Introduction

Heavy metals are a group of elements naturally released into the environment by various processes. Recent developments and human industrial activities, including mining, smelting, and the creation of synthetic compounds, have resulted in increased levels of heavy metals being released into the environment (Monachese et al. 2012). The problem of heavy metals is of great significance from both health and environmental viewpoints. These metals have hazardous and toxic properties, because they are non-biodegradable (Bhakta et al. 2012). Water and food are mainly contaminated by heavy metals (Monachese et al. 2012). Oxidative stress and altered physiological and biochemical characteristics are the primary mechanisms for heavy metal toxicity which can cause concern (Çolak et al. 2011). Lead is a toxic metal whose widespread use has resulted in extensive environmental pollution and health problems in many parts of the world. This accumulative toxic metal affects different body systems, including neurological, hematological, gastrointestinal, cardiovascular, and renal systems. Neurotoxic effects of lead, particularly in children and even at relatively low levels of exposure, can in some cases result in irreversible neurological damage (ATSDR 2010; Canas et al. 2014).

Lead can have toxic effects during short-time exposure (acute toxicity) or prolonged exposure to lower doses (chronic toxicity) (WHO 2011). The biological half-life of lead in blood is about 35 days, while it is about 2 years in the brain and decades in bone (Xu et al. 2011).

According to FAO and WHO definitions, probiotics are live microorganisms that have positive effects on health when consumed in sufficient amounts. Two common strains used in food products are Lactobacillus and Bifidobacterium (Halttunen et al. 2007). Previous research has shown that acid lactic bacteria can bind to heavy metals such as lead, cadmium, and copper and are able to remove them in vitro (Xu et al. 2011; Ibrahim et al. 2006). According to recent studies, probiotic dairy products should contain at least 106–107 CFU/ml of these bacteria at the time of consumption (Medici et al. 2004; Meng et al. 2008).

Research in the area of heavy metal removal by probiotics is limited to the study by Zhai in which he investigated the protective effects of these microorganisms against cadmium toxicity (Zhai et al. 2013). No research has been conducted on chronic lead toxicity in vivo.

In the current study, the effects of two bacteria strains, Bifidobacterium lactis BB-12 and Lactobacillus acidophilus LA-5, on lead accumulation in rat brain when simultaneously and individually consumed for 4 weeks were investigated. Since the main target of lead toxicity is the brain (Kumar et al. 2013) and since this metal can cross the blood-brain barrier and cause variable changes in several neurotransmitter systems and pathologies in humans and animals (Ferlemi et al. 2014), studying the effects of probiotics on the removal of lead accumulation from the brain is of great importance.

Materials and methods

Experimental animals were 64 male Wistar rats weighing 150 to 180 g and were purchased from the Laboratory Animal Center of Tehran Pasteur Institute. They were then transferred to the laboratory animal depository of the pharmacy faculty of Zanjan University of Medical Sciences. The animals were kept in cages equipped with water and food supplies and kept at 22 ± 2 °C with a humidity of 55 ± 5% and a 12-h on-and-off cycle during adaptation periods and experiments. All rats had free access to water and food throughout the experiment.

After a 1-week adaptation period, rats were divided randomly into eight different groups, and further experiments, categorized below, were performed for 4 weeks.

Experimental animals

  1. Group 1:

    Rats received a standard diet and safe drinking water.

  2. Group 2:

    Rats received a standard diet together with a solution of L. acidophilus and B. lactis along with safe drinking water.

  3. Group 3:

    Rats received a standard diet together with skim milk solution and safe drinking water.

  4. Group 4:

    Rats received a standard diet together with lead acetate solution.

  5. Group 5:

    Rats received a standard diet together with L. acidophilus solution and lead acetate solution.

  6. Group 6:

    Rats received a standard diet together with B. lactis solution and lead acetate solution.

  7. Group 7:

    Rats received a standard diet together with L. acidophilus and B. lactis along with lead acetate solution.

  8. Group 8:

    Rats received a standard diet together with skim milk solution and lead acetate solution.

Source of bacteria

Freeze-dried culture powder of L. acidophilus LA-5 and B. lactis BB-12 was bought from Pishgaman Pakhsh Sedigh Company (Tehran, Iran) branch of Hanssen DVS Company, Denmark.

Preparation of bacteria

According to mentioned studies, the bacteria solutions used in this study were prepared. 0.01 g freeze-dried culture powder of the mentioned bacteria (L. acidophilus LA-5 and B. lactis BB-12: Hanssen DVS, Denmark), usually used directly in the preparation of probiotic products and containing over 1011 CFU/g microorganism (Saarela et al. 2000), was dissolved in 1 ml normal saline solution and fed to the probiotic-receiving groups each day of the experiment.

Complete microbial counts (CFU/g) were taken from the bacteria powder-containing packages at the beginning of the first week, end of the second week, and end of the fourth week by culturing on MRS agar (de Man Rogosa Sharpe agar) at an incubation temperature of 37 °C for 72 h under anaerobic conditions. The obtained average was considered as the final colony-forming units per gram for each bacterium.

Since the freeze-dried powder of probiotic bacteria contains skim milk as its matrix, to remove the potential effect of this substance in the 8th group, rats received 0.01 g skim milk (Quelab, Canada) per day in a 1-cc normal saline solution prior to the gavaging of the lead acetate solution. In the skim milk control group, skim milk was also gavaged to rats.

Lead acetate solutions

In the group exposed to lead, 1 cc distilled water containing 100 ppm lead acetate (Merck, Germany) was gavaged to each rat on a daily basis as per a relevant study (Nwokocha et al. 2012). Lead was gavaged 1 hour after the bacteria solution or skim milk.

Sampling and measuring the amount of lead in the brain

At the end of the second week, the brains of three rats from each group were excised under anesthesia for sampling. The brains of all remaining rats were excised under anesthesia at the end of the fourth week. According to the digester device instructions (Sineo-MDS-10, China), 0.2 g of each sample brain was added to 8 ml HNO3 65% and 1 ml H2O2 35% in the cell device. Digestion was done during three heat stages according to the specifications listed in Table 1. These digested samples were kept refrigerated in closed test tubes until being injected into the polarography instrument and measured.

Table 1 Time, temperature, and power used in the digestion
Full size table

Device specifications

In this study, lead concentrations were measured using a polarography instrument (Metrohm 797, Sweis), according to application bulletin no. V86. Samples were prepared according to the device instructions, and their lead concentrations were measured three times via the ASV (Anodic Stripping Voltammetry) method. The working electrode was the hanging mercury drop electrode (HMDE), stirring rate (stirrer/RDE) was 2000 rpm, aeration time by nitrogen for deoxygenation of solution (purge time) was 300 s, deposition potential was −1.5 V and deposition time was 90 s, and start and end potentials were −1.5 and 0.5 V, respectively, where lead oxide potential was −0.38 V. It should be noted that the detectable range of lead in the polarography device was 0.1 μg/l–50 mg/l.

Cellular values of bacteria solutions

The results obtained from total microbial count (CFU/g) of bacteria powder used at three culture times are provided in Table 2.

Table 2 Values of bacteria used in this study
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Since 0.01 g bacteria powder was used for solution preparation, each of the rats in the Lactobacillus-receiving group received 5.3 × 109 CFU/ml and the B. lactis-receiving group received 3.1 × 1010 CFU/ml (these values are in accordance with standard values used in probiotics), and the group receiving a combination of the two bacteria received the sum of these two values at the same time.

Statistical analysis

The results were statistically analyzed in SPSS 11.5. Descriptive results were expressed as mean, median, and standard deviation. If data was normal, analysis of variance (ANOVA) (and post-hoc methods) or t test was used; otherwise, non-parametric methods such as the Mann-Whitney or Kruskal-Wallis were applied. Statistical significance was considered lower than 0.05.

Measurement of lead in brain samples

Table 3 shows the results of lead measurements in the brain samples of all groups at two sampling times together with the p values gained from comparing similar group data. The number of samples at the second week was three in each group and at the fourth week was five (except for the group that received only lead, which had two losses). Statistical analysis of this table revealed that the amount of lead in the group exposed to lead had no significant increase compared with control groups at both sampling times (p < 0.05). Comparing the control groups at two sampling times showed no reduction in the lead concentrations of the probiotic group at the fourth week compared with the second week (p < 0.05).

Table 3 Results of lead measurements in the brain samples of all groups
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At the second week (Fig. 1), the lowest amount of lead was seen in the Lactobacillus and lead group. Similarly, there was no significant difference in lead reduction between the group receiving Lactobacillus and the skim milk group (p < 0.05).

Fig. 1
figure 1

Results of lead measurements in the brain samples of all groups. Comparative lead levels between groups and in second and fourth weeks. Values are mean ± SEM values. Normal: normal control group, La+Bb: control group of combination LA-5 and BB-12, Sk: skim milk control group, Pb: Pb only, La+Pb: LA-5with Pb, Bi+Pb: BB-12 with Pb, La+Bi+Pb: combination LA-5 and BB-12 with Pb, Sk+Pb: skim milk with Pb (p < 0.05). Common letters indicate a significant difference among groups (p < 0.05)

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Results at the fourth week (Fig. 1) revealed that the groups receiving skim milk and Bifidobacterium had the highest concentrations of lead. The lowest amounts belonged to the group receiving both types of probiotics simultaneously and the Lactobacillus group. Significant drops in the amount of lead were seen in the group receiving skim milk and the group receiving both types of probiotics (p < 0.05).

Figure 1 present the lead-exposed and control groups at two sampling times. As can be observed in Fig. 1, lead concentrations were highest in the groups receiving skim milk and the Bifidobacterium group, while the lowest value was related to the group receiving both types of probiotics concurrently with Lactobacillus. There was a significant difference between lead concentrations in the group receiving skim milk and those in the group receiving both types of probiotics (p < 0.05).

No significant difference was observed between the amount of lead in the skim milk group and that in the lead group. Therefore, it is possible to confirm the lack of influence of skim milk in bacteria lyophilized powder on the amount of lead accumulation in this study.

Discussion

The present study is among only a few that address the effects of probiotic bacteria on heavy metal accumulation in vivo. It has dealt with the impact of two common strains of probiotic bacteria commonly used in the food industry as additives, L. acidophilus and B. lactis (Hathout et al. 2011), on the chronic accumulation of lead in brain tissue. The data investigation at the end of the study revealed that lead concentrations in the brains of rats from all three probiotic groups exposed to lead were lower than those of the lead alone group. This decrease, however, did not prove to be significant. The results of the present study regarding the effect of B. lactis on the reduction of lead accumulation in the brain are consistent with those of Zhai et al., who indicated that probiotic intake, particularly that of the L. plantarum strain, results in reduced heavymetal concentration in tissues (Zhai et al. 2013). The current study demonstrated that, at the second week, the L. acidophilus strain caused a significant decrease in brain lead levels in groups exposed to this metal.

Among the various methods applied to diminish lead toxicity, probiotic bacteria have attracted a great deal of attention in recent years, because they are part of the normal flora in the gastrointestinal tract. Furthermore, they have been classified as Generally Recognized as Safe (GRAS) (Kinoshita et al. 2013). Various studies have shown that probiotics, such as lactic acid bacteria and bifidobacteria, are effective agents that bind to toxic metals such as cadmium in vitro (Bhakta et al. 2012; Halttunen et al. 2007; Kinoshita et al. 2013); thus, they can be a suitable candidate for the biological removal of these toxins.

Some studies have proposed different mechanisms for the process of metal absorption in vitro, e.g., complex formation, ion exchange, absorption, flocculation, and particle deposition (Halttunen et al. 2007; Ibrahim et al. 2006).

The outer layer of lactic acid in the studied bacteria, similar to other gram-positive bacteria, consists of a thick layer of peptidoglycan, (lipo) teichoic acid, protein, and polysaccharide. These structures contain differently charged groups, such as the carboxyl, hydroxyl, and phosphate groups. Accordingly, these bacteria have numerous bonds with the capacity to connect to cation ions such as cadmium and lead. It has been further indicated that gram-positive bacteria have a negative electric charge around themselves, possibly accounting for the superficial binding of these bacteria to positive ions (Halttunen et al. 2007; Teemu et al. 2008).

Investigation of the second week also revealed that the amount of brain lead in each of the three probiotic groups exposed to lead was lower than that in the lead-alone group. The Lactobacillus group experienced the lowest amount of lead among the probiotic groups with a statistically significant difference (p < 0.05).

Halttunen et al. (2007) demonstrated that the trend of cadmium and lead absorption by the two strains of L. fermentum and B. longum in vitro together with other environmental factors, particularly in the presence of metal cations such as zinc (Zn), iron (Fe), calcium (Ca), and magnesium (Mg), is disrupted and can significantly decrease the binding capacity of metal ions to bacteria. This process has been attributed to the competition between these metals for bacteria binding positions. Halttunen also stated that, in addition to reversibility, washing masses consisting of bacteria and metal using distilled water, diluted acid solution, or diluted EDTA solution (as a furcator) can easily destruct bindings between this bacterium and metals. The capacity of bacteria to reabsorb metal also decreases significantly (Halttunen et al. 2007). Several studies suggested that metal binding and removal by bacteria are dependent on pH, which mostly increase linearly when pH grows from 4 to 7 (Zhai et al. 2013; Ibrahim et al. 2006). Lower pH values and the presence of other ions in the digestive system may have disrupted the performance of probiotics to reduce metal tissue aggregation in vivo and may account for their low effectiveness in the present study. The time interval between lead and bacteria binding up until its discharge from the digestive system might also provide the opportunity required for various factors such as those mentioned in the study by Halttunen et al. (2007) and result in binding removal.

According to Zhai and the current study, the precedence of bacteria solution intake over metal intake can significantly reduce the effectiveness of probiotics in binding to lead, because the ability of bacteria to bind and colonize in the digestive tract within the interval developed until the exposure to lead can reduce the number of free surfaces for binding and removing this metal in the body (Zhai et al. 2013). In this study, mice received probiotic bacteria regularly for a long time and were exposed to heavy metal (lead) on a daily basis. These conditions are similar to typical probiotic intake from food and human exposure to lead. This method can provide sufficient time for the probiotics to colonize in the digestive system. It can be concluded that this method is more closely similar to the natural trend of human exposure.

The lack of statistical significance in brain lead levels in the Bifidobacterium group after 4 weeks can be attributed to the fact that, regarding the lack of sufficient in vitro evidence, this bacterium might be loosely bound to lead or might need a longer intervention to have an effect on brain lead concentration.

In comparing the control groups (groups with no lead intake) with the lead group, it was observed that at the second week, the brain lead concentration was lower in the normal group than the other groups. It could be that the rats of this study were mature animals by the fourth week, and the possibility of lead absorption at earlier ages highlights the fact that aging is one of the important factors in reduced lead absorption (Collins et al. 1982; Toscano and Guilarte 2005).

The results of this study demonstrate that the process of lead removal by probiotics in in vivo digestive systems might be disrupted in some ways and is influenced by various factors such as bacterial strain or certain digestive system conditions, thereby reducing the role of these bacteria in diminishing lead accumulation in the brain. In general, considering the enhanced effect in the Lactobacillus-receiving group and the poor performance of the other groups together with useful physiological effects gained by consuming these bacteria, it appears that the application of Lactobacillus in the production of probiotic products can be useful in reducing damage caused by lead exposure.