1. Introduction
The presence of toxic metals in food products has become a global problem. The most important source of toxic metals for man is food of plant origin, and in particular cereals [
1], mostly with regard to the fact that they are the basis of nourishment throughout the world and are consumed most abundantly. The half-life of Cd in soft tissues is between 25–30 years [
2], while that of Pb is about 30 days [
3]. In 2012, the European Food Safety Authority (EFSA) [
1,
4] reduced the tolerable intake level for Cd and Pb. The dietary Cd tolerable weekly intake (TWI) was estimated at 2.5 μg/kg of body weight/week [
4], whereas the benchmark dose lower confidence limit (BMDL) for Pb was: BMDL
01 1.5 μg and BMDL
10 0.63 μg/kg of body weight/day in adults, and BMDL
10 0.5 μg/kg of body weight/day in children [
1]. The environmental exposure of humans to cadmium is approximately 5 mg/kg of body weight/week, and to lead, it is approximately 35 mg/kg of body weight/week [
5]. Studies in rats have shown substantially higher accumulation of Cd and Pb in organs when these metals are administered in water than in food [
6]. The distribution of Pb and Cd, depends primarily on their ability to penetrate biological membranes and to some extent on the affinity with the specific structure of the organism [
7]. Resorption depends on the form of Pb and Cd, metabolic activity of the organism, age and physiology [
8,
9,
10,
11].
Pb and Cd can cause both morphological lesions and disorders of cardiac muscle (contractility, conduction), lungs and brain damage as a result of oxidative stress [
12,
13]. Cd and Pb, despite not actively participating in redox reactions, are indirectly conducive to oxidative stress, generated, among other things, by increasing the peroxidation of lipids and inhibiting the activity of antioxidant proteins through binding them with sulfhydryl groups and disturbing calcium homeostasis [
14]. Under the influence of heavy metals, the concentration of reactive forms of oxygen, which can result from the release of transitory metals from places in which they naturally occur within a cell increases [
15,
16]. The study by Jedlińska-Krakowska [
17] revealed that the hearts of rats receiving Cd, Pb and simultaneously Cd and Pb in the form of water drinking solutions had higher concentrations of malondialdehyde, which is one of the end products of polyunsaturated fatty acid peroxidation in cells and thus a marker of oxidative stress in humans and animals. In rats poisoned with Cd and Pb, reduced activity of antioxidant enzymes was found [
18], which can show that antioxidant mechanisms become exhausted as the poisoning progresses. Cytotoxicity of xenobiotics is limited by antioxidant enzymes such as SOD (superoxide dismutase) and CAT (catalase) [
14]. The use of exogenous antioxidants increases the total antioxidant capacity of the body and reduces the likelihood of inducing oxidative damage [
19,
20].
Since it is impossible to eliminate Pb and Cd from food, research is carried out on methods of limiting its absorption by the body. These methods should be easily accessible and uncomplicated [
21,
22,
23,
24,
25,
26,
27,
28,
29]. Foods containing significant amounts of antioxidant components can be used in the daily diet to prevent the dangerous effects of toxic metals on the human body [
30,
31,
32,
33,
34,
35,
36,
37,
38,
39,
40]. Tea deserves particular attention. The most frequently drunk varieties are black and green tea [
41,
42]. Tea satisfies the conditions for classification as a functional food. In addition, it was found that tea infusions enhance detoxification processes in rats by binding toxic metals and intensifying their excretion from the body [
15]. Tea contains a number of substances which are capable of chelating metals, such as, tannic acid (TA) [
43], catechins, quercetin and others polyphenols [
44]. Polyphenols have strong antioxidant properties [
45], they chelate prooxidant metals and they are known as the agents of the pro-oxidant/antioxidant balance [
15,
46]. Moreover, studies have demonstrated that 60 min after drinking green tea, human blood serum contains three times more catechins than after drinking black tea [
47]; thus, the antioxidant potential in blood serum after drinking green tea is increased by 34%, while after drinking black tea, the increase is 29% [
48]. Drinking green tea in an amount of four brewing sachets per day for 12 weeks increased antioxidative capacity of saliva by 42% [
49]. Daily intake of 3 to 5 cups of green tea could provide about 250 mg of catechins [
50].
Extensive research was carried out investigating the impact of Pb and Cd both in people and in animals, which enabled approximate determination of toxic activity mechanisms. However, this research has mostly involved a model of poisoning with large doses of metals, which accelerates negative effects that, in terms of their intensity, reveal a whole range of responses of the body both in terms of destruction and defense mechanisms. However, the results of this research do not illustrate the actual interaction in the human environment. It should be remembered that people are mostly exposed to these factors occurring in low concentrations and their effects are revealed only when irreversible impairment occurs. It seems extremely significant to know the response of the body to toxins (including toxic metals) at small doses, which would enable effective protection against further adverse consequences or increase the effectiveness of therapies used.
Tannins are more effective antioxidants than, for example, vitamin C and E [
51]. Tannic acid (TA) in in vitro conditions is a very efficient chelator for Pb [
52]. It is known from our previous studies that a 2% TA drinking solution given to adult rats exposed to Pb and Cd in an amount not exceeding 18.5 mg Pb and 2.6 mg Cd/kg body weight/week is an effective way to reduce Cd accumulation in the brain; however, TA may not be as effective for Pb [
53]. This study aimed to verify whether TA and tea administered to rats orally in the form of a water solution will have any influence on the adverse effect of Cd and Pb in adolescent rats chronically exposed to Pb and Cd in doses not exceeding the levels of environmental exposure of humans. Using a rat model, this study aimed to: (1) determine the level of TA, most effectively reducing the adverse impact of Pb and Cd on the lungs, hearts and brains of adolescent rats during combined exposure: the degree of reduction of the accumulation of Pb and Cd in the analyzed tissues and the activity of antioxidant enzymes (SOD and CAT) and (2) check whether administering of black, green, red and white tea solutions containing the specific level of TA would decrease the absorption of Pb and Cd and increase the activity of SOD and CAT in organs; this study assumed that TA is one of the crucial components of tea determining the preventive effect of tea in relation to Pb and Cd.
4. Discussion
In this study, the effectiveness of TA and teas was evaluated based on the percent of reduction in the accumulation of Cd and Pb in lungs, hearts and brains, compared with the Control group, as a result of the chelating properties of polyphenols. It should be taken into account that Experiment 1 (TA) and Experiment 2 (tea) in the present study were not conducted at the same time, although both rats and the experimental environment were similar in both experiments. However, since the effectiveness of TA solutions and teas was assessed based on the degree of reduction (%) of the Cd and Pb content in the organs compared to the control group (receiving distilled water to drink), where the value was assumed to be 100%, the results of the effectiveness in both experiments were determined as comparable.
There is little information in the available literature on the effect of TA and tea infusions on the accumulation of Cd and Pb in the tissues of laboratory animals. Most studies refer to green tea which supports the detoxification of the body by inhibiting the absorption of toxic metals and facilitating their excretion from the body [
15]. This is due to the chelating action of polyphenols, mainly catechins, including EGCG that is abundant in green tea, quercetin and TA [
15,
52,
56,
57,
58,
59]. This study showed the highest effectiveness of reducing the absorption of Cd and Pb in the case of white and green tea solution, while black tea was the least effective. This should be attributed to the fact that white and green tea (non-fermented) contains more polyphenols, including catechins, capable of chelating metallic elements, than in black (fermented) and red (partially fermented) tea [
60]. However, the protective effect of teas on the body against toxic metals must be considered in the context of the summative effect of various active substances present in infusions, as their effect is summative. Since non-fermented white and green tea is considerably richer in antioxidant substances than other teas, the most favorable results achieved in our presented studies are not surprising. For this reason, it can be assumed that the better results in our own study were obtained using tea solutions rather than isolated TA. Although, the concentration of TA in the experimental solutions was much higher than in the teas: 5000 mg/L in the 0.5%TA group—25,000 mg/L in the 2.5%TA group compared to 770–1130 mg/L in the groups receiving tea infusions.
Catechins, believed to be the most important antioxidants in tea, and in particular green tea, were thoroughly studied. However, the very strong antioxidant properties of TA is completely underestimated. TA is characterized by a higher antioxidant capacity than other polyphenols and this capacity is not lower than that of BHA, BHT and α-tocopherol [
61]. TA in in vitro conditions inhibits the peroxidation of lipids almost 98% at the concentration of 15 μg/mL, while standard antioxidants (e.g., BHA and α-tocopherol) have similar results at the concentration of 45 μg/mL. TA also demonstrates a comparable capability of chelating metals [
61]. TA, degraded in the gut by bacteria and enzymes, is absorbed from the alimentary tract in animals, it is found in blood plasma [
62] and can have a chelating effect on the toxic metals present in blood and internal organs [
57]. Thus, TA is apparently one of the crucial components of tea determining the preventive effect of tea in relation to Cd and Pb. Studies on rats showed that TA has an effect preventing the absorption of Cd and Pb [
34] by binding these toxic metals and thus inhibiting their absorption into tissues. Kim et al. [
63] demonstrated that the tissues of mice poisoned with Cd and given TA contained less Cd. They administered Cd (20 mg/kg) and/or TA (0.5 mg/mL, 1 mg/mL, or 2 mg/mL) water per os to mice for 4 weeks. In turn, Peaslee and Einhellig [
56] showed that TA effectively inhibited Pb absorption in mice fed a diet containing both TA and Pb. Pekdemir et al. [
52] demonstrated that TA was a very effective chelator of Cd and Pb in vitro. Similarly, our previous studies showed the effectiveness of TA and/or tea infusions in reducing the accumulation of Cd and/or Pb in the following rat tissues: lungs, brain, heart, liver, kidneys, blood and bones [
15,
64,
65,
66,
67].
In these studies, TA and tea efficiently reduced the degree of accumulation of Cd in tissues, and Pb was less susceptible. Another study on rats showed that Pb was a metal more resistant than Cd to being bound by TA [
30], where the use of TA solutions with the concentration from 0.5% to 2% in adult rats simultaneously exposed to Cd and Pb resulted in a statistically significant reduction of Cd absorption in brains but had no effect on the level of Pb. This could be due to the fact that Pb reveals a considerably stronger affinity with thiol groups [
62], which do not occur in polyphenolic compounds, than with hydroxyl groups present therein. It is noticeable that the effectiveness of TA and teas in relation to Cd and Pb was higher in the second experimental period than in the first one. The results obtained suggest that long-term continuing administration of polyphenols increases its effectiveness as a chelator for Cd and Pb. Moreover, it cannot be excluded that if the term of the experiment was longer (>12 weeks), the effects for Pb would be more explicit. Further investigation is required to clarify this problem, since available results obtained by other authors, who unfortunately are scarce, indicate that Pb is a metal poorly susceptible to the chelating effect of orally administered antioxidants.
Teas were the least effective in the case of lungs compared to other organs. The results referred to both Cd and Pb. This can mean that drinking tea will not considerably change, for example, the adverse impact of smoking tobacco that is one of the most important sources of Cd and Pb for humans and simultaneously the most important cause of lungs cancer [
68]. The polyphenols present in tea reveal strong anticarcinogenic properties [
69,
70], which in this case may not be fully utilized.
The system of endogenous antioxidant enzymes is supported by exogenous antioxidants. Supposedly, the decreased likelihood of a reaction between metals and critical biomolecules and inducing of oxidative damage is connected with the effect of antioxidant compounds contained in food or supplements [
15,
71]. The authors’ own research has demonstrated that the activity of SOD and CAT in the lungs, hearts and brains of the examined rats increased, which can provide evidence that TA and other polyphenols reduces the toxic effect of Pb and Cd on the activity of these metals. The highest increase in activity was recorded in rats receiving a 2% TA solution and green tea infusion. Polyphenols, including TA, have strong antioxidant properties [
61,
72]; they chelate prooxidant metals and they are known as the agents of the pro-oxidant/antioxidant balance [
15]. Research by Guliasar et al. [
73] demonstrated that the use of antioxidant substances (such as melatonin and taurine) significantly reduced the concentration of Cd in the brains and lungs of rats receiving a water drinking solution containing 200 μg/mL of Cd over 3 months. Significantly, the results for the heart were more favorable than for the brain, which is particularly important since the studies by the above-named authors revealed that the accumulation of Cd in hearts was several times higher than in brains. In turn, the authors’ own research shows that the organ most susceptible to the detoxicating effect of TA was the brain, where the accumulation of Cd was higher than in other tissues. The results of the authors’ own research and of the study by Guliasar et al. [
73] can evidence that substances chelating toxic metals, when administered orally, are more effective for higher concentrations of these metals in the tissues. In addition, based on the results of our previous studies, it can be supposed that drinks containing TA, for example tea, more effectively reduce the accumulation of Cd in tissues when drunk along with consuming food contaminated with Cd than when drunk in between meals [
34].
Exposing rats to Cd and Pb reduces the activity of antioxidant enzymes, which points to a decrease in the antioxidant potential of the body as a result of supplying factors enhancing cellular oxidation [
15,
71]. The present study showed an increase in the SOD and CAT activity in the lungs, brain and hearts of rats receiving tea, which was probably related to the enhancement of the antioxidant capacity of the organism through the supply of exogenous antioxidants. Our earlier research demonstrated an increase in the level of endogenous antioxidants only upon long-term exposure [
65,
66], which indicates that positive effects can only be achieved through the regular consumption of tea. El- Sayed et al. [
46] revealed that green tea extract administered to rats receiving Cd in the form of a water-based solution containing 0.4% CdCl
2 reduced the degree of peroxidation of lipids in testicles, thus preventing damage. According to Khalaf et al. [
74], green tea extract administered to rats poisoned with Pb in the amount of 100 mg/kg of body weight over 15 days increased the activity of antioxidant enzymes, including SOD, in the brain. Wei and Meng [
75] and Areba et al. [
76] revealed that in rats poisoned with Pb, the activity of antioxidant factors after the use of EGCG was increased.