INTRODUCTION:

Cardiovascular disease is the prominent contributor of deaths worldwide. A cohort study in 2013 discovered the relationship between increasing cadmium (Cd) levels in human body and cardiovascular disease¹. In Sweden, heart failures were also associated with low dose Cd exposure². An average Cd dose of 1. 53 µg/L in blood is potentially capable to cause myocardial heart disease and hypertension³. Cd is a toxic heavy metal substance that can easily contaminate human body and environment⁴.

This toxic metal substance enters the human body through inhalation or oral route then accumulated in human body⁵ and causes long term health problems^6,7.

Cd indirectly triggers reactive oxygen species (ROS) production by releasing active redox metal, which causes oxidative stress through the fenton reaction^8,9.

Vascular disorders are often initiated by endothelial dysfunction. This symptom is characterized with impaired endothelial cell homeostasis, decreased antioxidant levels, and increased antithrombotic and adhesion molecules. Typical signs of endothelial dysfunction are decreased levels of nitric oxide (NO)¹⁰ and increased lipid concentration¹¹.

Malondealdehyde (MDA) is used in this study as the indicator of the lipids peroxydation¹² which affected toward cell and membrant damages¹³ Cd toxicity due to ROS production and oxidant damage generates several problems, such as complex disorders of homeostasis of calcium (Ca), copper (Cu), and zinc (Zn), lysosom damage, lipid peroxydation, impaired mitochondrial function, abnormal DNA repair, and apoptosis¹⁴.

Vitamin C plays many roles in human body, such as metabolism, connective tissue, immune system, that can be used as prevention as well as therapy against diseases¹⁵. Vitamin C is an hydrophilic non enzymatic antioxidant that serves as a free radical scavenger¹⁶. Some studies provided the relationship between the effectiveness of vitamin C in chelating heavy metals substances. Vitamin C impedes the electrophysiology changes caused by lead induction¹⁷. Vitamin C also has the ability to improve mitochondrial function due to the arsenic induction¹⁸.

This study aimed to determine the effects of vitamin C towards the viability of endothelial cells, and levels of NO and MDA in CdCl₂-induced human umbilical vein endothelial cells (HUVEC).

MATERIAL AND METHODS:

Study Design:

The study used two control groups, non CdCl₂-induced HUVEC without vitamin C, and HUVEC induced with 24.154 µg/L CdCl₂, and three treatment groups CdCl₂-induced HUVECs supplemented with 50 µM, 100 µM, and 200 µM vitamin C, respectively. Treatments were performed for 48 hours. The result were indicated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromidefor (MTT) assay, NO, and MDA levels. All experiments were performed in 5 independent replications. This study was approved by Ethic Committee of Faculty of Medicine Brawijaya University (No.133A/ EC /KEPK-S2/04/2017).

Isolation and culture of Human Umbilical Vein Endhotel Cells (HUVECs):

The cord was taken from section caesarian (CS) mothers and babies meeting inclusion and exclusion criteria. The inclusion criteria were healthy mother with haemoglobin levels above 11, per vaginam or SC labour, and healthy infant (body weight >2,5 kg, Apgar score 7–9). Exclusion criteria included mother with hypertension, diabetes, cardiovascular disease, pre-eclampsia, and hyperlipidemia. Umbilical cord was incubated (37^OC ,8 minutes) in type 2 colagenase (Sigma) to isolate endothelial cells.

The cells were placed in culture medium containing 100 iu/ml M199 (Sigma), 100 µg/ml Penicillin, streptomycin (Gibco) and 10% FBS (Gibco 26140-087). The cells were cultured in 24- and 96-well plates containing 0.2% gelatin (Sigma G1393) and incubated at37^oC, 5% CO₂ environment (New Bruwstich Scientific).

The culture medium was changed every 48 hours. The cells turned to monolayer within 3-4 days. The 70% confluent cells were treated by replacing the culture medium and adding 24.154µg/L of CdCl₂(Sigma 28811-1ML-F) for CdCl₂group. In addition, three treatment groups were given Vitamin C (TCI) concentration 50µM, 100µM and 200µM respectively. For control groups, culture media was replaced without the addition of CdCl₂ and vitamin C. All groups were incubated for 48 hours.

Measurement Parameters:

MTT Assay:

Ten microliters of MTT were added to cells in 96-well plates and incubated for 4 hours until purple crystals appeared. Before being examined in microplate reader, detergent ((Trevigen 4890-25-K) was added, and purple precipitates were observed. The absorbance was measured at a wavelength of 570 nm (Bio-Rad Laboratories, USA).

NO Level:

NO levels were determined by harvesting the HUVEC culture medium and performing Griess reaction with NO KIT (Cayman). NO densities in each well were read at a wavelength of 550 nm using spectrophotometer (BMG Lab Tech, Germany).

MDA level:

The levels of MDA were determined using thiobarbituric acid (TBA) (Bioassay System) with calorimetric procedure. The harvested cells were sonicated and 10% of trichloroacetic acid (TCA) was added. Cells were incubated for 5 minutes on ice before being centrifuged at 14000 rpm for 5 minutes. Supernatant was collected in an Eppendorf tube. Standards were created according to manufacturer’s instructions. TBA was added on the samples and the standards and incubated in 100°C water baths for 60 minutes. Then, the samples were moved to microplates and read at 535 nm (BMG Lab Tech, Germany).

Data Analysis:

The data analysis was performed using IBM SPSS23 software (IBM Corporation, USA). Association of normal and homogenously distributed data was analyzed by one way ANOVA, followed by least significant difference (LSD) test. Association of non homogeneous and abnormally distributed data was analyzed using Kruskal Wallis, continued by Mann-Whitney test. The data normality was tested using Shapiro Wilk. Levine test was used for homogenity.

RESULTS:

The Effect of Vitamin C towards Cell Viability of CdCl₂-induced HUVEC

Figure 1.Viability measurement of Vitamin C’s Effect on HUVECs Induced with CdCl₂_..

Control (Without CdCl₂), CdCl₂ (24.154 µg/L), CdCl₂ + Vit C 50 µM, CdCl₂ + Vit C 100 µM, CdCl₂ + Vit C 200 µM. Data are presented as mean ± SEM. The treatmeant were conducted for 48 hours

The percentage of cell viability was decreased in the group with the exposure of 24.154 µg/L CdCl₂ (66,13 ± 46,89%) with no significant difference ( p < 0.05) compared to normal (100.00 ± 0.00%) (Figure 1). The addition of 100 and 200 µM of Vitamin C resulted in the significant increase of cell viability compared to positive control (195.16 ± 59.69% vs 148.39 ± 48.52, (p < 0.05 ).

The Effect of Vitamin C on NO levels of CdCl₂-induced HUVEC:

Figure 2. The Measurement of NO levels with the Effect of Vitamin C on HUVECs induced with CdCl₂.

Control (Without CdCl₂), CdCl₂ 24.154 µg/), CdCl₂ + Vit C 50 µM, CdCl₂ + Vit C 100 µM, CdCl₂ + Vit C 200 µM. The data are presented as mean ± SEM. Vitamin C increased the NO levels. The treatmeant were conducted for 48 hours

NO levels in the group receiving were decreased insignificantly compared to the normal or treatment group (Figure 2). NO levels in positive and negative control groups were not significantly different (8.413 ± 1.572 vs 5.433 ± 2.385). The addition of vitamin C on CdCl₂-induced HUVEC was able to improve NO levels compared to positive control with no significant difference (p < 0.05).

The Effect of Vitamin C on MDA levels of CdCl₂-induced HUVEC:

Figure 3. Measurements of MDA levels. The effect of Vitamin C on HUVECs induced with CdCl₂.

Control (without CdCl2), CdCl2 24.154 µg/L, CdCl₂+ Vit C 50 µM, CdCl₂ + Vit C 100 µM, CdCl₂ + Vit C 200 µM. The data are presented as mean ± SEM. The higher the concentration of Vitamin C, the lower MDA levels obtained. Yet, the MDA levels were increased again after additional higher concentration of Vitamin C. The treatmeant were conducted for 48 hours

MDA levels of the group receiving CdCl₂ induction were increased compared to normal or treatment group (Figure 3). The levels of controlled MDA were 0.42±0.13 and in the control group were 0.32±0.13 µM. Vitamin C 50 and 100 µM were able to reduce the MDA levels on the CdCl₂ induced HUVEC by0.28 ± 0.13 µM and 0.21 ± 0, 19 µM, respectively.

DISCUSSION:

Based on this study, the cell viability was decreased after 48 hours of CdCl₂ induction as well as the levels of NO, followed with the increasing levels of MDA. The induction of Cd caused increased in ROS through various mechanisms, such as through Fenton reaction, oxidative stress by releasing active redox metal from the cytoplasm and membrane proteins, and increased free iron ions¹⁹. Cd binding with semi-ubiquinon and cytochrome b mitochondrial complex III disrupts electron transport chain and forms the O₂^- radicals²⁰. Cellular gluthathione (GSH) are decreased and sulfhydryl protein binding are diminished due to Cd toxicity, which causes an imbalance of redox reactions. Thus, it increases the production of O₂^-, H₂O₂ and OH radicals. ²^1,²²

Vitamin C, chemically 2-oxo-L-threo-hexono-1,4- lactone-2,3-enediol, is an antioxidant and essential micronutrient which is needed by human body to prevent free radical ^23,24. Vitamin c can be found easily in food, specifically in fresh fruits such as Lemon, Guava, Grape etc^25,26,27 This substance roles as the cells protector by transfoming into singlet oxygen quencher, peroxyl radical scavenger^28. By releasing the hydrogen, Vitamin C can neutralize the free radical substance²⁹. It protects the cells from free radical both in single or combination therapy.^30,31

Cd Induction on cellular levels can disrupt proliferation, cell cycle, and even cause cell death³². Cell death due to Cd induction usually starts with the functional change of endothelial cells, which are characterized by increased endothelial permeability, leading to impaired endothelial integrity, which serves on vascular dysfunction³³. In accordance to previous studies^34,³⁵^,, our study also displayed that Cd induction decreases endothelial cells viability (Figure 1). This is in accordance with in vivo research toward mouse’s testical and liver which induced with cadmium cloride^36,37. Various mechanisms were used to uncover the causes of cell death due to Cd induction, including DNA damage²², ROS production ³⁸, increased of ceramides and calpain activation³⁹, mitochondria depolarization⁴⁰, and endoplasmic reticulum stress⁴¹. The treatment groups receiving 50, 100, and 200 µM vitamin C showed higher cell viability compared to control group (Figure 1). Therefore, it can be assumed that vitamin C supplementation can prevent Cd-induced cell death. A study conducted on Cd-induced mice testicles and given vitamin C pretreatment showed that the number of cell death is lower than those without vitamin C pretreatment⁴². Vitamin C has an unique characteristic. On low concentrations, it serves as antioxidants to prevent oxidation and inhibit apoptosis. On the other hand, on higher concentration, vitamin C on CdCl₂-induced HUVEC was shown to decrease cell viability. The authors hypothesized that high concentration of vitamin C has pro-oxidant properties, in line with a previous study⁴³, where vitamin C concentrations of 0.25 – 1 mM was able to induce apoptosis in acute myeloid leukemia (AML) cell line.

Blood vessel regulation is controlled by NO, which is produced by HUVEC through endothelial nitric oxide synthase (eNOS)⁴⁴. The eNOS inhibition by endothelial dysfunction can lead to endothelial dysfunction⁴⁵. The study showed that Cd induction led to increased levels of H₂O₂ and O₂, yet contrary to NO levels⁴⁶. Calsium channel were blocked by Cd induction, which in turn inactivates eNOS⁴⁷. In this study, CdCl₂ induction of 24.154 µg/L had lowered NO levels of HUVEC (Figure 2). Exposure to low dose Cd of 100 and 200 nmol/L caused a decrease in NO levels⁴⁸. Cd induction increases the O₂^- level, which acts as NO scavenger by forming peroxy nitrite. The additional of vitamin C at certain doses can decrease superoxide production. The possible mechanisms is that vitamin C functions as antioxidant, GSH maintenance, and is involved in redox balance⁴⁹. In our study, vitamin C pre-treatment on CdCl₂-induced HUVEC at concentrations of 50,100,200 µM caused increase of NO levels compared to control group (Figure 2). The addition of vitamin C can enhance eNOS production by preventing the formation of uncoupled eNOS, so peroxynitrite cannot be formed⁵⁰. Vitamin C enhances serine phosphorylation that activates eNOS by Akt or adenosine monophosphate-activated protein kinase (AMPK) upregulation and prevents protein phosphatase 2A (PP2A) by reducing dephosphorylation ⁵¹.

One of the oxidative stress manifestations is lipid peroxidation, where nucleic acids, lipids and proteins react with lipid peroxyl radicals and oxidation process occurs on the substrate. Cell membranes composed of poly unsaturated fatty acids are particularly susceptible to oxidative attack, which resulted in change in permeability, membrane fluidity, and cellular metabolic functions. Lipid peroxidation is formed through the interaction of sulfhydril (SH^-) from GSH caused by oxidative stress induced by cadmium⁵². In accordance with the study ⁵³^,⁵⁴, we found an increase on MDA levels on endothelial cells induced by Cd compared to normal group (Figure 3). In accordance to the study⁵⁵, the addition of 50 and 100 µM Vitamin C was able to decrease MDA levels of CdCl₂-induced HUVEC (Figure 3). This also agrees with in vivo research toward wistar rats’ cornea tissue which suffered oxidative stress caused by mobile phone radiation. Here, Vitamin C treatment can reduce MDA levels on the rats’ cornea⁵⁶. However, the vitamin C concentration of 200 µM increased MDA levels instead (Figure 3). It was possible, since at higher doses, because Vitamin C can induce H₂O₂, resulting in lipid peroxidation⁴³^,⁵⁷

Based on these results, we concluded that vitamin C in particular concentrations was able to increase cells viability and NO level, and decrease MDA levels on CdCl₂-induced HUVEC. This study implies the importance of the optimal dose of vitamin C supplementation for the prevention cardiovascular diseases.

ACKNOWLEDGEMENT:

The authors of this research would like to express our gratitude towards Permata Bunda Hospital for providing us the opportunity to take samples for this research. We are also very grateful to our technical assistants: Mr. Wahyuda Ngatiril Lady.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 10.11.2017 Modified on 24.12.2017

Research J. Pharm. and Tech. 2018; 11(3): 899-904.

DOI: 10.5958/0974-360X.2018.00166.X