Lead is a heavy metal natural element with toxic properties, widely distributed in the environment, and still mined and added in some parts of the world to many different products including paints, eye cosmetics, and aviation fuel; it may also still be present in older water pipes. It is the cause of one of the most serious global health problems wherever communities are involved in activities such as lead smelting, gold mining, and lead battery manufacturing and recycling (28).
Lead is not an essential metal and has no biological role or function in the body, so it uses the transport proteins of other metals, such as calcium (Ca) and magnesium (Mg), to pass through the cell membrane. The membrane transport protein, known as divalent metal transporter 1 (DMT1) and divalent cation transporter 1 (7), is present in neurons and cerebral capillary endothelial cells and is thought to non-selectively transport divalent metal ions, including Mn2+, Fe2+, Co2+, Ni2+, and Cu2+ (27). Most of the effects of lead in neural tissue come from the substitution of Ca2+ and Mg2+ by this metal. The blood–brain barrier (BBB) has long been known as a target for lead toxicity. Under conditions of lead exposure, an increase in pinocytotic activity and changes in the characteristic endothelial tight junctions resulted in microvascular damage to it (30). Successfully crossing the BBB, inorganic compounds of lead accumulate significantly in the hippocampus and cause memory and learning disorders (17).
The hippocampus is the locus of synaptic routes thought to be the main ones for encoding new memories. A decrease in hippocampal long-term potentiation (a phenomenon of synaptic plasticity by which recent activity strengthens synapses in a persistent way) leads to impaired memory, whereas an increase in it correlates with improved memory and learning (12). Learning and memory are major functions of the brain that are affected by several factors, including some which are dietary and environmental (lead exposure being one), and some which are genetic. Many supplements on the market claim to improve these functions, through various metabolic approaches. One, vitamin C, has been found to be less abundant in individuals with impaired cognition (24). Another, magnesium L-threonate, is commonly used as a storage supplement to increase magnesium levels in plasma and cerebrospinal fluid (CSF) and has been suggested as having the capacity to improve short-term synaptic plasticity and long-term potentiation (21). These supplements are investigated for their supportiveness of cognitive functions after lead intoxication in the present study.
The accumulation of reactive oxygen species (ROS) is controlled
The two compounds vitamin C and magnesium L-threonate were assessed for their capacity to alleviate memory and learning deficiencies in a murine model intoxicated with the most bio-available form of lead, lead acetate.
Total catalase activity was determined spectrophotometrically with a UV-VIS 1240 Mini Spectrophotometer (Shimadzu, Kyoto, Japan) by following the decline in absorbance at 240nm as H2O2 (E = 36 M−1 cm−1) was catabolised according to the method of Nilsson (14). The accuracy and sensitivity of the method were estimated by measuring bovine catalase levels at various activity levels, from 3 to 450 units (IU).
Catalase activity levels were calculated as mean values per mg protein of the samples. In all organ samples, we observed a significant (P < 0.05 for liver, pancreas, and brain and P < 0.001 for kidney) difference in CAT activity between the control group and group Pb. Group Pb also showed a significantly increased CAT activity in liver, pancreas and kidney samples when compared with groups Pb+vit C, Pb+MgT and MgT. Brain catalase activity was increased in groups Pb+vit C and Pb+MgT compared with group Pb. The increased effect of lead acetate toxicity in the brain after treatment with vit C and MgT is shown by increased levels of catalase activity in the brain tissue compared with the group intoxicated with lead acetate and not administered supplements (Table 1).
Catalase activity level (CAT IU/ mg protein) in Swiss albino mouse liver, kidney, pancreas and brain tissue after administration of lead acetate
Group | ||||||
---|---|---|---|---|---|---|
Control (n = 10) | Pb (n = 10) | Pb+Vit C (n = 10) | Pb+MgT (n = 10) | MgT (n = 10) | ||
Organ | Liver | 222.5 ± 45.61 | 501.8 ± 70.10* | 229.3 ± 54.13 | 250.2 ± 81.52 | 204.9 ± 50.02 |
Pancreas | 18.5 ± 8.98 | 51.2 ± 16.12* | 31.3 ± 17.54 | 26.3 ± 13.22 | 37.3 ± 16.75 | |
Kidney | 97.3 ± 34.56 | 269.1 ± 55.89** | 124.6 ± 43.54 | 105.3 ± 52.23 | 130.5 ± 42.54 | |
Brain | 3.9 ± 2.86 | 8.1 ± 3.05* | 9.3 ± 3.67* | 8.9 ± 3.11* | 4.4 ± 2.26 |
Vit C – vitamin C; MgT – magnesium L-threonate; * P < 0.05, ** P < 0.001 compared to control
The daily measured body weight and water and food intake (Table 2) of the animals did not change significantly during the treatment.
Average values of body weight and food and water intake during the 40 days of treatment
Group | Initial average body weight (g) | Terminal average body weight (g) | Average daily food intake (g) | Average daily water intake (mL) |
---|---|---|---|---|
Control | 35.24 | 35.05 | 6.89 | 6.21 |
Pb | 33.34 | 36.97 | 7.01 | 6.07 |
Pb+Vit C | 32.24 | 35.69 | 6.51 | 6.11 |
Pb+MgT | 33.52 | 35.91 | 6.78 | 6.15 |
MgT | 34.04 | 37.12 | 7.12 | 6.52 |
Vit C – vitamin C; MgT – magnesium L-threonate
After dissection, the organs were weighed, and the results are presented in Table 3. After statistical calculation, there were no significant differences in organ weight.
Average organs weights in grams (liver, kidney, pancreas and brain) of mice (
Group | ||||||
---|---|---|---|---|---|---|
Control (n = 10) | Pb (n = 10) | Pb+Vit C (n = 10) | Pb+MgT (n = 10) | MgT (n = 10) | ||
Organ | Liver | 1.02 ± 0.29 | 0.89 ± 0.08 | 0.93 ± 0.16 | 0.92 ± 0.49 | 1.11 ± 0.26 |
Pancreas | 0.20 ± 0.10 | 0.17 ± 0.07 | 0.23 ± 0.07 | 0.15 ± 0.10 | 0.17 ± 0.05 | |
Kidney | 0.38 ± 0.16 | 0.50 ± 0.08 | 0.56 ± 0.12 | 0.55 ± 0.10 | 0.43 ± 0.10 | |
Brain | 0.35 ± 0.03 | 0.36 ± 0.05 | 0.35 ± 0.02 | 0.36 ± 0.02 | 0.36 ± 0.04 |
Vit C – vitamin C; MgT – magnesium L-threonate
We also measured the lead concentration in the brain. The concentration levels of lead in different groups of mice are shown in Table 4.
Lead concentration (mg/kg) in the brains of mice treated with different combinations of chemicals
Group | |||||
---|---|---|---|---|---|
Parameter | Control (n = 10) | Pb (n = 10) | Pb+Vit C (n = 10) | Pb+MgT (n = 10) | MgT (n = 10) |
Pb | 0.35 ± 0.001 | 1.58 ± 0.009* | 1.67 ± 0.039* | 0.94 ± 0.035* | 0.41 ± 0.025 |
Vit C – vitamin C; MgT – magnesium L-threonate; * P < 0.05 compared with control
Lead concentration in the brains of animals intoxicated with lead acetate was significantly higher than in the brains of control group animals. The highest levels of lead were found in group Pb+vit C. On the other hand, group Pb+MgT showed a significantly lower concentration of lead in the brain than groups Pb and Pb+vit C.
When we analysed correlations we found high correspondence between CAT activity in the brain and the number of entries into the platform zone and latency to the first entry into the platform zone (0.91 and 0.67, respectively). A very high correlation values (0.87) was found also between lead concentration in the brain and the memory parameters mentioned above.
Our research findings are consistent with studies by other authors. Catalase is one of the most important enzymes for antioxidant protection of cells. It is found mainly in hepatocytes and erythrocytes, but is present in almost all cell types in the animal body. Hepatocytes and erythrocytes were the cells in which the measured CAT activity was highest (10). When rats were treated
Brain CAT activity was also augmented after acute lead acetate administration in mice (2). Research on rats showed the effect of lead in relation to the activity of SOD, CAT, and other antioxidant enzymes important for the prevention of lipid peroxidation (22). Other authors (18) also reported increased levels of CAT activity in the livers, pancreas and kidneys of mice intoxicated with lead. The hepatotoxicity of heavy metals derives from their suppressive action on the levels of proteins and glutathione and facilitative effect on the synthesis of ROS species such as H2O2, OH− and O22−. These products can cause damage to the cell membrane by peroxidation of lipids (23). Vitamin C supplementation significantly decreased GSH content, and SOD and CAT enzyme activity in lead-intoxicated animals (5, 15). Other studies showed that treatment with vitamin C decreases the level of antioxidant enzymes (3).
Changes in the activity of antioxidant enzymes are one of the first indicators of intoxication of the organism. As shown by CAT activity, vitamin C had a protective effect in the groups intoxicated with lead, with the exception of brain tissue. These results can be explained by the antioxidant effect of vitamin C and action as a chelating agent for metals in intoxicated organisms. As our research showed, magnesium L-threonate, a substance used as a dietary supplement to improve cognitive abilities (21), did not affect other organs of the body adversely.
Vitamin C had no antioxidant or protective role in the brains of the lead acetate group mice (Table 1, Figs 1–4). This lack of effect was demonstrated by higher levels of CAT activity in the brains of animals in the vitamin C-treated group than in those of the mice in the lead acetate poisoned and unsupplemented group (Table 1). Moreover, the results of learning and memory tests showed that the treatment of lead-intoxicated animals with vitamin C did not improve these parameters, but on the contrary, worsened them. Treatment of lead-intoxicated animals with vitamin C increased the concentration of lead in the brain (Table 2). Vitamin C has the main role in DMT1 overexpression (16), and the substrate profile of DMT1 includes the metal cations Fe2+, Cd2+, Co2+, Mn2+, Ni2+, VO2+, and Pb2+. Divalent metal transporter 1, which is a cation membrane transporter, was involved in the transcellular transport of lead across the BBB (26). It is suggested that the increased protein transporters in the BBB are the mechanism for the increased lead concentrations in the brain tissue of the groups that were intoxicated with lead and treated with vitamin C.
The levels of lead concentration in the Pb+vit C group were significantly higher than in the Pb group, suggesting that vitamin C played no role in reducing the amount of lead in the brain. There is a possibility that magnesium played a role in the transport of lead to the intestinal wall. This may have led to this decrease in lead uptake and the lower concentration of lead in the brain in the MgT group.
The present results of learning and memory tests are consistent with the reports of other authors. The effects of lead in the impairment of hippocampal function and on nervous system function were also reported by other researchers (1, 17). Low levels of lead exposure caused a significant dose-dependent inhibition of proliferation of neural stem cells originating from the rat ventral mesencephalon and striatum and decreased the number of microtubule-associated protein 2-positive neurons differentiated from neural stem cells originating from these regions (8). Chronic lead exposure reduced neurogenesis in the dentate gyrus of the adult rat hippocampus (29). In this study, we found impairment of learning and memory in mice that were treated with lead acetate, lead acetate and vitamin C, and lead acetate and magnesium L-threonate. Learning and memory investigation in the group treated with magnesium L-threonate yielded results which were not significantly increased and which in this aspect were not as other authors reported (21).
The pharmaceutical supplements vitamin C and magnesium L-threonate played a significant detoxifying role (assessed by catalase activity) in all organs exposed to lead except the brain. Vitamin C and magnesium L-threonate have a large involvement in membrane transport of metallic ions, and contribute to intensifying the transportation of lead into the brain tissue (Table 4). The negative effects of vitamin C treatment on brain function with lead exposure are also supported by the results of learning and memory assessment. In the groups treated with lead acetate alone or lead acetate plus vitamin C, measurements showed decreased learning and memory abilities in mice. Moreover, besides the learning and memory parameters, the lead concentration parameter and CAT activity evaluation results in the brain followed the same trend.
Lead is one of the most dangerous heavy metal intoxicants of the body and especially the brain. Our findings for learning, memory and catalase activity show that even in small concentrations it has a great effect on the basic functions of the murine brain. Similar effects of lead were found in other organs (the liver, pancreas and kidney) where vitamin C as an antioxidant supplement had a detoxifying effect in lead-intoxicated animals. Although it is known as a detoxifying supplement, vitamin C had an adverse effect on lead neuro intoxication, increasing the concentration and toxicity of the heavy metal. A similar influence to that of vitamin C was also found for magnesium L-threonate. Further lead intoxication research will explain the molecular mechanism of these effects of vitamin C in the brain and to this end the continuation of research on the agency of vitamin C in the transport of lead through the BBB is worthwhile.