ID9263 can bind and remove heavy metals such as lead, cadmium and copper from polluted water in vitro [15,16]. Also, the positive effect of L. plantarum CCFM8661 on reducing the toxicity of lead in mice was demonstrated by Tian et al. [17]. Al-Wabel et al. [18] used synbiotic fermented milk against lead acetate contamination in rats and found out the effective role of this diet on the liver by increasing the activity of the antioxidant enzymes.
The purpose of the present study is to compare the effect of two probiotic, L. plantarum and B. coagulans, in combination with the prebiotics, inulin, to prevent the toxic effects of cadmium in rats.

Preparing suspension of probiotic bacteria
Two probiotic bacteria used throughout the study were B. coagulans and L. plantarum. Lyophilized probiotic B. coagulans was generously supplied by the Pardis Roshd Mehregan Company, Shiraz, Iran. B. coagulans spores were prepared according to the method of Abhari et al. [19]. The bacteria were grown aerobically in nutrient yeast extract salt medium (NYSM) agar. After incubation at 37°C for 24 h, a single colony was obtained, inoculated into NYSM broth and kept in a shaker incubator at 37°C for 48 h. Bacterial cells was achieved by centrifugation at 3000×g for 20 min and then heated at 80°C for 15 min to kill the vegetative cells. Finally, spore suspension of the bacterium was prepared at a concentration of 1 × 10 9 spore/ml in sterile saline and kept in the refrigerator up to a week [19].
L. plantarum CNR273 was taken from the culture collection of the Department of Food Science and Technology, Shiraz University, Iran. It was cultivated aerobically in De Man Rogosa Sharpe (MRS) agar (Difco, Detroit, MI, USA) at 37°C for 48 h. A single colony was inoculated into MRS broth and incubated at 37°C with shaking for 48 h. The bacterial pellets were attained by centrifugation at 3000×g for 20 min. The viable bacterial cells were prepared at a concentration of 1 × 10 9 CFU/mL after appropriate serial dilution and plating in MRS agar. This method was carried out as described by Jafarpour et al. [20]. The bacterial suspension was kept in the refrigerator up to a week.

Preparation of cadmium
Cd-exposed groups received cadmium chloride (CdCl 2 ) (Merck, Darmstadt, Germany) solution (200 μg/mL) at a dose of 200 μg/rat/day. The cadmium solution at the mentioned dose was prepared by the method described by Nwokocha et al. [21]. The molecular weight of CdCl 2 was divided by the molecular weight of cadmium (183.32/112.41) to obtain 1.63 g as the weight of 1 part of cadmium in CdCl 2 . 0.326 g of cadmium chloride was dissolved in 1 L of distilled water to achieve a concentration of 200 ppm. The CdCl 2 solution was fed daily to each rat by gavage.

Animals and treatment
Twenty-four male Wistar rats weighing approximately 170 ± 10 g were used. Animals were purchased from the Razi Vaccine and Serum Research Institute, Shiraz, Iran and kept in standard polypropylene cages under the following conditions: temperature (23 ± 2°C), relative humidity (38%), and exposure to a 12 h light/dark cycle with ad libitum access to food and tap water.
After an acclimatization period of 1 week, the animals were randomly divided into four groups (n = 6/group). The rats were identified by marking their tails. Three rats of each group were treated for 21 days and the treatment of the other three were continued for 42 days. They were weighed individually every week by using a balance scale (Mettler Toledo® scale, model Spider 2). Table 1 summarizes the profile of rat groups and fed diets.

Collection of blood and organ samples
On days 21 and 42, three rats from each treatment group were sacrificed under an atmosphere of 100% diethyl ether anaesthesia after overnight fasting. Two blood samples were immediately obtained via the right side of the heart for biochemical assay. One sample was transferred to an EDTA tube and the second one was transferred to a plain tube without anticoagulant and allowed to stand for 2 h. Thereafter the samples were centrifuged at 2000×g for 10 min to harvest plasma and serum, respectively. A small amount of the samples were also heparinized and used for preparation of red blood cell hemolysate for SOD and GPx assay.
Livers and kidneys of sacrificed rats were removed from the bodies, washed in ice-cold saline, and stored at −80°C for further measurements.

Superoxide dismutase (SOD) assay
Liver samples (0.5 g) were weighed and homogenized on ice and in cold 0.5 M phosphate buffer (pH 7.2) by using a homogenizer (Yellow Line DI18, Ikawerke, Germany). The homogenates were then centrifuged at 3000×g for 15 min at 4°C (Hettichmikro 200R, Tuttlingen, Germany) and the supernatants were stored at −80°C until use.
Blood and liver SOD activity was evaluated with SOD detection RANSOD kit (Randox Lab., Crumlin United Kingdom) according to the manufacturer's instructions. SOD levels were recorded at 505 nm through a standard curve and expressed as unit per gram of hemoglobin or liver tissue.

Glutathione peroxidase (GP X ) assay
The activity of GP X was evaluated with GPx detection RANSEL kit (Randox Lab., Crumlin United Kingdom) according to the manufacturer's instructions. GP X levels were measured spectrophotometrically at 340 nm and expressed as unit per gram of hemoglobin (U/g Hb).
One unit (U) of GP X activity was defined as the amount of enzyme that converts 1 μmol of NADPH to NADP + per minute.

Measurement of lipid peroxidation (MDA)
To evaluate lipid peroxidation in serum, a modified HPLC method was used based on the reaction of malondialdehyde (MDA) with thiobarbituric acid (TBA) to form a colored MDA-TBA adduct [22]. Forty μL of the sample was diluted with 100 μL of H 2 O and mixed with 20 μL of 2.8 mmol/L butylated hydroxyl toluene (BHT) in ethanol, 40 μL of 81 g/L sodium dodecyl sulfate and 600 μL of TBA reagent (8 g/L TBA diluted 1:1 with 200 ml/L acetic acid adjusted to pH 3.5 with NaOH). The mixture was immediately heated (60 min at 95°C) and cooled with running water; 200 μL of H 2 O and

Serum biochemical analyses
All biochemical parameters including alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine, blood urea nitrogen (BUN) and bilirubin were measured by commercial kits (Pars Azmoon Co., Tehran, Iran). All the enzyme activities were measured at 37°C and the results have been presented in units per litre [23]. Biochemical analyses were measured using a standard autoanalyser with veterinary software (Cobas-Mira, ABX-Diagnostics, Japan).

Cadmium measurement
Approximately 0.5 g of liver and kidney samples were taken from the experimental rats at day 42, weighed with an accuracy of 0.01 g and homogenized using a blade mixer until the appropriate consistency was reached.
Then the homogenized tissues were digested in 10 mL of concentrated nitric acid at boiling bath (about 96°C) for 18 h [24]. The concentration of cadmium was then determined by atomic absorption mass spectrophotometry (Shimadzu-AA-670, Japan).

Animal ethics
All the procedures conducted in this experiment follow the ethical guidelines of animal welfare approved by the Ethics Committee of the School of Veterinary Medicine, Shiraz University, Shiraz, Iran (Ethical approved number: 1392/905659).

Statistical analysis
The results are expressed as mean ± SD with a significance level of p < 0.05. Statistical analysis for significant differences among group means were tested by analysis of variance, followed by Duncan's post hoc test with the help of SPSS 16.0 Windows software. Figure 1 shows the changes to the body weight of the different groups during the experimental time. There was a gradual increase in the body weight of all groups over 6 weeks but a significant reduction was observed in the body weight of the cadmium group at the start of the experiment (p < 0.05). After a period of three weeks, the cadmium treated animals exhibited sluggish behaviour and had the lowest weight amongst the other three treatment groups. Data indicates that the treatment of the animals with synbiotics (198.25 ± 8.88 and 207.75 ± 6.34 g,  respectively, for ILp + Cd and IBc + Cd groups) significantly (p < 0.05) increased their body weights compared with cadmium group (168.75 ± 8.58 g).

Effects on biochemical properties
The effects of probiotics and inulin as a prebiotic on oxidative stress indicators (SOD, GP X and MDA) are shown in Table 2. At day 42 of the experiment, cadmium treated rats showed a significant decrease in the activity of the enzyme SOD both in the blood and liver (254.2 ± 1.60 and 2.55 ± 0.04 U/g, respectively), compared to other groups. Rats treated with Cd + synbiotic diet showed a significant increase in the activity of blood SOD (290.50 ± 3.20 and 287.00 ± 4.2 U/g Hb, respectively, for ILp + Cd and IBc + Cd groups) and liver SOD (2.74 ± 0.02 and 2.69 ± 0.03 U/g liver tissue, respectively, for ILp + Cd and IBc + Cd groups) when compared to cadmium treated rats (p < 0.05). No significant differences were observed with regard to MDA contents in the treatment groups during the 42 days. Similarly, GP X exhibited no differences between groups and also during the different times.
Serum activities of ALT, AST and total bilirubin were significantly higher in the cadmium treated group when compared to other groups (p < 0.05) (Fig. 2). Also, the data showed that probiotics along with inulin had significantly lower ALT and AST values in comparison with the cadmium treated group (p < 0.05) but that they had no effect on total bilirubin compared to the Cd group (Fig. 2).
Creatinine and BUN levels in the control group at day 21 and 42 of treatment were 0.72 and 0.80, 18.38 and 19.91 mg/dL, respectively (Table 3). Both factors were significantly higher in cadmium induced rat (p < 0.05). Administration of L. plantarum and B. coagulans together with inulin in cadmium induced rat caused a marked decrease in the level of creatinine, at day 42 of experiment, from 1.11 to 0.91 and 0.99 mg/dL, respectively (p < 0.05) ( Table 3). The level of BUN also significantly decreased in these groups from 24.55 to 20.29 and 20.67 mg/dL respectively (p < 0.05) ( Table 3).

Effects of synbiotic diet on heavy metals contents in the liver and kidney
The results of the cadmium content of both the liver and kidney of the treated groups are presented in Fig. 3. When a synbiotic diet was administered together with the cadmium, it provided to significantly lower metal accumulation compared to the cadmium group (p < 0.05). In ILp + Cd and IBc + Cd groups, the cadmium level of the livers were significantly decreased from 23.36 ± 2.27 to 5.44 ± 0.04 and 5.43 ± 0.27 μg/g, respectively (p < 0.05). A similar trend was also seen in the cadmium level of the kidney (Fig. 3).

Discussion
Heavy metal poisoning has become a major health concern in industrialized countries. Cadmium is one of the most toxic heavy metal and is so harmful that strategies must be devised to reduce its levels and hence toxicity. Various studies have reported that specific microorganisms such as LAB are able to absorb several metal ions and help to eliminate them [25]. In the present study, two probiotic bacteria (B. coagulans and L. plantarum) in combination with inulin were used to reduce the toxic effects of cadmium. Herein, we observed that two symbiotic diets could offer significant protection against cadmium toxicity in vivo by decreasing cadmium in the liver and kidney, and thus preventing alterations in the levels of SOD, ALT, AST, BUN and creatinine. There are reports presented that indicate B. longum 46 and L. fermentum ME3 can bind the toxic cathionic heavy metals, cadmium and lead from water [15]. Tian et al. [17] noted that L. plantarum CCFM8661 had the potency to alleviate the lead toxicity in mice. In addition to cadmium reduction, the protective effects on the antioxidant enzymes and oxidative stress were studied.
It was proposed that animals experiencing continuous exposure to heavy metals usually lose weight [21]. In this study we observed that cadmium administration resulted in remarkable reduction in body weight of rats but at first three weeks of experiment weight losses were significantly reversed by synbiotic treatment. Similar types of findings were observed by Horton et al. [26] and Tatara et al. [27].
The SOD is an important antioxidant defence mechanism that plays an extremely important role in the protection of all aerobic life-systems against oxygen toxicity. The main function of this enzyme is to accelerate the dismutation of the toxic superoxide produced during oxidative energy processes to hydrogen peroxide and molecular oxygen and reduces the oxidative stress. We realized that the synbiotic diets resulted in increasing the SOD enzyme against cadmium toxicity. AL-Hashem [28] reported significant reduction in SOD activity of rats that were exposed to aluminium chloride. Similar assumption was proposed by Tian et al. [17].
In the present study, despite the accumulation of cadmium in the liver (23.36 ± 2.27 μg/g) and kidney (16.77 ± 0.48 μg/g) of the rats which were taken 200 mg/day of CdCl 2 for 42 days, concentrations of the GPx and MD as major markers of oxidative stress were not raised compared to the control group.
Serum ALT and AST are the most commonly used biochemical markers of liver damage and are considered sensitive indicators of hepatic injury. The marked treatment increased the activities of serum ALT and AST induced liver injury due to metals exposure [29]. We perceived that the activities of serum ALT and AST increased due to cadmium toxicity. In contrast, in synbiotic groups, liver injury improved as a result of decreased serum ALT and AST levels. The report of Al-Wabel et al. [18] that utilization of synbiotic fermented milk containing Lactobacillus acidophilus and Bifidobacterium bifidum decreased serum ALT and AST levels in rats exposed to lead acetate is in agreement with our findings.
From the results, both AST and ALT values decreased in synbiotic treatment groups but AST was even lower than the control group. The baseline of AST activity is usually in a defined range due to hepatic and muscular cells death in control group [30]. It seems, the death trend of hepatic and muscular cells and the AST level were significantly reduced in the rats treated with synbiotics. This finding suggests the protective effect of synbiotic diet. To detect the renal damage the creatinine and BUN concentrations in serum were examined, as they are recognized as biomarkers of renal injury and the amplification of these biomarkers is usually correlated with impairment of renal function [31,32]. In the Cd group the values of creatinine and BUN increased but in the ILp + Cd and IBc + Cd groups their values decreased. Therefore our study showed that synbiotic treatment decreased elevated creatine and BUN level induced by cadmium exposure. Ulutas et al. [33] reported renal cell injury by cadmium, Cr resulted in elevation of serum urea and creatinine in metal treated rats compared to the negative control.
Our results showed that, although under continuous exposure to cadmium, the concentration of this heavy metal increased in the kidney and liver and that a synbiotic diet played a crucial role in the reduction of metal accumulation. Also, comparison between two probiotics showed both L. plantarum and B. coagulans had a major effect against cadmium toxicity and no significant differences were observed between them. Previous research has indicated that diet may influence heavy metal uptake and/or excretion [34]. Gram -positive bacteria, particularly Bacillus and LAB spp., have high adsorptive capacity of heavy metals due to high peptidoglycan and teichoic acid content in their cell walls [35,36]. Surface anionic groups in gram-positive bacteria are effective in the removal of heavy metals. Reduction of cadmium accumulation in the liver and kidney and increased levels in the stool of challenged groups indicated the ability of the probiotic bacteria to bind with cadmium and reduce the absorption of this heavy metal through the intestine by defecation. These observations are also in agreement with earlier studies by Tian et al. [17], Majlesi et al. [37] and Zhai et al. [38].