|Year : 2018 | Volume
| Issue : 1 | Page : 12-18
Vitamin B12 insufficiency in excess folic acid downregulates methylenetetrahydrofolate reductase gene and increases homocysteine, tumor necrosis factor-alpha, and oxidative stress in hepatocytes
Tejas Shah1, Kalpana Joshi2, Sanjay Mishra1
1 Dr. Prabhakar Kore Basic Science Research Centre, KLE Academy of Higher Education and Research, KLE University, Belagavi, Karnataka, India
2 Department of Biotechnology, Sinhgad College of Engineering, SP Pune University, Vadgaon Budruk, Pune, Maharashtra, India
|Date of Web Publication||17-Jan-2018|
Dr. Sanjay Mishra
Dr. Prabhakar Kore Basic Science Research Centre, KLE University, Nehru Nagar, Belagavi - 590 010, Karnataka
Source of Support: None, Conflict of Interest: None
OBJECTIVE: Folate and Vitamin B12 (B12) are crucial for normal growth, development, and cellular functions. The aim of this study was to determine the effect of required and excess folic acid (FA) concentration and imbalance of FA and B12 caused due to folate fortification on hepatocytes health and function in vitro.
MATERIALS AND METHODS: Human hepatocellular carcinoma (HepG2) cells were cultured in FA and B12 deficient media for 15 days and further were supplemented with required FA: (6 μM) and excess FA: (60 μM) individually and in combination with B12: (500 nM). We assessed HepG2 proliferation, viability, tumor necrosis factor-alpha (TNFα) and methylenetetrahydrofolate reductase (MTHFR) mRNA expression, homocysteine, and malondialdehyde levels.
RESULTS: Supplementation of B12 with FA increased proliferation as compared to only FA supplemented group. FA and B12 deficient cells and excess FA supplementation resulted in decreased viability. Supplementation of B12 at both FA concentrations increased (P < 0.05) cell viability. In the presence of B12 oxidative stress, TNFα mRNA and TNFα levels in cell lysate significantly decreased (P < 0.05) in both FA supplemented cells as compared to excess FA cells. Homocysteine levels significantly decreased (P < 0.05) in cells supplemented with normal FA + B12 as compared to excess FA supplemented cells and FA and B12 deficient cells. Supplementation of excess FA significantly downregulated (P < 0.05) MTHFR mRNA levels as compared to required FA cells. Combination of FA + B12 significantly upregulated (P < 0.01) MTHFR mRNA levels as compared to excess FA.
CONCLUSION: These results accord the promoting effect of B12 with FA in developing nations like India where B12 deficiency is common while the concentration of folate is adequate.
Keywords: Folate, homocysteine, human hepatocellular carcinoma, methylenetetrahydrofolate reductase, tumor necrosis factor-alpha, Vitamin B12
|How to cite this article:|
Shah T, Joshi K, Mishra S. Vitamin B12 insufficiency in excess folic acid downregulates methylenetetrahydrofolate reductase gene and increases homocysteine, tumor necrosis factor-alpha, and oxidative stress in hepatocytes. Indian J Health Sci Biomed Res 2018;11:12-8
|How to cite this URL:|
Shah T, Joshi K, Mishra S. Vitamin B12 insufficiency in excess folic acid downregulates methylenetetrahydrofolate reductase gene and increases homocysteine, tumor necrosis factor-alpha, and oxidative stress in hepatocytes. Indian J Health Sci Biomed Res [serial online] 2018 [cited 2020 Jun 4];11:12-8. Available from: http://www.ijournalhs.org/text.asp?2018/11/1/12/223419
| Introduction|| |
Vitamin B12 (B12) and folic acid (FA) plays a vital physiological role in one carbon metabolism. B12 acts as a coenzyme to various reactions in metabolism: One carbon related substrate metabolism, synthesis, and stability of nucleic acids and DNA methylation which in turn modulates gene expressions. B12 deficiency is though common in India due to vegetarian diet pattern and poor consumption of dairy products is overlooked.Report states, among Indian males with different socioeconomic status B12 deficiency rates were 81% in urban middle class, 68% in rural, and 51% of males in slum region. Many developing nations like India have adopted FA fortification program to address the required 400 mg of folate, although in India strength of folate available is 1 mg, 2.5 mg, and 5 mg. There is an ongoing debate on possible adverse outcomes such as cardiovascular diseases, risk of cancer, adverse pregnancy outcomes, and masking of B12 deficiency due to folate fortification., Studies in animals and humans provide insight that excess FA supplementation may be associated with adverse outcomes, although limited work has been carried out to determine whether an exposure of excess FA and imbalance of FA and B12 has any pathophysiological consequences.
Tumor necrosis factor-alpha (TNFα) resembles as an important cytokine in the progression of various liver diseases and injuries. Previously, synergistic elevation of cytokines with oxidative stresses and lipid peroxides has been related to nonalcoholic fatty liver disease. The oxidative stresses exacerbate hepatic impairment through induction of irreversible lipids alteration, mutations in proteins and DNA contents and thereby regulating pathways that control protein expression, genes expression, cell apoptosis, and perisinusoidal cells/hepatic stellate cells activation. Oxidative stress is considered to be an important pathophysiological mechanism in initiation and progression of hepatic diseases and injuries.
Homocysteine rises with either folate or B12 deficiency because folate and B12 are looped within the metabolism of methionine and homocysteine. Methylenetetrahydrofolate reductase (MTHFR) enzyme plays a vital role in the regulation of 5-methyl-tetrahydrofolate for remethylation of homocysteine. Disturbed remethylation is caused by deficiency in MTHFR due to mutations in their genes and result in hyperhomocysteinemia or homocystinuria. Selhub et al. associated lower serum B12 concentrations and high plasma folate with elevated homocysteine levels in humans, a possible biochemical inference for excess FA intake exacerbating the clinical exhibition of B12 deficiency.
Authors further concluded that these observations provide a possible biochemical explanation for excess FA intake exacerbation of the clinical manifestations of Vitamin B12 deficiency.
Thus, the aim of the present study was to determine whether excess FA exposure and imbalance in FA and B12 would negatively impact primary indicators of hepatocyte health and function. Using human hepatocellular carcinoma (HepG2) cells, the effect of normal and excess FA and in combination with B12 was assessed on cell proliferation, viability, TNFα levels in cell lysate, TNFα mRNA levels, oxidative stress, homocysteine, and MTHFR mRNA levels.
| Materials and Methods|| |
Chemicals and reagents
Dulbecco's modified Eagle's medium (DMEM-D2429), 3-(4, 5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT), Trizol, FA, and Vitamin B12 (B12) were purchased from Sigma–Aldrich (St. Louis, MO, USA). Fetal bovine serum (FBS), antibiotic– antimycotic 100X solution was procured from Gibco and ThermoScientific, respectively (United States of America). RNeasy mini kit for isolation of total RNA was purchased from Qiagen (Hilden, Germany). High capacity cDNA kit and SYBR Green was procured from ThermoScientific (Massachusetts, United States of America). Analytical grade reagents were used for experimental purpose.
HepG2 cells were obtained from National Centre for Cell Science Pune, India, and were cultured in DMEM medium with 10% FBS and 1% antibiotic-antimycotic solution under an atmosphere of 5% CO2 at 37°C with 95% humidity. Cells were passaged for every 3–4 days with fresh media for 15 days to deprive them of folate completely. Cells were serum starved overnight in serum free media, before any experimental procedures. Concentration for FA and B12 were chosen based on the previous study and were prepared by diluting in distilled water. The five experimental treatments were as follows: (1) Untreated cells (FA and B12 deficient), (2) normal FA (FA: 6 μM), (3) excess FA (FA: 60 μM), (4) normal FA + normal B12 (FA: 6 μM + B12: 500 nM), and (5) excess FA + normal B12 (FA: 60 μM + B12: 500 nM).
Cytotoxicity and cell proliferation study
HepG2 cells were plated in 96-multiwell culture plates at 1 × 105 cells per well. To study cytotoxicity and proliferation, 24 h after cell adhesion, the medium was discarded, and media as per experimental treatments was added. After 48 h, the cells in well were washed with phosphate buffer solution (PBS) and 20 μL of the MTT staining solution (5 mg/mL in PBS) was added and the plate was incubated at 37°C. After 4 h, 100 mL of di-methyl sulfoxide (DMSO) was added in wells to dissolve the formazan crystals, and absorbance was recorded at 492 nm using enzyme-linked immunosorbent assay (ELISA) plate reader.
HepG2 cells viability was assessed using a trypan blue dye exclusion assay. Cells were seeded (5 × 105 cells/well) in a 24 well plate and incubated for 48 h with the experimental treatments. Cells were harvested and assessed using hemocytometer (Rohem, India). Cell viability was measured as the number of viable cells divided by the total number of cells expressed as a percentage.
HepG2 cells 5 × 105 were seeded and incubated for 48 h with experimental treatments. The anti-inflammatory cytokine was measured using TNFα ELISA kit in cell lysates (Krishgen Biosystems, Mumbai, India) as per manufacturers instructions.
Measurement of lipid peroxidation
HepG2 cells 5 × 105 were seeded and incubated for 48 h with experimental treatments. Cells were harvested and suspended in 500 μl PBS. The extent of lipid peroxidation was calculated by the levels of malondialdehyde (MDA) measured using the thiobarbituric acid reactive substances assay at 535 nm on microplate reader as previously described.
Measurement of homocysteine levels
Homocysteine levels were estimated in cell lysate using Homocysteine ELISA kit (Cell Biolabs, Inc.). HepG2 cells 5 × 105 were seeded and incubated for 48 h with experimental treatments. Cells were harvested and suspended in 500 μl in ice cold PBS and were centrifuge at 2500 rpm for 10 min and were sonicated. After centrifugation at 12,000 for 15 min, the supernatant was removed and stored for homocysteine analysis as per manufacturer's instruction. The readings were taken at 450 nm using ELISA plate reader.
Quantitative polymerase chain reaction
The cellular RNA was extracted using RNeasy mini kit according to manufacturer's instruction with slight modification. High-capacity cDNA reverse transcription kit was used to transcribe total RNA to cDNA as per manufacturer's instruction (Thermo Fisher Scientific, Massachusetts, USA). Quantitative polymerase chain reaction (PCR) was performed on ABI step one plus RT-PCR (Applied Biosystem), reaction condition and primers for βactin (housekeeping gene), TNFα and MTHFR gene were used as previously described., The results were analyzed by relative quantification, using ΔΔCt method.
All values are expressed as mean ± standard deviation from at least three independent experiments performed in triplicates. One-way-ANOVA followed by Tukey's post hoc test was performed for comparison of data using GraphPad PRISM software v5.0 (San Diego, CA, USA). The value of P < 0.05 was considered statistically significant.
| Results|| |
Effect of folic acid and Vitamin B12 concentrations on human hepatocellular carcinoma proliferation
Cell deprived of FA and B12 had comparatively slower proliferation rate as compared to cells treated with FA and with B12 combination for 48 h [Figure 1]a. Supplementation of B12 with both normal and excess FA concentration significantly increased (P< 0.05) cell proliferation as compared to untreated cells.
|Figure 1: Effect of folic acid and Vitamin B12 concentrations on human hepatocellular carcinoma proliferation and cell viability. Proliferation of human hepatocellular carcinoma (a) and viability (b) per total cell count in folic acid and B12 deprived cells and following 48 h treatment with folic acid and combination of folic acid and B12. Data are presented as mean ± standard deviation (n = 9). Statistical significance was determined using one-way ANOVA followed by Tukey's multiple comparisons post hoc analysis (*P < 0.05)|
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Effect of folic acid and Vitamin B12 concentrations on human hepatocellular carcinoma cell viability
Deprivation of FA and B12 resulted in decrement of HepG2 viability [Figure 1]b. Interestingly, we found excess FA supplementation cells had significantly lesser viability (P< 0.05) than normal FA supplemented cells. Cells cultured in combination with B12 demonstrated increase in viability at normal FA + B12 (~59%) and at excess FA + B12 (~44%) as compared to untreated and excess FA cells. Significant viability (P< 0.05) was achieved in both B12 treated groups as compared to excess FA treated cells.
Effect of folic acid and Vitamin B12 concentrations on tumor necrosis factor-alpha levels
TNFα mRNA levels and cell lysate TNFα levels were highly increased in excess FA: 60 μM supplemented group as compared to other experimental groups [Figure 2]a and [Figure 2]b. Supplementation of B12 significantly decreased (P< 0.05) TNFα mRNA and cell lysates levels in both normal and excess FA supplemented group as compared to excess FA group, indicating probable regulation of inflammation.
|Figure 2: Effect of folic acid and Vitamin B12 concentrations on human hepatocellular carcinoma cells tumor necrosis factor-alpha levels and oxidative stress. Tumor necrosis factor-alpha mRNA levels (a) tumor necrosis factor-alpha cell lysate levels (b) and oxidative stress (c) in folic acid and B12 deprived cells and following 48 h treatment with folic acid and combination of folic acid and B12. Data are presented as mean ± standard deviation (n = 6). Statistical significance was determined using one-way ANOVA followed by Tukey's multiple comparisons post hoc analysis (*P < 0.05)|
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Effect of folic acid and Vitamin B12 concentrations on lipid peroxidation
Deprivation of FA and B12 and supplementation of excess FA cells had increased MDA levels though statistically significance was seen [Figure 2]c. Supplementation of excess FA: 60 μM showed significant increase (P< 0.05) in MDA levels as compared to other experimental group. Both FA concentration in the presence of B12 showed statistical decrement (P< 0.05) in MDA levels, implying reduction in free radicals and oxidative stress in the cells.
Effect of folic acid and Vitamin B12 concentrations on homocysteine levels
Both FA-B12 deficient cells and excess FA treated cells exhibited higher levels of homocysteine levels [Figure 3]a. Supplementation of normal folate in the presence of B12 significantly regulated (P< 0.05) homocysteine levels as compared to untreated and excess folate supplemented group.
|Figure 3: Effect of folic acid and Vitamin B12 concentrations on human hepatocellular carcinoma cells homocysteine and methylenetetrahydrofolate reductase mRNA levels. Homocysteine levels (a) and methylenetetrahydrofolate reductase mRNA levels (b) in folic acid and B12 deprived cells and 48 hours treatment with folic acid and combination of folic acid and B12. Data are presented as mean ± standard deviation (n = 6). Statistical significance was determined using one-way ANOVA followed by Tukey's multiple comparisons post hoc analysis (*P < 0.05, ** P < 0.01)|
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Effect of folic acid and Vitamin B12 concentrations on methylenetetrahydrofolate reductase transcript levels
MTHFR mRNA levels were significantly lower (P< 0.05) in the untreated cells and excess FA supplemented group as compared to normal FA supplemented group [Figure 3]b. Supplementation of B12 in both excess and normal FA supplemented group significantly increased (P< 0.01) MTHFR levels as compared to untreated and excess FA group.
| Discussion|| |
In the present study, HepG2 cells were supplemented with physiological (6 μM) and excess levels (60 μM) of FA and also in combination of B12 (500 nM) to explore the effect of excess FA supplementation and imbalance of FA and B12 in real-time situation. Folate and Vitamin B12: the methyl donors are center pieces of the one carbon metabolism and play a key part in transmethylation reactions thereby affecting epigenetic mechanism. Cells deprived of folate resulted in nucleotide imbalance and accumulated in S phase and slow DNA synthesis and damage was seen due to increased uracil misincorporation. Shift in S phase accumulation and proliferation is retained by the addition of folate back., Interestingly, B12 plays an important role in synthesis of DNA and deficiency of B12 leads to uracil misincorporation by the same mechanism as folate deficiency. In this study, we found that deprivation of FA and B12 in HepG2 cells (untreated cells) as compared to experimental groups resulted in slow growth of cells and lesser cell viability.
In agreement with recent findings on trophoblastic cell lines,, viability of HepG2 cells decreased significantly (P< 0.05) when supplemented with excess FA: 60 μM as compared to normal FA: 6 μM. Recent evidence suggests that higher concentration of FA inhibits proliferation of vascular endothelial cells and is mediated by folate receptor. Folate plays a crucial role in nucleotide synthesis, and perhaps may regulate cellular immunity and responsiveness. Evidence from human and animal studies suggest that an imbalance in folate and B12 altered NK cytotoxicity and other immunological parameters., This finding suggests that increase in FA intake was inversely associated with cytotoxicity and un-metabolized levels of FA in plasma. Supplementation of B12 along with FA: 6 μM and FA: 60 μM increased cell proliferation and viability significantly as compared to only FA supplemented group. Evidence exhibit the aggravation of B12 deficiency at supra physiological FA concentrations, we found that supplementation of B12 at FA: 6 μM had significant proliferation and viability than FA: 60 μM + B12.
Earlier studies have shown that hepatocytes are more susceptible to TNFα-induced apoptotic killing. TNFα activates NFκB signaling mediating the transcription of various cytokines involved in key cellular physiological functions such as proliferation and differentiation, cell adhesion, inflammatory regulation, and anti-apoptotic determinants. Previously, B12 deficiency and imbalance in FA and B12 ratio was associated with an increased TNFα levels., In line with previous findings, our results depicted that TNFα mRNA and cell lysate levels significantly increased in HepG2 cells supplemented with excess FA as compared to other experimental groups. Supplementation of B12 at both FA concentration reduced TNFα mRNA transcript and cell lysate levels significantly.
The previous finding suggests that oxidative stress mediates TNFα-induced mitochondrial DNA damage and dysfunction in cardiac myocytes. It has been evident that the predominant cause for cellular dysfunction, oxidative damage of biomolecules and endothelial dysfunctioning is due to increased free radicals lead. Oxidative stress is upstream of TNFα activation in various pathophysiological conditions, and thus, it is difficult to separate oxidative stress and TNFα. Interestingly, we found MDA levels were significantly higher in groups with higher expression of TNFα mRNA levels, especially in excess FA supplemented group. Synergistic supplementation of FA + B12 significantly reduced MDA levels and results are in agreement with previous findings.,
Homocysteine metabolism is closely linked to metabolism of folate and B12. Recently, it was shown that treatment with high homocysteine (2 mM) in HepG2 cells suspended proliferation owing to increased IGF binding protein-2, which has a growth-inhibiting function and leads to endoplasmic reticulum stress, decrease protein synthesis, and apoptosis. In this study, FA and B12 deficient cells and excess FA supplemented cells depicted high homocysteine levels and showed low proliferation and viability. Combination of FA and B12 significantly reduced homocysteine levels. Reduced homocysteine levels might have modulated methyl balance and reduced glutathione formation thereby increasing the susceptibility of hepatocytes to oxidative events.
Since MTHFR is a key enzyme of folate interconversion, we studied the effect of excess FA and imbalance between FA and B12 on MTHFR gene. Basically, MTHFR upholds the delicate balance between utilization of folate for nucleotide synthesis and methionine synthesis, later its substrate is required for synthesis of thymidine, thereby playing a key role in cell growth and division. In the deficient FA and B12 cells and excess FA supplemented cells there was downregulation of MTHFR mRNA. The media deficient of FA and B12 probably led to a poor inflow of methyl groups for the homocysteine remethylation. This lowering of remethylation may be due to MTHFR enzyme, and probably may have down regulated MTHFR mRNA expression. Supplementation of B12 at both normal and excess FA concentrations significantly increased MTHFR mRNA levels and results are in agreement with previous in vivo findings.
| Conclusion|| |
The study results for the first time exhibit the unfavorable effect caused due to imbalance in FA and Vitamin B12 on HepG2 cells as indicated by the decreased proliferation, viability, mRNA level of the key gene encoding enzyme of the one carbon cycle: MTHFR and increase in homocysteine levels, oxidative stress, and cytokine level, which, however, needs additional and more extensive investigation. Supplementation of B12 reduced the detrimental effect of excess FA on HepG2 cells. Thus, it may be thoughtful to include B12 in FA fortification programs in developing nations like India, where B12 deficiency is seen due to the dietary pattern of vegetarianism and ovo-lacto-vegetarianism.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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