Effect of an Extract from Aronia melanocarpa L. Berries on the Body Status of Zinc and Copper under Chronic Exposure to Cadmium: An In Vivo Experimental Study

In an experimental model of low-level and moderate environmental human exposure to cadmium (Cd), it was investigated whether the consumption of a polyphenol-rich Aronia melanocarpa L. berries (chokeberries) extract (AE) may influence the body status of zinc (Zn) and copper (Cu). The bioelements’ apparent absorption, body retention, serum and tissue concentrations, total pool in internal organs, excretion, and the degree of binding to metallothionein were evaluated in female rats administered 0.1% aqueous AE or/and Cd in their diet (1 and 5 mg/kg) for 3–24 months. The consumption of AE alone had no influence on the body status of Zn and Cu. The extract administration at both levels of Cd treatment significantly (completely or partially) protected against most of the changes in the metabolism of Zn and Cu caused by this xenobiotic; however, it increased or decreased some of the Cd-unchanged indices of their body status. Based on the findings, it seems that rational amounts of chokeberry products may be included in the daily diet without the risk of destroying Zn and Cu metabolisms; however, their potential prophylactic use under exposure to Cd needs further study to exclude any unfavourable impact of these essential elements on the metabolism.


Copper 1
The administration of AE alone decreased the apparent absorption (AbsCu) and retention (RetCu) of copper (Cu) and increased its faecal excretion (FECu) after 10 months (Figures 7 and S1).
The administration of AE under 10-month exposure to the 1 mg Cd/kg diet declined the AbsCu and RetCu, unaffected by Cd alone, compared to the group treated with Cd alone and the control group, and it increased the FECu (Figures 7 and S1). Moreover, the co-administration of the 1 mg Cd/kg diet and AE increased the UECu compared to the control group after 17 months and compared to the control group and Cd1 group after 24 months ( Figure 7).

Copper 2
The administration of AE alone resulted in an increase in the heart and decrease in the serum, spleen and stomach concentration of Cu after 3 months, a decline in the duodenal tissue after 10 months, a decrease in the duodenal tissue and kidney, as well as an increase in the bone tissue at the femoral distal epiphysis after 17 months .
Compared to the control group, the administration of AE under exposure to the 1 mg Cd/kg diet declined this heavy metal-unaffected Cu concentration in the serum after 3 months and did not influence its increased serum concentration after 17 months ( Figure 11). The consumption of AE under the low exposure to Cd resulted in a decrease or increase in Cu concentration in some tissues (liver, spleen, brain, stomach, duodenum, femoral muscle, bone tissue) unchanged by Cd alone compared to the control group and/or the Cd1 group at some time points (Figures 8-10). The extract intake caused a decrease (bone tissue at the femoral distal epiphysis after 3 months, duodenum after 17 months; Figures 5 and 10) in Cd alone-unchanged Cu concentration in some tissues compared to the control group (in the case of duodenum after 17 months also compared to the Cd1 group), as well as in the case of femoral muscle (after 3 months) and brain (after 24 months) compared only to Cd1 group (Figures 9 and 10). Moreover, the co-administration of AE resulted in an increase in Cu concentration in the brain and stomach after 10 months (Figures 8 and 9), bone tissue at the femoral distal epiphysis after 17 months ( Figure 10) and spleen after 24 months ( Figure 8) compared to the control group (in the case of brain after 10 months, stomach and bone tissue at the femoral distal epiphysis also compared to Cd1 group), as well as in the case of brain after 3 months and liver after 17 months compared only to Cd1 group (Figures 8 and 9). The extract administration had no impact on the decreased stomach Cu concentration after 3 and 17 months of exposure to the 1 mg Cd/kg diet ( Figure 9).
The administration of AE to the animals fed with the 5 mg Cd/kg diet influenced the serum Cu concentration at all time-points ( Figure 11). Three-and 10-month intake of the extract decreased the Cd alone-unchanged Cu concentration in the serum compared to the control group and compared to the Cd5 group after the shorter co-administration. Seventeen-month consumption of the extract partially protected from this xenobiotic-caused increase in the serum concentration of this bioelement, while its 24-month intake increased the serum Cu level compared to the control group and Cd5 group ( Figure 11). The extract consumption under the moderate exposure to Cd increased, compared to the control group and Cd5 group, the Cd alone-unaffected Cu concentration in the heart after 3 months ( Figure 9) and duodenum after 24 months ( Figure 5). Moreover, the 24-month administration of the AE to the animals exposed to the 5 mg Cd/kg diet increased, but only compared to the Cd5 group, the Cd-unchanged Cu concentration in the spleen ( Figure 8) and heart ( Figure 9). The extract administration did not provide protection regarding the exposure to the 5 mg Cd/kg dietinduced decrease in Cu concentration in the spleen after 3 months (Figure 8), stomach after 3 and 17 months (Figure 9), duodenum after 17 months ( Figure 5), and bone tissue at the femoral distal epiphysis after 3 months (Figure 10), as well as the 3-month treatment-caused increase in this bioelement concentration in the bone tissue at the femoral diaphysis ( Figure 10).

Copper 3
The extract consumption decreased the Cd-unchanged sum of the content of this element in the liver and kidneys and its total pool after 3 and 24 months, compared to the control group and the Cd5 group, except for the total Cu pool in internal organs after 3 months, which was lower only compared to the Cd5 group (Figures 11 and S4). Moreover, the administration of AE under exposure to the 1 and/or 5 mg Cd/kg diet at some time points changed (increased or decreased) the content of Cu in some internal organs (liver, spleen and brain), unaffected by this toxic metal, in comparison to the control and/or appropriate Cd group (Figures 11, S4, and S5).  Table S2. Effect of the extract from the berries of Aronia melanocarpa (AE) on cadmium (Cd) concentration in the liver and kidney of rats exposed to this toxic metal. 1, 2.

Cd5 + AE Effect of Cd + AE Effect of AE Effect of Cd + AE Effect of AE 3 months Liver
1 The rats received 0.1% aqueous AE or not and Cd in diet at the concentration of 0, 1, and 5 mg/kg. 2 Detailed data on Cd concentration in the liver and kidney in all experimental groups have already been presented [7]. In this table only changes compared to the control group (↑, a factor of increase, *** p < 0.001), and the respective group that received Cd alone ( † p < 0.05, † † p < 0.01, † † † p < 0.001; ↘, a factor of decrease) are indicated. ↔, without change (p > 0.05) compared to the respective group treated with Cd alone. Cd concentration in the liver in the control group reached 0.035 ± 0.003 μg/g, 0.023 ± 0.001 μg/g, 0.014 ± 0.002 μg/g, and 0.014 ± 0.001 μg/g after 3, 10, 17, and 24 months, respectively, whereas its kidney concentration was 0.037 ± 0.003 μg/g, 0.050 ± 0.002 μg/g, 0.047 ± 0.003 μg/g, and 0.084 ± 0.013 μg/g, respectively. Table S3. The daily intake of zinc (Zn) and copper (Cu) with diet in particular experimental groups during the 5-day balance study. 1, 2, 3.

Main Effect of Cd
The rats received 0.1% aqueous AE and Cd in diet at the concentration of 5 mg/kg. 2 In the case when a one way-analysis of variance (Anova, Duncan's multiple range test) revealed any influence of the co-administration of Cd and AE on the investigated parameter, a two-way analysis of variance (Anova/Manova, test F) was conducted in aim to discern possible interactive and independent impact of Cd and AE on this parameter. The results of the Anova/Manova analysis are presented as F values and the level of statistical significance (p). F values having p < 0.05 were considered statistically significant ( * p < 0.05, ** p < 0.01, *** p < 0.001). NS -not statistically significant (p > 0.05). The rats received 0.1% aqueous AE and Cd in diet at the concentration of 1 or 5 mg/kg. 2 In the case when a one way-analysis of variance (Anova, Duncan's multiple range test) revealed any influence of the co-administration of Cd and AE on the investigated parameter, a two-way analysis of variance (Anova/Manova, test F) was conducted in aim to discern possible interactive and independent impact of Cd and AE on this parameter. The results of the Anova/Manova analysis are presented as F values and the level of statistical significance (p). F values having p < 0.05 were considered statistically significant ( * p < 0.05, ** p < 0.01, *** p < 0.001). NS-not statistically significant (p > 0.05).
---6.60 * 8.61 ** 4.57 * 1 The rats received 0.1% aqueous AE and Cd in diet at the concentration of 1 or 5 mg/kg. 2 In the case when a one way-analysis of variance (Anova, Duncan's multiple range test) revealed any influence of the co-administration of Cd and AE on the investigated parameter, a two-way analysis of variance (Anova/Manova, test F) was conducted in aim to discern possible interactive and independent impact of Cd and AE on this parameter. The results of the Anova/Manova analysis are presented as F values and the level of statistical significance (p). F values having p < 0.05 were considered statistically significant (* p < 0.05, ** p < 0.01, *** p < 0.001). NS -not statistically significant (p > 0.05). Table S9. Main and interactive effects of cadmium (Cd) and the extract from the berries of Aronia melanocarpa (AE) on copper (Cu) concentration in the serum and tissues of rats exposed to the 1 mg Cd/kg diet. 1, 2 . The rats received 0.1% aqueous AE and Cd in diet at the concentration of 1 mg/kg. 2 In the case when a one way-analysis of variance (Anova, Duncan's multiple range test) revealed any influence of the co-administration of Cd and AE on the investigated parameter, a two-way analysis of variance (Anova/Manova, test F) was conducted in aim to discern possible interactive and independent impact of Cd and AE on this parameter. The results of the Anova/Manova analysis are presented as F values and the level of statistical significance (p). F values having p < 0.05 were considered statistically significant ( * p < 0.05, ** p < 0.01, *** p < 0.001). NS -not statistically significant (p > 0.05). Table S10. Main and interactive effects of cadmium (Cd) and the extract from the berries of Aronia melanocarpa (AE) on copper (Cu) concentration in the serum and tissues of rats exposed to the 5 mg Cd/kg diet. 1, 2 .

Tissue Cu concentration
The rats received 0.1% aqueous AE and Cd in diet at the concentration of 1 mg/kg. 2 In the case when a one way-analysis of variance (Anova, Duncan's multiple range test) revealed any influence of the co-administration of Cd and AE on the investigated parameter, a two-way analysis of variance (Anova/Manova, test F) was conducted in aim to discern possible interactive and independent impact of Cd and AE on this parameter. The results of the Anova/Manova analysis are presented as F values and the level of statistical significance (p). F values having p < 0.05 were considered statistically significant ( * p < 0.05, ** p < 0.01, *** p < 0.001). NS -not statistically significant (p > 0.05).  The rats received 0.1% aqueous AE and Cd in diet at the concentration of 1 or 5 mg/kg. 2 In the case when a one way-analysis of variance (Anova, Duncan's multiple range test) revealed any influence of the co-administration of Cd and AE on the investigated parameter, a two-way analysis of variance (Anova/Manova, test F) was conducted in aim to discern possible interactive and independent impact of Cd and the AE on this parameter. The results of the Anova/Manova analysis are presented as F values and the level of statistical significance (p). F values having p < 0.05 were considered statistically significant (* p < 0.05, ** p < 0.01, *** p < 0.001). NS -not statistically significant (p > 0.05). Zn/ (MT x 7), the pool of MT-unbound Zn; Cu/(MT x 12), the pool of MT-unbound Cu; Cd/(MT x 7), the pool of MTunbound Cd; Me/(Me-MT), the pool of MT-unbound metals (Zn, Cu and Cd).    Figure S1. The body retention of zinc (RetZn) and copper (RetCu) in particular experimental groups. The rats received cadmium (Cd) in diet at the concentration of 0, 1, and 5 mg/kg and/or 0.1% extract from the berries of Aronia melanocarpa (AE; "+", received; "-", not received). Data represent mean ± SE for eight rats (except for seven animals in the AE, Cd1, and Cd5 groups after 24 months). Statistically significant differences (Anova, Duncan's multiple range test): * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control group; † † p < 0.01, † † † p < 0.001 vs. respective group receiving Cd alone; ‡ p < 0.05, ‡ ‡ p < 0.01, ‡ ‡ ‡ p < 0.001 vs. respective group receiving the 1 mg Cd/kg diet (alone or with AE) are marked. Numerical values in bars indicate percentage changes compared to the control group (↓, decrease; ↑, increase) or the respective group receiving Cd alone (↘, decrease; ↘, increase). Figure S2. Zinc (Zn) content in the kidney, liver, and heart in particular experimental groups. The rats received cadmium (Cd) in diet at the concentration of 0, 1, and 5 mg/kg and/or 0.1% extract from the berries of Aronia melanocarpa (AE; "+", received; "-", not received). Data represent mean ± SE for eight rats (except for seven animals in the AE, Cd1, and Cd5 groups after 24 months). Statistically significant differences (Anova, Duncan's multiple range test): * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control group; † p < 0.05, † † p < 0.01 vs. respective group receiving Cd alone; ‡ p < 0.05, ‡ ‡ p < 0.01, ‡ ‡ ‡ p < 0.001 vs. respective group receiving the 1 mg Cd/kg diet (alone or with AE) are marked. Numerical values in bars indicate percentage changes compared to the control group (↓, decrease; ↑, increase) or the respective group receiving Cd alone (↘, decrease; ↗, increase). Figure S3. Zinc (Zn) content in the spleen and brain in particular experimental groups. The rats received cadmium (Cd) in diet at the concentration of 0, 1, and 5 mg/kg and/or 0.1% extract from the berries of Aronia melanocarpa (AE; "+", received; "-", not received). Data represent mean ± SE for eight rats (except for seven animals in the AE, Cd1, and Cd5 groups after 24 months). Statistically significant differences (Anova, Duncan's multiple range test): * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control group; † p < 0.05 vs. respective group receiving Cd alone; ‡ p < 0.05 vs. respective group receiving the 1 mg Cd/kg diet (alone or with AE) are marked. Numerical values in bars indicate percentage changes compared to the control group (↑, increase) or the respective group receiving Cd alone (↘, decrease; ↗, increase). Figure S4. Copper (Cu) content in the kidney, liver, and heart in particular experimental groups. The rats received cadmium (Cd) in diet at the concentration of 0, 1, and 5 mg/kg and/or 0.1% extract from the berries of Aronia melanocarpa (AE; "+", received; "-", not received). Data represent mean ± SE for eight rats (except for seven animals in the AE, Cd1, and Cd5 groups after 24 months). Statistically significant differences (Anova, Duncan's multiple range test): * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control group; † p < 0.05, † † p < 0.01, † † † p < 0.001 vs. respective group receiving Cd alone; ‡ p < 0.05, ‡ ‡ p < 0.01, ‡ ‡ ‡ p < 0.001 vs. respective group receiving 1 mg Cd/kg diet (alone or with AE) are marked. Numerical values in bars indicate percentage changes compared to the control group (↓, decrease; ↑, increase) or the respective group receiving Cd alone (↘, decrease; ↗, increase). Figure S5. Copper (Cu) content in the spleen and brain in particular experimental groups. The rats received cadmium (Cd) in diet at the concentration of 0, 1, and 5 mg/kg and/or 0.1% extract from the berries of Aronia melanocarpa (AE; "+", received; "-", not received). Data represent mean ± SE for eight rats (except for seven animals in the AE, Cd1, and Cd5 groups after 24 months). Statistically significant differences (Anova, Duncan's multiple range test): * p < 0.05, ** p < 0.01 vs. control group; † p < 0.05, † † p < 0.01 vs. respective group receiving Cd alone; ‡ p < 0.05, ‡ ‡ ‡ p < 0.001 vs. respective group receiving 1 mg Cd/kg diet (alone or with AE) are marked. Numerical values in bars indicate percentage changes compared to the control group (↓, decrease; ↑, increase) or the respective group receiving Cd alone (↘, decrease; ↗, increase).