Inhibitory effect of hydroxymethylfurfural in viability of BALB/C mice splenocytes

Abstract

This study was
designed to discover if  hydroxymethylfurfural (HMF) exposure modifies
proliferation of cell and DNA damage in BALB/c mice
splenocytes. Mitogenesis in T cells and B cells was induced via Concanavalin
A (Con A) and lipopolysaccharide (LPS),
respectively, under air containing 5% carbon dioxide.
The colorimetric tetrazolium assay was used to evaluate cell
proliferation. DNA
damaging consequences was evaluated via measurement of 8-OHdG level as in BALB/c mice splenocytes. Spleen cells proliferation elicited by ConA, was dramatically suppressed
by HMF at the concentrations of 25, 50 and 100 mM, however there was not any
significant difference between different concentrations of HMF. The same result
was observed following treatment with LPS and HMF in different concentrations.
8-OHdG concentration was elevated significantly in HMF treated groups compared
with untreated control and mitogens. In conclusion, HMF was found to
have immunosuppressing and DNA damaging properties in mMolar concentrations in
mice splenocytes.

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Key
words: food processing, food safety,
mutagenicity, 5-hydroxymethylfurfural, DNA damage

 

 

 

 

 

 

 

 

 

 

 

 

Introduction

One of the most serious disease which has been the leading cause
of death globally, is cancer 1.
Cells of the human body is continuously exposed to oxidative stress. Eight-hydroxy-2-deoxyguanosine
(8-OHdG) is one of the most prevalent oxidative DNA damage products.  Carcinogenesis has been correlated with 8-OHdG
formation based on reports of various studies 2.

Hydroxymethyl
furfural (HMF), a furan derivative, 3
formed by the degradation of sugars and carbohydrates through maillard reaction
or caramelization 4. The
concentration of HMF in some heat-treated foods including bread 5, dried fruits 6, honey 7, breakfast cereals 8,
coffee and instant coffee 9 is
high. Depend on the country and its culture,
consumption of these food items and therefore exposure to HMF is different. For
instance, consumption of bread which is baked traditionally under direct
heating, and subsequently HMF exposure is high in Iran 5. Hence, in vitro and in vivo studies focusing
on toxicity and metabolism of HMF is necessarily recommended. Some studies have
reported it’s cytotoxic, mutagenic and DNA damaging effects 10-13; However,
a 2 year animal study declared that different doses of 5-HMF had no neoplastic
effect 14. Interestingly, in
recent studies this compound has been characterized to exert antioxidant properties
with promising potential in cancer chemoprevention 15, decrease of oxidative stress elicited by sugar 16, anti-hypoxia 17 and anti-
allergic effects 18.

Till
now, there is neither no data showing correlation between exposure to HMF and
risk of cancer, nor experimental evidence available regarding HMF-mediated cell
proliferation and DNA damage in model cells. In this study, we decided to
examine the effect of HMF in different concentrations on cell proliferation and
DNA damaging consequences via measurement of 8-OHdG level as in BALB/c mice
splenocytes.

Method and materials

Preparation
of Blood serum and Spleen cell suspension

BALB/c mice without any specific pathogen at
6 week of age, weighing 15-20 g were anesthetized using nembutal and blood was collected from inferior
vena cava. Then, mice were killed and spleen cell suspensions were obtained
following the procedure described by Alizadeh, etal. with some modifications 19.
Briefly, spleens were removed and smashed by a plunger of syringe through a mesh
screen in RPMI-1640 (Sigma, USA)/Antibiotic-Antimycotic medium (GIBCO BRL
Life., USA). They were then washed by centrifugation at 4°C. Elimination of red
blood cells in the cell suspensions was carried out using lysis buffer (5 ml of
0.83% NH4Cl in Tris-HCl (pH 7.4)) at 37°C for 5 minutes and 3 subsequent washing with
media was done. All procedures were operated under strict sterile conditions.

Cell
proliferation and cell viability assays

Viability
of the cells was evaluated via mixing 100 ?l of the resultant solution with
equal volume of a solution of 0.4% trypan blue. Finally, a suspension of spleen
cells (5 × 106 cells/well) in a 96-well plate was prepared in
RPMI-1640/antibiotic-antimycotic medium and 10% fetal bovine serum. Two µg/dl of concanavalin A (Con A) as T cell
mitogen or 10 µg/dl of lipopolysaccharide
(LPS) as B cell mitogen
was used under air containing 5% carbon dioxide. Cell growth in absence and
presence of HMF at concentrations 0, 25, 50 and 100 mM for 72 h was determined
by the colorimetric tetrazolium (MTT) assay. MTT reagent was added and
incubated for further 3 hours at 37ºC in humidified 5% CO2/air
atmosphere. Then, the media were removed and 200 ?L of DMSO were added to each well to
dissolve the formazan product by shaking for 30 min. Finally, the absorbance
was determined at 570 nm.

 Treatments were as follows:

1)      Cells
+ medium

2)      Cells
+ medium+ 2 µg/dl
of concanavalin A (Con A) or 10 µg/dl
of lipopolysaccharide
(LPS)+ 0 mM HMF

3)      Cells
+ medium+ 2 µg/dl
of concanavalin A (Con A) or 10 µg/dl
of lipopolysaccharide
(LPS) + 25 mM HMF

4)      Cells
+ medium+ 2 µg/dl
of concanavalin A (Con A) or 10 µg/dl
of lipopolysaccharide
(LPS)+ 50 mM HMF

5)      Cells
+ medium+ 2 µg/dl
of concanavalin A (Con A) or 10 µg/dl
of lipopolysaccharide
(LPS)+ 100 mM HMF

All of the treatments
were in triplicate.

DNA
damage evaluation

Extracted mice
splenocytes were cultured in 96-well culture plates. After the cells were
doubled, one ml of fresh medium and HMF in different concentrations (0, 25, 50
and 100 mM) were added to the culture wells. Absolute medium was considered
served as control 20. The treatments were similar to the above
mentioned ones. After 72 h, the supernatants of cultures were collected to
measure 8-OHdG concentration as oxidative DNA damage indicator via enzyme-linked
immunosorbent assay (ELISA) analysis (Glory science, China) following the
instructions made by manufacturers. Briefly, cells were transferred into two ml
microtubes and centrifuged for 20 minutes at 2000-3000 g. The supernatants were
removed and the cells were diluted with PBS to 1*106 cells after
counting. Freeze-defreeze cycle was repeated four times to help release of
8-OHdG. Finally cells were centrifuged for 20 minutes at 2000-3000 g and clear
supernatants were collected to measure 8-OHdG level by using the kit.

Statistical
analysis

The
normality of distribution of data was tested by the Kolmogorov-Smirnov test.
One-way analysis of variance (ANOVA), together with Tukey model was employed to
assess differences among groups. P value less than 0.05 was considered
statistically significant. The statistical analyses were performed with the use
of SPSS version 20.0 software (SPSS Inc, Chicago, IL, USA).

Results

Lymphocytes are considered as an essential component of mammalian
immune system cells and our study showed that the proliferation of different
subpopulations of lymphocytes were stopped by HMF at varying levels. As shown in
figure 1, the effects of HMF on lymphocyte viability
were assessed using stimulant mitogen (Con A: T-cell mitogen and LPS: B-cell
mitogen). Con A or LPS induced splenocyte proliferation, was significantly
suppressed by HMF at the concentrations of 25, 50 and 100 mM; However, there
was not statistically significant difference between groups.

8-OHdG concentration, oxidative DNA damage indicator, was elevated
significantly in HMF treated groups, also Con A or LPS + HMF treated groups;
however it was not statistically significant in treated cells with LPS or Con A
mitogens, individually compared with control.

Discussion

In
this study, harmful actions of 5-HMF on mice splenocytes was evaluated in vitro. Lymphocytes proliferation is an indicator of immunopotentiation
21. Proliferation of spleen lymphocyte was induced by ConA in vitro to evaluate T
lymphocyte activity, while B lymphocyte activity was examined after induction
by LPS 22. In vitro lymphocyte proliferation assay, 5-HMF had
directly antimitogenic effect on both intact splenocytes and stimulated
splenocytes via conA and LPS. These data indicated 5-HMF was a potent
immunosuppressing substance. Moreover, it showed that 5-HMF in different
concentrations induce a significant oxidative DNA damage to splenocytes in
presence or absence of mitogens.

Several
studies have examined the effect of 5-HMF in various cell lines. Ding etal
revealed that 5-HMF increased the LO2 cell viability. Therefore, it
had a protective effect against H2O2-elicited LO2
hepatic cell injury 23, 24. Hypoxic
injury in ECV304 cells was attenuated with 5-HMF 17. Similarly,
ameliorative effects of 5-HMF was observed on alcoholic liver oxidative injury
in mice 25. 5-HMF had a protective effect in HUVEC treated with high
glucose. Since, it increased the viability of injured cells with glucose.
Moreover, it inhibited expression of JNK1 and JNK2/3 and high activity of IL-8
induced by glucose, a downstream activator of P-Akt 16. In a dose
dependent manner, 5-HMF at the concentration of 0.3–5.0 ?g/mL did not show any cytotoxicity, although KU812 cells
viability was reduced remarkably at the dose of 10.0 ?g/mL18.
Similar to this study, Li et al showed that 5-HMF
in different concentrations had no cytotoxic effects on RAW 264.7, HL-60, and
MRC-5 cells, while the highest concentration (100 ?M) could influence cell
viability 26. In the present study,
the decreased cell viability in HMF treated cells could be due to its extremely
high concentration compared to previous studies.
Production of H2O2 due to high concentration of HMF and affected
by its apoptotic activity 10 might cause a cell damage23.
Production of 8-OHdG, one of the main oxidative stress biomarkers, was
statistically significant between groups in the present study; although the
involved mechanism was not evaluated. Overall antioxidant and
antiproliferative activities against 2 type colon cancer cells were not altered
after treatment with increasing concentrations of 5-HMF (from 1 to 13 ?g/mL)
and olive oil, a source of 5-HMF. A higher concentration of 5-HMF (300 ?g/mL),
300 times greater than the amount found in olive leaves, inhibited
proliferation in both of the cell lines 27. Evaluation of antiproliferative
effect of 5-HMF in different cell lines indicated that proliferation of human
melanoma A375 cells was influenced more than other cell lines after treatment
with 5-HMF by activation of apoptosis in A375 cell and G0/G1
cell cycle arrest 15.

Based on several studies, various cells display different
sensitivity towards the cytotoxicity of the 5-HMF 20. Humans are
more sensitive to toxic effects of HMF than rats, when exposed orally 28.
However,
consumption of dried fruits with different HMF levels showed an inverse
association with changes of 8-OHdG in a sample of
healthy volunteers 29. Spleen cells of mice immunized with
OVA following exposure to HMF orally, led to inhibition of IL-4 production
without influencing IFN-? which could be considered as a promising agent in
preventing (Th2) cytokine-dominant disease30.

Based
on the controversial conclusions in
previous studies on the biological effects of 5-HMF,
the cytoprotective effects of the compound are not established. The 5-HMF
induced DNA damage in different cell lines with different sulfotransferase
(SULT) activities. It was considered a weak genotoxic in Hep-G2 cell line.
Similar results were observed with animal studies. Growth of aberrant crypt
foci (ACF) in rats after
administration of caramelized sugar was higher than the control group 31.
The Min/+mice treated with 5-HMF or SMF did not have any effect on the adenomas
size, although 5-HMF increased the number of small intestinal adenomas and SMF
increased the flat ACF number in the large intestine 32. High doses
of 5-HMF induced nephrotoxic and hepatotoxic effects in wild-type mice and
transgenic mice expressing human SULT 33. Differences
in expression levels of receptors, metabolism, chemical structure, and enzyme
activity of the 5-HMF might be responsible for the variation in the
susceptibility of cells to the compound.

Conclusion

In
conclusion, 5-HMF was found to have immunosuppressing properties in mice
splenocytes. We also found that 5-HMF induces DNA damage in mMolar
concentrations. Further studies are needed to understand the mechanism involved in the immunosuppressing effect of 5-HMF.

Acknowledgment

This
study was supported by a grant from the Nutrition Research Center of Tabriz
University of Medical science.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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Figure
1- cytotoxicity effect of 5-HMF on mice splenocytes by MTT assay. The percent cell viability was calculated relative to the
untreated control. * Significantly different from the LPS control (*: p

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