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


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.

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














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.

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.

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

Method and materials

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.

proliferation and cell viability assays

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.

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.


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).


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.


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.

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.
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.

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, 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.


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


















1.         Siegel
R.L, Miller K.D, Jemal A. Cancer statistics, 2016. CA: a cancer journal for
clinicians; 66 (1): 7-30: (2016).

2.         Arunachalam
R, Reshma A.P, Rajeev V, Kurra S.B, Prince M.R.J, Syam N. Salivary
8-Hydroxydeoxyguanosine–a valuable indicator for oxidative DNA damage in
periodontal disease. The Saudi Journal for Dental Research; 6 (1):
15-20: (2015).

3.         Ya B,
Zhang L, Zhang L, Li Y, Li L. 5-Hydroxymethyl-2-furfural prolongs survival and
inhibits oxidative stress in a mouse model of forebrain ischemia. Neural
regeneration research; 7 (22): 1722: (2012).

4.         van
Putten R-J, van der Waal J.C, de Jong E, Rasrendra C.B, Heeres H.J, de Vries J.G.
Hydroxymethylfurfural, a versatile platform chemical made from renewable
resources. Chemical reviews; 113 (3): 1499-597: (2013).

5.         Ghadimi
S.S, Alizadeh M, Esfanjani AT, Hezaveh S.J.G, Vayghan H.J. ASSESMENT OF DIETARY
Journal of Food Science; 26 (2): 169-75: (2014).

6.         Rahimzadeh
N, Alizadeh M, Hezaveh S.J.G. Estimated Bioaccessibility to
5-hydroxymethylfurfural from Frequently Consumed Dried Fruits in Iran. Journal
of Chemical Health Risks; 4 (3): (2014).

7.         Biluca
F.C, Della Betta F, de Oliveira G.P, Pereira L.M, Gonzaga L.V, Costa A.C.O et
al. 5-HMF and carbohydrates content in stingless bee honey by CE before and
after thermal treatment. Food chemistry; 159: 244-9: (2014).

8.         Rufián-Henares
J, Delgado-Andrade C, Morales F. Analysis of heat-damage indices in breakfast
cereals: Influence of composition. Journal of cereal science; 43 (1):
63-9: (2006).

9.         del
Campo G, Berregi I, Caracena R, Zuriarrain J. Quantitative determination of
caffeine, formic acid, trigonelline and 5-(hydroxymethyl) furfural in soluble
coffees by< sup> 1 H NMR spectrometry. Talanta; 81 (1):
367-71: (2010).

10.       Janzowski
C, Glaab V, Samimi E, Schlatter J, Eisenbrand G. 5-Hydroxymethylfurfural:
assessment of mutagenicity, DNA-damaging potential and reactivity towards
cellular glutathione. Food and chemical toxicology;  38 (9): 801-9: (2000).

11.       Severin
I, Dumont C, Jondeau-Cabaton A, Graillot V, Chagnon M-C. Genotoxic activities
of the food contaminant 5-hydroxymethylfurfural using different< i> in
vitro bioassays. Toxicology letters;  192 (2): 189-94: (2010).

12.       Zhang X.M,
Chan C.C, Stamp D, Minkin S, Archer M.C, Bruce W.R. Initiation and promotion of
colonic aberrant crypt foci in rats by 5-hydroxymethy1-2-furaldehyde in
thermolyzed sucrose. Carcinogenesis;  14 (4): 773-5: (1993).

13.       Florian
S, Bauer?Marinovic M, Taugner F,
Dobbernack G, Monien B.H, Meinl W et al. Study of 5?hydroxymethylfurfural and
its metabolite 5?sulfooxymethylfurfural
on induction of colonic aberrant crypt foci in wild?type mice and transgenic
mice expressing human sulfotransferases 1A1 and 1A2. Molecular nutrition
& food research; 56 (4): 593-600: (2012).

14.       Program
N.T. NTP toxicology and carcinogenesis studies of 5-(Hydroxymethyl)-2-furfural
(CAS No. 67-47-0) in F344/N rats and B6C3F1 mice (gavage studies). National
Toxicology Program technical report series; 554: 7: (2010).

15.       Zhao L,
Chen J, Su J, Li. L, Hu S, Li B et al. In vitro antioxidant and
antiproliferative activities of 5-hydroxymethylfurfural. Journal of
agricultural and food chemistry; 61 (44): 10604-11: (2013).

16.       Cao G,
Cai H, Cai B, Tu S. Effect of 5-hydroxymethylfurfural derived from processed
Cornus officinalis on the prevention of high glucose-induced oxidative stress
in human umbilical vein endothelial cells and its mechanism. Food chemistry;
 140 (1): 273-9: (2013).

17.       Li M.M,
Wu L.Y, Zhao T, Xiong L, Huang X, Liu Z.H et al. The protective role of 5-HMF
against hypoxic injury. Cell Stress and Chaperones; 16 (3): 267-73: (2011).

18.       Yamada
P, Nemoto M, Shigemori H, Yokota S, Isoda H. Isolation of 5-(hydroxymethyl)
furfural from Lycium chinense and its inhibitory effect on the chemical
mediator release by basophilic cells. Planta medica; 77 (05): 434-40: (2011).

19.       Alizadeh
M, Ota F, Hosoi K, Kato M, Sakai T, Satter MA. Altered allergic cytokine and
antibody response in mice treated with Bisphenol A. The Journal of Medical
Investigation; 53 (1, 2): 70-80: (2006).

20.       Durling
LJ, Busk L, Hellman BE. Evaluation of the DNA damaging effect of the
heat-induced food toxicant 5-hydroxymethylfurfural (HMF) in various cell lines
with different activities of sulfotransferases. Food and chemical toxicology;
47 (4): 880-4: (2009).

21.       Sun Y,
Sun T, Wang F, Zhang J, Li C, Chen X et al. A polysaccharide from the fungi of
Huaier exhibits anti-tumor potential and immunomodulatory effects. Carbohydrate
polymers; 92 (1):577-82: (2013).

22.       Yi Y,
Zhang M.W, Liao S.T, Zhang R.F, Deng Y.Y, Wei Z.C et al. Structural features
and immunomodulatory activities of polysaccharides of longan pulp. Carbohydrate
Polymers; 87 (1): 636-43: (2012).

23.       Ding X,
Wang M.Y, Yao Y.X, Li G.Y, Cai B.C. Protective effect of
5-hydroxymethylfurfural derived from processed< i> Fructus
Corni on human hepatocyte LO2 injured by hydrogen peroxide and its
mechanism. Journal of ethnopharmacology;  128 (2):373-6: (2010).

24.       Wang M.Y,
Zhao F.M, Peng H.Y, Lou C.H, Li Y, Ding X et al. Investigation on the
morphological protective effect of 5-hydroxymethylfurfural extracted from
wine-processed Fructus corni on human L02 hepatocytes. Journal of
ethnopharmacology; 130 (2):424-8: (2010).

25.       Li W,
Qu X.N, Han Y, Zheng S.W, Wang J, Wang Y.P. Ameliorative effects of
5-hydroxymethyl-2-furfural (5-HMF) from Schisandra chinensis on alcoholic liver
oxidative injury in mice. International journal of molecular sciences; 16
(2): 2446-57: (2015).

26.       Li Y.X,
Li Y, Qian Z.J, Kim M.M, Kim S.K. In vitro antioxidant activity of 5-HMF
isolated from marine red alga Laurencia undulata in free-radical-mediated
oxidative systems. J Microbiol Biotechnol; 19 (11): 1319-27: (2009).

27.       Herrero
M, Castro-Puyana M, Rocamora-Reverte L, Ferragut J.A, Cifuentes A, Ibáñez E.
Formation and relevance of 5-hydroxymethylfurfural in bioactive subcritical
water extracts from olive leaves. Food research international; 47 (1): 31-7:

28.       Ulbricht
R.J, Northup S.J, Thomas J.A. A review of 5-hydroxymethylfurfural (HMF) in
parenteral solutions. Fundamental and Applied Toxicology; 4 (5): 843-53:

29.       Mousavi
R, Alizadeh M, Saleh-Ghadimi S. Consumption of 5-hydroxymethylfurfural-rich
dried fruits is associated with reduction in urinary excretion of 8-hydroxy-2?-deoxyguanosine:
a randomized clinical trial. European Food Research and Technology; 242 (5):677-84:

30.       Khodaei
H, Alizadeh M. Inhibition of IL-4 but not IFN-? production by splenocytes of
mice immunized with ovalbumin after oral administration of
5-hydroxymethylfurfural. Food and Agricultural Immunology; 1-8: (2016).

31.       Corpet
D.E, Stamp D, Medline A, Minkin S, Archer M.C, Bruce W.R. Promotion of colonic
microadenoma growth in mice and rats fed cooked sugar or cooked casein and fat.
Cancer research; 50 (21): 6955-8: (1990).

32.       Svendsen
C, HUSØY T, Glatt H, Paulsen J.E, Alexander J. 5-Hydroxymethylfurfural and
5-sulfooxymethylfurfural increase adenoma and flat ACF number in the intestine
of Min/+ mice. Anticancer research; 29 (6):1921-6: (2009).

33.       Bauer-Marinovic
M, Taugner F, Florian S, Glatt H. Toxicity studies with 5-hydroxymethylfurfural
and its metabolite 5-sulphooxymethylfurfural in wild-type mice and transgenic
mice expressing human sulphotransferases 1A1 and 1A2. Archives of toxicology;
86 (5):701-11: (2012).











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 < 0.05,  ANOVA -test).                   Figure 2- 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 con A control (*: p < 0.05, ANOVA -test).