3.3. Anti-Cd Toxicity of RG and Se-RG and Effect on Lipid Peroxidation
The kidney is the critical organ affected by chronic Cd exposure and toxicity. Cd accumulates in the kidney because of its preferential uptake by the receptor in the renal proximal tubule and accumulates in the human kidney for a relatively long time, from 20 to 30 years [27
]. The exposed level of Cd can cause chronic difficulties, thus leading to damage of kidney filtering mechanisms and kidney dysfunction [4
]. HEK293 cells, human embryonic kidney cells, were used as a demonstrative model to examine the effect of CdCl2
toxicity and the protective role of extracts against CdCl2
. The level of CdCl2
which could be used to induce the cytotoxicity was examined. Figure 2
showed that the viability of HEK293 cells was significantly decreased (p
< 0.05) while exposed to higher level of CdCl2
. Cd is not able to generate radical itself but the toxicity was related to the generation of reactive oxygen species (ROS) such as superoxide ions, hydrogen peroxides and hydroxyls radicals and therefore induced oxidative stress and DNA damage by initiation of the lipid peroxidation [6
].The estimated half maximal concentration (CC50
) dose of CdCl2
in HEK293 was indicated as 68.5 µmol/L and this level could be used as a suitable dose for the evaluation of anti-cadmium toxicity properties of the RG and Se-RG.
The experiment on anti-cadmium toxicity properties was designed to assess the effect of RG and Se-RG against CdCl2
exposure on the cell viability and lipid peroxidation at different time orders, as each substance may alleviate the toxicity from Cd induction differently [10
]. Results reveal that RG and Se-RG significantly increased (p
< 0.05) the percentage of cell viability during pre-treatment and co-treatment but not during post-treatment with Cd compared to the cells treated with Cd at CC50
level alone (Figure 3
The highest concentration of both extracts (10,000 µg/mL) exerted the highest ability to protect HEK293 cells against Cd toxicity. Thus, the pathological evaluation of cell morphology treated with the extracts at this concentration was observed as shown in Figure 4
. The morphological changes while treating the cells with Cd were detected. A majority of the cells were broken and floated into the media while the rest were weakened and lost their cell structure. Although the cells in the condition of extracts pre-treatment illustrated some lost and unusual cells morphology, the cells remained strengthened in their frame similarly to web shape. The changes of cells in co-treatment conditions were also detected as they were swollen and changed to a circle-like shape, but they preserved their structure. These data suggested that pre-treatment and co-treatment of RG and Se-RG with Cd could improve the Cd-induced pathological damage of kidney cells better than the Cd-treated and can potentially protect against kidney cell damage. Living organisms contain lipid as the main structure of cellular membranes. Cd could induce the damaging effects to the cells from the lipid peroxidation process [28
]. Therefore, the extent of lipid peroxidation by-products produced like malondialdehyde (MDA) can imitate the extent of cells oxidative damage initiated by Cd [29
]. TBARS assay is a well-established method use as an index of lipid peroxidation and lipid hydroperoxides. When the cells exposed to Cd, the MDA content was markedly increased, thus suggesting the increased in oxidative stress of kidney cells (Figure 5
). However, outcomes indicated that during pre-treatment and co-treatment of both RG and Se-RG, the level of MDA in HEK293 was significantly reduced compared to Cd-treated cells (p
The role of phenolic compounds in the extracts was considered as having the major effects on the protective role against Cd-induced damage. It could be explained that the extracts rich in polyphenols compounds possess the inhibition of lipid peroxidation chain reaction by stabilizing the hydroxyl radicals and lipid peroxyl radicals, thereby lowering the extent of oxidative damage to the lipid cell membrane and lower level of MDA [30
]. Moreover, phenolic compounds as antioxidant molecules could propose the role of upregulating the antioxidant protection system by stimulating the production of antioxidant enzymes including super oxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx). As a result, strengthening the immunity and lowering the damage caused by Cd during pre-treatment and co-treatment to the cells [10
]. RG and Se-RG contained abundant polyphenols such as flavone glycosides. Therefore, the protective role of the extracts could be related mainly to these groups of compounds. Similar results also indicated the protective effect of bioflavonoids, for example, quercetin against Cd-induced oxidative stress-related renal dysfunction in rats by attenuating the Cd-induced biochemical alterations in serum, urine and tissue pathological changes via a decrease in lipid peroxidation rate [32
]. Flavonoid, namely catechin from green tea, has also been proved to protect against bone metabolic disorders in cadmium-poisoned rats [33
]. While focusing on the effect of high Se, Se-RG extracts revealed marginally higher protective properties against Cd toxicity over the RG in pre-treatment and co-treatment conditions. Se was used as antidote agent to a range of heavy metal toxicities including Cd, Hg, and Ag [11
]. Generally, studies have indicated the beneficial effect of Se on antioxidant status and lipid peroxidation when pre-exposed and co-exposed to Cd [34
]. Lipid peroxidation occurred because of Cd exposure; moreover, a significant decrease in the antioxidant composition factors, such as glutathione (GSH) levels, the activities of glutathione peroxidase (GPx) and thioredoxin reductase (TrxR), was also stated [34
]. Se compounds have been generally known as a major cofactor of GPx and TrxR, thus, Se could logically promote the greater level of these antioxidant enzymes activity and play a role in managing the radicals occurring in the cells. Se could also present protective effects on mitochondria dysfunction by blocking the ROS generation, a possible inhibition of Cd-induced mitochondrial membrane collapse [37
3.4. DNA Protective Properties
Comet assay or single cell gel electrophoresis is a standard rapid method for detecting DNA damage in individual cells. The percentage of DNA in tail and tail length was analyzed as the measure of primary DNA damage [24
]. Therefore, comet assay is useful for this study to evaluate the DNA protective role of RG and Se-RG extracts against Cd. The highest concentration of both was used (10,000 µg/mL) as they protect the highest number of percent cell viability. Figure 6
illustrates the capture of comet cells of each treatment during pre-treatment, co-treatment, and post-treatment of RG and Se-RG compared to the Cd-treated and control. Figure 7
shows the parameters of the comet cells included % DNA in tail and tail length.
Comet cells of the control of every treatment displayed a circle-like shape in which the whole nuclei and DNA were beautifully stained with the fluorescent color. The condition of Cd-treated cells indicated the presence of a clouded comet tail in which the % DNA in tail and tail length were increased significantly (p < 0.05) because of Cd-induced the oxidative damage to cells. In both of pre-treatment and co-treatment conditions, the RG and Se-RG treated cells significantly exhibited the reduction in the % DNA in tail and tail length compared to the Cd-treated group (negative control), and thus illustrated a DNA protective effect. The results on comet assay parameters of each treatment were correlated to the content of MDA production.
The damaging of DNA could be a subsequent effect on the production of high ROS and lipid radicals induced by Cd. The role of flavone glycosides as a natural antioxidant in the RG and Se-RG may influence the protective property by possibly up-regulating the level of the antioxidant defense system and abolishing oxidative DNA damage via the donation of electrons to reactive metabolites and rendering them inactive to prevent the interaction to the DNA. The experiment on the protective role of flavonoid compounds as an excellent radical scavenger to reduce the DNA damage in human blood lymphocyte is also consistent with this result [38
]. Se-RG showed higher ability on the reduction in the tail length and % DNA in the tail of the comets compared to the RG. This indicated that Se in combination with the polyphenols could provide an extra protection and promote a protective role for the kidney cells. Se as the cofactor of various endogenous enzymes works in the antioxidant system could support the activity on the destructive of ROS. Fischer, et al. [39
] suggested another possible role of Se, especially in the organic form, of protecting DNA damage via induction of p53 DNA repair pathway and transactivation of p53-regulated effector genes.
In the post-treatment condition, although the addition of the RG and Se-RG showed no significant effect on an improvement in cell viability of HEK293 cells, a minor role of DNA protection can visibly be seen. In the Cd-treated condition, most DNA in cells were broken down as indicated by the size of the comet head being obviously decreased from the control. DNA fragments were spread into the surrounding area as detected from the blurred green background. However, the addition of the extracts could not save the cell viability from Cd exposure, and the intensive black background remained to be observed. This might indicate a slight reduction in the number of DNA fragments in the surrounding area as both RG and Se-RG indicated a reduction in % DNA in the tail of comets compared to Cd-treated cells. Moreover, the addition of Se-RG indicated significant reduction in the tail length (p < 0.05), and thus showed better DNA protective properties.