Research Article
Induction of Macrophage Cell-Cycle Arrest and Apoptosis by Humic Acid
Hsin-Ling Yang1, Hsin-Ju Cho1, Ssu-Ching Chen2, K.J. Senthil Kumar5, Fung-Jou Lu3,
Chia-Ting Chang1, You-Cheng Hseu4,5,*
1
2
Department of Life Sciences, National Central University, Chung-Li 32001, Taiwan
3
4
Institute of Nutrition, China Medical University, Taichung 40402, Taiwan
Institute of Medicine, Chun Shan Medical University, Taichung 40201, Taiwan
Department of Health and Nutrition Biotechnology, Asia University, Taichung 41354, Taiwan
5 Department
of Cosmeceutics, College of Pharmacy, China Medical University, Taichung 40402,
Taiwan
*Correspondence to: You-Cheng Hseu, Department of Cosmeceutics, College of Pharmacy, China
Medical
University,
91
Huseh-Shih
Road,
ychseu@mail.cmu.edu.tw
1
Taichung
40402,
Taiwan.
E-mail:
ABSTRACT
Humic acid (HA) in drinking well water is associated with blackfoot diseases and various cancers.
We were the first to report that acute humic acid exposure (25-200 µg/mL for 24 h) induces
inflammation in RAW264.7 macrophages. In this study, we found that prolonged (72 h) HA
exposure (25-200 µg/mL) induced cell-cycle arrest and apoptosis in cultured RAW264.7 cells. We
observed that exposing macrophages to HA causes G2/M phase arrest through reductions in cyclin
A/B1, Cdc2, and Cdc25C levels. Furthermore, treating macrophages with HA resulted in a
sequence of events marked by apoptotic cell death, such as a loss of cell viability, morphological
changes, inter-nucleosomal DNA fragmentation, and sub-G1 accumulation. Moreover, HAinduced apoptosis was associated with mitochondrial dysfunction, cytochrome c release, caspase-3
and -9 activation, and Bcl-2/Bax dysregulation. Our investigation also revealed that HA induces
Fas, caspase-8, -4, and -12 activities within macrophages. These data suggest that HA-induced
apoptosis is mediated by mitochondrial, death receptor, and ER stress pathways. In addition, HA
up-regulates p53 expression and induces DNA damage (genotoxicity) as shown by the Comet
assay. These data provide an important new insight that HA can affect the immune system through
macrophages.
Key words: humic acid; macrophage; G2/M arrest; apoptosis; DNA damage
2
INTRODUCTION
Humic substances, which occur in the forms of humic acid, fulvic acid, and humin, have been
found in half of the world's drinking well water. HA is classified in a group of high-molecularweight polymers that are primarily derived from the decomposition of dead plants. HA is a darkbrown, carbon-rich material that mostly exists in peat, soil, and well water [Hartenstein, 1981].
The presence of these materials in the drinking water supply causes problems because they act as
precursors for undesirable trihalomethane formation during the chlorination process, the
consequences of which may be detrimental to human health [Man et al., 2013].
HA has been implicated as one of the etiological factors in the peripheral vasculopathy of an
outbreak of blackfoot disease that occurred on the Southwest Coast of Taiwan in the 1970s [Hseu
et al., 2002a]. Epidemiologic and geochemical studies uncovered the presence of high
concentrations of HA (approximately 200 ppm) in artesian well water in BFD endemic areas [Lu,
1990]. HA intake by an average resident in these areas was estimated to be as high as 400 mg/day
[Huang et al., 1995]. HA contamination of well water consumed by the inhabitants of this region
is considered to be one of the possible causes of the blackfoot disease outbreak [Lu, 1990].
Notably, the signs and symptoms of blackfoot disease are similar to those of arteriosclerosis and
Buerger’s disease [Wang et al., 2007]. Increased mortality from cardiovascular and cerebrovascular diseases has also been associated with blackfoot disease [Wang et al., 2007]. HA has
been found in the gastrointestinal tract of humans and animals and could be circulated in the blood
[Hu et al., 2010]. It is plausible that consuming excessive amounts of HA from well water
adversely affected the health of inhabitants, which led to the pathogenesis and progression of
blackfoot disease. However, the underlying pathophysiological mechanisms are poorly
understood.
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Macrophages are one of the principal immune effector cell types, and they play vital roles in
inflammation, host defense, and reactions against a spectrum of autologous and foreign invaders
[Zhang et al., 2010]. However, when their control mechanisms stop working, macrophage
inflammatory responses may result in persistent swelling, pain, and eventual tissue injury [Karin
et al., 2006]. Macrophages release various inflammatory molecules when they are activated by
endotoxins, and they are also known to be very sensitive to changes in environmental conditions
including toxic chemical exposures [Fujiwara and Kobayashi, 2005]. Increasing evidence
indicates that heavy metals or toxic chemicals induce apoptosis in macrophages [Sakurai et al.,
1998; Sakurai et al., 2006; Tabas, 2010]. Taken together, these data indicate the usefulness of
macrophages for examining the influence of chemical materials on mammalian immune systems.
In our previous study, we used an in vitro model of macrophage inflammation in which HA
(25-200 µg/mL for 24 h) activates macrophages to produce pro-inflammatory molecules by
activating their transcriptional factors, including NF-κB and AP-1 [Hseu et al., 2014]. However,
there was no information available for the cytotoxic and genotoxic effects of HA in macrophages.
In the present study, we used mouse macrophage cell line RAW264.7 as an in vitro model to
evaluate the possible cytotoxic effects of HA (25-200 µg/mL for 72 h) by studying cell-cycle
arrest and apoptosis. We believe that the present study will improve our understanding of HA
involvement in cardiovascular and blackfoot diseases.
MATERIALS AND METHODS
Chemicals and Reagents
Dulbecco’s
modified
Eagle’s medium
(DMEM), fetal bovine serum (FBS),
and
penicillin/streptomycin were obtained from Gibco/BRL Life Technologies Inc. (Grand Island, NY,
4
USA). 3-[4,5-dimethyl-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) was obtained from SigmaAldrich (St. Louis, MO, USA). Antibodies against cytochrome c, Bcl-2, Bax, Fas, FasL, cyclin B1,
Cdc2, p53, p-p53, and β-actin were obtained from Santa Cruz Biotechnology Inc (Heidelberg,
Germany). PARP rabbit polyclonal antibody was purchased from Roche (Mannheim, Germany).
Antibodies against cyclin A, Cdc25C, caspase-9, caspase-3, PARP, and Bid were obtained from
Cell Signaling Technology Inc (Danvers, MA, USA). Antibody against caspase-4 from Biomol
Inc (Montgomery, PA, USA) and antibodies against caspase-8 from NeoMarkers Inc (Fremont,
CA, USA). Antibody against caspase-12 was purchased from Millipore (Billerica, MA, USA).
4’,6-Diamidino-2-phenylindole dihydrochloride (DAPI) was purchased from Calbiochem (La
Jolla, CA, USA). All other chemicals were of the highest grade commercially available and
supplied either by Merck & Co., Inc (Darmstadt, Germany) or Sigma-Aldrich.
Preparation of Synthetic HA
To better define the chemical components associated with the adverse effects assumed to result
from the consumption of contaminated artesian well water, synthetic HA was synthesized from
monomeric protocatechuic acid and, thus free of other inorganic contaminants, was used for this
study according to the published procedure [Hseu et al., 2008], with slight modifications. For
oxidative polymerization, 1 g of protocatechuic acid in 100 mL of distilled water was oxidized
with sodium periodate for 24 hours in a water bath at 50 C with gentle shaking. After
centrifugation at 3000 × g, the supernatant was acidified to pH 1.0 using 0.1 N HCl. The acidified
solution was again centrifuged, and the precipitate was treated with 0.1 N NaOH to solubilize the
HA. The obtained HA was further purified by using absorption chromatography with XAD-7 resin
and fractionated by Sephadex G-25 chromatography, as described previously [Hseu et al., 2008].
5
Then the HA solution was ultrafiltered through a Molecular/Por membrane (which excludes
particles of <500 Da MW). The resultant HA (with MWs of 500 Da to several tens of thousands of
Daltons) was collected and subjected in this study.
Cell Culture and Cell Viability Assay
A murine macrophage (RAW264.7) cell line was obtained from the American Type Culture
Collection (ATCC, Rockville, MD, USA) and cultured in DMEM containing 4 mM glutamine and
10% heat-inactivated FBS in a humidified atmosphere with 5% CO2. Cell viability was assessed
by MTT colorimetric assay as previously described [Erhayem and Sohn, 2013]. In brief, 8 × 104
cells/well were cultured in a 24-well culture plate for 72 h with or without HA (25-200 µg/mL).
After incubation, the culture supernatant was removed and 400 µL of 0.5 mg/mL MTT in PBS was
added to each well and incubated at 37 °C for 4 h. MTT-generated farmazan crystals were
dissolved in 400 µL of isopropanol, and the colorimetric absorbance was measured at 570 nm
(A570) with an enzyme-linked immunosorbent assay (ELISA) microplate reader (Bio-Tek
Instruments Inc., Winooski, VT, USA).
Cell-Cycle Analysis
Cellular DNA content was determined by flow cytometry with propidium iodide (PI)-labeled
cells. In brief, macrophages (1 × 106 cells/dish) were cultured in 10 cm culture dishes. After being
treated with HA, the cells were harvested, washed and suspended in PBS and fixed in ice-cold
70% ethanol at -20 °C overnight. Following incubation, the cells were re-suspended in PBS
containing 1% Triton X-100, 0.5 mg/mL RNase, and 4 g/mL PI at 37 °C for 30 min. A
FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA, USA) equipped with a single
6
argon-ion laser (488 nm) was used for flow cytometric analysis. Forward and right-angle light
scatter, which are correlated with the size of the cell and the cytoplasmic complexity, respectively,
were used to establish size gates and exclude cellular debris from the analysis. The DNA content
of 1 × 104 cells per analysis was monitored with the FACSCalibur system. The cell-cycle was
determined and analyzed by using ModFit software (Verity Software House, Topsham, ME, USA).
Apoptotic nuclei were identified as subploid DNA peaks, and they were distinguished from cell
debris on the basis of forward light scatter and PI fluorescence.
Apoptosis Determination
Apoptotic cell death was measured by using terminal deoxynucleotidyl transferase-meditated
dUTP-fluorescein nick end-labeling (TUNEL) with a fragmented DNA detection kit (Roche,
Mannheim, Germany) by following the supplier’s instruction. Macrophages (8 × 104 cells/well)
were seeded on a 24-well culture plate and treated with HA (100 and 200 μg/mL) for 72 h.
Following HA treatment, the cells were washed twice with PBS, fixed in 2% paraformaldehyde
for 30 min and then permeabilized with 0.1% Triton X-100 for 30 min at room temperature. The
cells were then incubated with TUNEL reaction buffer in a 37 °C humidified chamber for 1 h in
the dark, then rinsed twice with PBS and incubated with DAPI (1 µg/mL) at 37 °C for 5 min;
stained cells were visualized under a fluorescence microscope. The fluorescence intensity under
each condition was quantified using a squared section of fluorescence-stained cells with analySIS
LS 5.0 soft image solution (Olympus Imaging America Inc., Corporate Parkway Centre Valley,
PA, USA), and the fold-increase of fluorescence intensity is directly proportional to apoptotic
cells, was compared with that of the un-treated control cells, which were arbitrarily assigned a
value of 1-fold.
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Western Blot Analysis
The macrophages (1 × 106 cells/dish) in a 10 cm dish were incubated with various
concentrations of HA (25-200 μg/mL) for 72 h. After incubation, the cells were washed once in
PBS and detached. The cells were suspended in lysis buffer (10 mM Tris-HCl [pH 8.0], 0.32 M
sucrose, 1% Triton X-100, 5 mM EDTA, 2 mM DTT, and 1 mM phenylmethyl sulfonyl fluoride)
and then centrifuged at 15,000 × g for 30 min at 4 °C. Total protein content was determined by
using Bio-Rad protein assay reagent (Bio-Rad, Hercules, CA, USA) with BSA as a standard.
Equal amounts (50 μg) of denatured protein samples were loaded into each lane and separated by
SDS-PAGE. The membranes were incubated with primary antibodies for overnight, followed by
either horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse antibodies for 2 h. The
blots were detected with an ImageQuant LAS 4000 mini (Fujifilm) with a SuperSignal West Pico
chemiluminescence substrate (Thermo Scientific, IL, USA).
Fluorescent Imaging of Mitochondrial Activity
Fluorescent mitochondrial imaging was accomplished by using MitoTracker Green FM
(Molecular Probe, Eugene, OR, USA) as directed by the manufacturer. MitoTracker is a green
fluorescent mitochondrial stain that appears to localize to mitochondria regardless of
mitochondrial membrane potential. Cells (3 × 104 cells/well) were seeded on a 24-well plate and
treated with different concentrations of HA (25-200 μg/mL) for 72 h. After HA treatment, the cells
were fixed in 2% paraformaldehyde in PBS for 15 min and then incubated with 1 μM MitoTracker
for 30 min. A 1 μg/mL DAPI stain was applied for 5 min and stained cells were visualized by
using a fluorescence microscope at 400 × magnification.
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Single-Cell Gel Electrophoresis Assay (comet assay)
The comet assay is an uncomplicated and sensitive technique for the detection of DNA damage
at the level of the individual eukaryotic cell [Zhao et al., 2009]. Macrophages (1  106 cells/dish in
10 cm dish) were incubated with increasing concentrations of HA (25-200 µg/mL) for 72 h at 37
°C. Cells were then suspended in 1% low-melting-point agarose in PBS (pH 7.4) and pipetted onto
superfrosted glass microscope slides that had been pre-coated with a layer of 1% normal melting
point agarose (which was warmed to 37 °C prior to use). The agarose was allowed to set at 4 °C
for 10 min, and then, the slides were immersed in lysis solution containing 2.5 M NaCl, 100 mM
EDTA, 10 mM Tris, and 1% Triton X-100) at 4 °C for 1 h. The slides were then placed in single
rows inside a 30-cm wide horizontal electrophoresis tank containing 0.3 M NaOH and 1 mM
EDTA (pH 13.4) at 4 °C for 40 min to allow the separation of the two DNA strands (alkaline
unwinding). Electrophoresis was performed in the unwinding solution at 30 V (1 V/cm), 300 mA
for 30 min. The slides were then washed three times for 5 min each with 0.4 M Tris (pH 7.5) at 4
°C before they were stained with DAPI (1 µg/mL). DAPI-stained nucleoids were examined under
a UV microscope using a 435 nm excitation filter at 200 × magnification.
The apparent damage was not homogeneous, and visual scoring of the cellular DNA on each
slide was based on the characterization of 100 randomly selected nucleoids. DNA damage in
RAW264.7 cells, which is specified as DNA strand breaks including double and single-strand
variants at alkali-labile sites, was analyzed under an alkaline condition (pH 13.4). Comet-like
DNA formations were categorized into five classes (0, 1, 2, 3 or 4) representing increasing DNA
damage in the form of a “tail”. Each comet was assigned a value according to its class. The overall
score for 100 comets ranged from 0 (100% of comets in class 0) to 400 (100% of comets in class
4), and the overall DNA damage in the cell population can be expressed in arbitrary units [Zhao et
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al., 2009]. The observation and analysis of the results were always performed by the same
experienced observer. The observer was blinded and had no knowledge of the slide identity.
Statistics
In vitro results are presented as mean ± standard deviation (mean ± SD). All study data were
analyzed using analysis of variance, followed by Dunnett’s test for pair-wise comparison.
Statistical significance was defined as p < 0.05 for all tests.
RESULTS
HA Inhibited the Growth and Survival of RAW264.7 Cells
Macrophages, which play a key role in inflammation, host defense, and reactions against a
spectrum of autologous and foreign invaders, are crucial for innate immunity [Zhao et al., 2009].
To investigate the effects of HA on RAW264.7 cell survival, cells were exposed to increasing
concentrations of HA (25, 50, 100, and 200 μg/mL) for 72 h, and the resulting cell viability was
observed with an optical microscope. As shown in Fig. 1A, RAW264.7 cells demonstrated clear
structural evidence of cell death, including cell shrinkage, cytoplasmic vacuolization, and
detachment from the substratum, after being exposed to HA for 72 h. An MTT colorimetric assay
was performed to further confirm HA-induced cell death. Figure 1B shows that HA significantly
(p < 0.05) decreased cell viability in a dose-dependent manner, with an IC50 value of 96 µg/mL.
More precisely, the cell viability decreased by 84 ± 2%, 71 ± 5%, 29 ± 11%, and 10 ± 2% of the
control group after being exposed to 25, 50, 100, and 200 µg/mL of HA, respectively, for 72 h
(Fig. 1B). These results indicate that HA had an inhibitory effect on the proliferation and survival
of RAW264.7 cells.
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HA Induced Cell-Cycle Arrest at the G2/M Transition Phase
The DNA content profile for HA-treated RAW264.7 cells (25-200 μg/mL for 72 h) was
obtained by using flow cytometric analysis to measure the fluorescence yielded by PI binding to
DNA. As shown in Fig. 2A, HA exposure caused a progressive and sustained accumulation of
cells in G2/M transition phase. Furthermore, the percentage of S and G2/M phase cells increased,
and those in the G1 phase decreased after HA treatment (Fig. 2B). In addition, cells with lower
DNA staining relative to diploid analogs were considered apoptotic cells. Figure 2C shows that
HA treatment resulted in a remarkable accumulation of subploid cells, or the so-called sub-G1
phase, from 2.6 to 41%. Our findings suggest that HA promotes cell growth inhibition by inducing
cell-cycle arrest at G2/M phase in macrophages.
HA Down-Regulates Cyclin A, Cyclin B1, Cdc2, and Cdc25C Expression in Macrophages
We investigated the effects of various cyclins and CDKs involved in cell-cycle regulation in
RAW264.7 cells to examine the molecular mechanism(s) and underlying changes in cell-cycle
patterns caused by HA treatment. Cells were treated with HA (25–200 μg/mL) for 72 h. Dosedependent reductions in cyclin A, cyclin B1, mitotic cyclin-dependent kinase Cdc2, and mitotic
phosphatase Cdc25C expression were observed (Fig. 2D). These results imply that HA inhibits
cell-cycle progression by reducing the levels of cyclin A, cyclin B1, Cdc2, and Cdc25C in
RAW264.7 cells.
HA Induced Apoptotic DNA Fragmentation in Macrophages
To characterize the type of cell death observed in HA-exposed cells, we examined whether the
cell death was caused by apoptosis. RAW264.7 cells were exposed to 100 and 200 µg/mL of HA
11
for 72 h, and the apoptotic DNA fragmentation of RAW264.7 cells was visualized by using a
TUNEL assay. Apoptotic DNA fragmentation was detected by labeling the 3'-OH ends of
fragmented DNA with dUTP-fluorescein, and TUNEL-positive cells were counted as apoptotic
cells. The dose-dependent increase in the number of TUNEL-positive cells (green) showed that
HA induced apoptosis in RAW264.7 cells (Fig. 3A). Apoptotic DNA fragmentation was
apparently increased to 5.7 ± 1.6-fold and 7.1 ± 0.8-fold by 100 and 200 µg/mL of HA,
respectively (Fig. 3B). These data confirm that HA induces apoptosis in RAW264.7 cells.
HA Induced the Release of Cytochrome c, the Activation of Caspases-9 and -3, and the
Cleavage of PARP in Macrophages
Cytosolic and mitochondrial cytochrome c levels were examined by western blot analysis. The
results revealed that HA treatment (25–200 g/mL for 72 h) increased the cytochrome c
expression levels in the cytoplasm in a dose-dependent manner, whereas the cytochrome c levels
in the mitochondria were dose-dependently decreased (Fig. 4A). Cytochrome c is reportedly
involved in activating the caspases that trigger apoptosis. Therefore, we investigated the roles of
caspase-9 and caspase-3 in the cellular response to HA. As shown in Fig. 4A, treating RAW264.7
cells with HA induced proteolytic cleavage in procaspase-9 and -3 into their active forms because
PARP-specific proteolytic cleavage by caspase-3 is considered to be a biochemical characteristic
of apoptosis. Following the addition of HA, the 115 kDa PARP protein is cleaved into an 85 kDa
fragment as shown in Fig. 4A. These data suggest that HA-induced apoptosis was mediated by a
mitochondria-dependent pathway.
Mitochondrial Membrane Permeability in Response to HA Treatment
12
To further confirm that HA-induced apoptosis was associated with a loss of mitochondrial
membrane potential, we examined the HA-induced mitochondrial injury in RAW264.7 cells by
using the Mito-Tracker assay kit. Mito-Tracker is a green fluorescent dye that stains mitochondria
in live cells, and its accumulation is membrane potential-dependent. As shown in Fig. 4b, a bright
green fluorescence was observed in the control cells, whereas 72 h of HA treatment significantly
reduced the intensity of green fluorescence from 100% to 74 ± 6% and 51 ±18% by 100 and 200
µg/mL of HA, respectively. The capacity for Mito-Tracker uptake by the mitochondria in control
cells is higher than that of HA-treated cells (Fig. 4B). These data directly indicated that
mitochondrial function is severely impaired by HA in RAW264.7 cells.
HA Mediated Bcl-2 and Bax Protein Dysregulation
The balance between the proapoptotic and antiapoptotic members of the Bcl-2 family
presumably determines a cell's fate [Singh, 2007]. Therefore, Bcl-2 and Bax protein levels were
studied in cultured RAW264.7 cells to examine their involvement in HA-mediated apoptosis. As
shown in Fig. 5 A and B, incubating RAW264.7 cells with HA caused a dramatic reduction in the
level of anti-apoptotic Bcl-2, a potent cell death inhibitor, and increased the level of a proapoptotic
Bax protein, which heterodimerizes with Bcl-2 and thereby inhibits Bcl-2 activity. These results
strongly indicate that HA significantly induced Bcl-2 and Bax dysregulation, which enhanced
apoptosis in RAW264.7 cells.
HA Activates Fas-Mediated Apoptosis through the Activation of Caspase-8 and the Cleavage
of Bid
To further assess whether HA (25–200 μg/mL for 72 h) promoted apoptosis via a receptor-
13
mediated pathway, the Fas protein levels in RAW264.7 cells were determined by western blot
analysis. These results showed that HA treatment significantly stimulates Fas protein expression
in a dose-dependent manner (Fig. 6A). To further verify whether the activation of caspase-8 is
associated with Fas expression in response to HA treatment, the results showed that HA
significantly induced the proteolytic cleavage of procaspase-8 (Fig. 6A). Furthermore, the
expression levels of pro-apoptotic Bid protein, which produces the truncated Bid fragment (tBid)
upon cleavage by caspase-8, were also measured. Our data indicated that HA treatment
significantly induced Bid cleavage in RAW264.7 cells (Fig. 6A). These data provide additional
evidence that HA-induced apoptosis was also mediated by the death receptor pathway.
ER Stress was involved in HA-Induced RAW264.7 Cell Apoptosis
To demonstrate the role of endoplasmic reticulum (ER) stress in HA-induced apoptosis,
RAW264.7 cells were incubated with HA (25–200 μg/mL) for 72 h. Caspase-4 or -12 reportedly
act as initiator caspases in the human ER stress-induced apoptotic pathway [Binet et al., 2010].
Our result showed that HA treatment induced the proteolytic cleavage of procaspase-4 and
procaspase-12 in RAW264.7 cells in a dose-dependent fashion (Fig. 6B). Therefore, we conclude
that ER stress was induced by HA, which triggers apoptosis in RAW264.7 cells.
p53 and p-p53 Protein Expression Induction by HA
Tumor suppressor gene p53 acts as a transcription factor that regulates DNA repair, cell
proliferation, and cell death. p53 could function as a sensor for DNA damage that could, in turn,
arrest the cell-cycle for DNA repair or up-regulate pro-apoptotic factors, resulting in increased
susceptibility to apoptosis. Moreover, p53 tumor suppressor induction has been implicated in cell
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growth and apoptosis. Therefore, the effects of HA (25–200 μg/mL) on p53 and p-p53 protein
levels were assessed in RAW246.7 cells over 72 h. We observed that HA treatment caused a
significant increase in both p53 and p-p53 protein levels in a dose-dependent manner (Fig. 6C).
This result suggests that HA-mediated p53 activation triggers mitochondria-dependent apoptosis
in RAW264.7 cells.
Genotoxicity Induction by HA
DNA damage, as represented by DNA single strand breaks, was reflected by an increase in tail
moments. DNA damage was evaluated by using the comet assay, in which the tail length is an
important quantitative parameter. Therefore, the HA effect (25-200 µg/mL for 72 h) on cellular
DNA damage induction was evaluated by using a single-cell gel electrophoresis comet assay. A
total toxicity scale was generated by considering the comet length (Fig. 7A). Our results show that
a dose-dependent increase in comet length was observed after HA treatment (25-200 µg/mL) for
72 h (Fig. 7B), which clearly indicates that HA treatment enhanced DNA damage in RAW264.7
cells.
DISCUSSION
Humic substances have been found in half of the world's well water [Seo et al., 2013]. Humic
substances are generally classified into humic acids, fulvic acids, and humin on the basis of their
pH and solubility in water [Man et al., 2013]. HA has been suggested as an etiological factor in the
development of vascular diseases in blackfoot disease-endemic regions of Taiwan. In our previous
study, we provided an experimental support for the hypothesis that acute exposure to
environmental HA may trigger an inflammatory response, which has been implicated as a possible
15
factor for the development of atherosclerosis and blackfoot disease. HA may be involved in
atherosclerosis through the induction of pro-inflammatory mediators (TNF-α, IL-1β, NO, PGE2,
iNOS, and COX-2), the activation of NF-κB/AP-1 cascades via ROS generation and the AKT and
MAPK signaling pathways in murine macrophages (Fig. 8) [Erhayem and Sohn, 2013]. In this
study, we further explored the role of long-term HA exposure in inducing G2/M arrest and
mitochondrial-, death receptor-, and ER stress-mediated macrophage apoptosis (Fig. 8).
Macrophage apoptosis occurs throughout all stages of atherosclerosis. In late lesions, a number
of factors may contribute to defective phagocytic clearance in apoptotic macrophages, leading to
secondary necrosis in these cells and a pro-inflammatory response [Tabas, 2010]. Our findings not
only suggested that HA possesses potential immunotoxicity but they also advanced current
understanding of the probable molecular mechanisms of HA-induced G2/M cell-cycle arrest and
apoptosis in macrophages. Considering the widespread use of HA in the world and the ubiquitous
presence of HA in drinking well water, it would be worthwhile to engage in more comprehensive
studies and epidemiological investigations to understand the potential for HA to contribute to
atherosclerosis [Lu, 1990; Hseu et al., 2000; Hseu et al., 2002a; Hseu et al., 2002b; Hseu and
Yang, 2002; Hseu et al., 2008]. Therefore, HA-induced macrophage apoptosis has also been
suggested as an underlying mechanism in the development of atherosclerosis in blackfoot diseaseendemic regions of Taiwan.
Eukaryotic cell-cycle progression involves the sequential activation of CDKs, the activation of
which is dependent on their association with cyclins [Bloom and Cross, 2007]. Among the CDKs
that regulate cell-cycle progression, CDK2 and Cdc2 kinases are primarily activated in association
with cyclin A and cyclin B1 during G2/M phase progression [Bloom and Cross, 2007]. The
phosphorylation of Cdc2 suppresses Cdc2/cyclin A and B1 kinase complex activity. Cdc2
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dephosphorylation is catalyzed by Cdc25C phosphatase, and this reaction is believed to be the
rate-limiting step for entry into mitosis [Lim and Kaldis, 2013]. In this study, flow cytometry
analysis clearly demonstrates that HA treatment had a profound effect on cell-cycle progression,
as evidenced by cell accumulation during the G2/M phase transition. We assumed that this cellcycle blockade was associated with the inhibition of cell-cycle regulatory proteins and their kinase
activity. Our results imply that the expression levels of cyclin A/B, Cdc2, and Cdc25C are downregulated by HA in macrophages, which is consistent with G2/M arrest.
There is evidence that HA-induced macrophage apoptosis occurs via mitochondrial, death
receptor, and ER stress pathways. Mitochondrial dysfunction, including the loss of mitochondrial
membrane potential (ΔΨm) and the release of cytochrome c from the mitochondria into the cytosol,
are associated with apoptosis [Ly et al., 2003]. Cytosolic cytochrome c activates procaspase-9 and
subsequently has a downstream effect on caspases including caspase-3, which triggers apoptosis
[Jiang and Wang, 2004]. In this study, exposing macrophages to HA induced mitochondrial
membrane damage and released cytochrome c into the cytoplasm, and it also activated
procasepase-9 and procaspase-3. In mammalian cells, the Bcl-2 gene family contains a number of
anti-apoptotic proteins including Bcl-2 and Bcl-xL, which are thought to be involved in resistance
to conventional cancer treatment. In contrast, the pro-apoptotic proteins from the same gene
family, including Bax, can induce apoptotic cell death. Therefore, apoptosis largely depends on
the balance between anti-apoptotic and pro-apoptotic protein levels [Kuwana and Newmeyer,
2003]. These data indicate that HA treatment disturbs the Bcl-2/Bax ratio and thereby leads to
macrophage apoptosis. In addition, the prolonged activation of PARP may also lead to DNA
damage by up-regulating cellular NAD and ATP levels [Soldani and Scovassi, 2002]. This study
also shows that HA can activate PARP DNA repair enzymes in macrophages. Membrane death
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receptors, including Fas, are activated by their respective ligands and engage adaptor molecules
and caspases, including proximal caspase-8. Activated caspase-8 further stimulates caspase-3 via a
mitochondria-dependent cascade [Schmitz et al., 2000]. In the mitochondrial apoptosis pathway,
caspase-8 proteolytically activates pro-apoptotic protein Bid, which targets mitochondrial
membrane permeabilization and represents the primary link between extrinsic and intrinsic
apoptotic pathways [Schug et al., 2011]. In this study, we found that HA treatment significantly
increased Fas activity, and it also activates caspase-8 and Bid within macrophages. ER stressinduced apoptosis has its own signaling pathway. This pathway is independent from mitochondria
and death receptors and is thought to be mediated by caspase-12 [Li et al., 2006]. Stimulated
caspase-12 reportedly further activates caspase-9 independent of Apaf-1, followed by the
activation of caspase-3. Human caspase-4 is involved in the ER stress-induced cell death pathway
and is an alternative to caspase-12 [Szegezdi et al., 2006]. Current data supports the idea that HAinduced apoptosis is also mediated by the ER stress pathway as evidenced by the increased
activation of caspase-4 and -12 in macrophages.
ROS generation is one of several proposed mechanisms of action for HA-induced toxicity. We
have also demonstrated that HA exposure can up-regulate ROS and/or RNS production and
apoptosis in various human cells, which may contribute to inflammation and atherosclerosis [Hseu
et al., 2002a; Hseu et al., 2002b]. Therefore, the generation of ROS induced by HA may trigger
the mitochondrial pathway, e.g., by activating p53 expression. p53 could act as a sensor for DNA
damage that could, in turn, arrest the cell-cycle for DNA repair or up-regulate pro-apoptotic
factors, resulting in increased susceptibility to apoptosis. These results suggest that p53 may help
to mediate HA-induced cell-cycle arrest and/or apoptosis in macrophages. The reason p53 upregulation is induced by HA remains unclear; however, the role of increased ROS generation and
18
DNA damage must be considered.
Substantial effort has been devoted to elucidating the molecular basis of blackfoot disease in
terms of HA-related atherogenic potential; however, no mechanism has been unequivocally
established to date. On the basis of the results of the present study, the mechanism through which
HA induced G2/M cell-cycle arrest and apoptosis in macrophages has been better clarified. Indeed,
the induction of macrophage apoptosis by HA is associated with mitochondrial, death receptor,
and ER stress pathways. Considering the ubiquitous environmental presence of HA, the present
study provided new information on the potential physiological and immunological effects caused
by chronic and long-term exposures to HA.
AUTHOR CONTRIBUTIONS
You-Cheng Hseu, Fung-Jou Lu, and Ssu-Ching Chen designed the experiment. Hsin-Ju Cho and
Chia-Ting Chang conducted all the experiments. K. J. Senthil Kumar and You-Cheng Hseu
prepared the manuscript. Hsin-Ling Yang organized the data and prepared the tables and figures
supplied in the manuscript.
ACKNOWLEDGMENTS
This work was supported by grants NSC-101-2320-B-039-050-MY3 and NSC-99-2320-B-039035-MY3 from the National Science Council and China Medical University, Taiwan.
19
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Figure Legends
Fig. 1. HA inhibited the growth of RAW264.7 macrophages. Cells were treated with increasing
concentrations of HA (25-200 g/mL) or vehicle alone (PBS) for 72 h. (A) Cell morphology was
observed under a phase contrast microscope at 200 × magnification. (B) Cell viability was
determined by the MTT assay. Each value is expressed as the mean ± SD (n=3). *Significant
difference in comparison to control group (p <0.05).
Fig. 2. HA induced sub-G1 accumulation and G2/M arrest in macrophages. (A) Cells were treated
with HA (100 and 200 g/mL) for 72 h, stained with PI and analyzed for their sub-G1 and cellcycle phases by using flow cytometry. (B) Apoptotic nuclei were identified as a subploid DNA
peak and distinguished from cell debris on the basis of forward light-scattering and PI
fluorescence. (C) Cellular distribution (percentage) in different phases of the cell-cycle (G1, S and
G2/M) after HA treatment. (D) The effects of HA (25-200 g/mL) on the protein levels of cyclin
A, cyclin B1, Cdc 2, and Cdc25C were monitored for 72 h by western blot analysis. The relative
band intensity is shown just below the gel data. The results are presented as the mean  SD of
three assays. *Significant difference in comparison to the control group (p <0.05).
Fig. 3. HA induced apoptotic DNA fragmentation in macrophages. (A) TUNEL assay of cells
exposed to HA (100 g/mL) for 72 h. The average number of apoptosis-positive cells in
microscopic fields (magnification  400). (B) The histogram shows the fold-increase of apoptotic
cells calculated by fluorescence intensity. The results are presented as the mean  SD of three
assays. *Significant difference in comparison to the control group (p <0.05).
25
Fig. 4. HA induced apoptosis via a mitochondria-dependent pathway. (A) Western blot analysis of
apoptosis-related proteins (mitochondrial and cytosolic cytochrome c, caspase-9, and -3, and
PARP) in macrophages exposed to HA (25-200 g/mL) for 72 h. The relative band intensity is
shown just below the gel data. (B) HA effect on macrophage mitochondrial activity. The
mitochondrial activity assessment was performed by tracking the uptake of Mito-Tracker FM
(green CMXRos) into the mitochondria. After the HA (25-200 g/mL for 72 h) treatment,
RAW264.7 cells were incubated for 30 min with 1 μM Mito-Tracker at 37C. DAPI (1 g/mL)
was stained for 5 min and examined by fluorescence microscopy (magnification  400) as
described in the materials and methods section.
Fig. 5. Effect of HA on Bax/Blc-2 ratio in macrophages. (A) Western blot analysis of antiapoptotic
Bcl-2 and proapoptotic Bax protein levels after exposing the macrophages to HA. (B) Relative
changes in Bcl-2 and Bax protein bands were measured by using densitometric analysis. The
results are presented as the mean  SD of three assays. *Significant difference in comparison to
the control group (p <0.05).
Fig. 6. HA induces apoptosis in macrophages via death receptor, ER stress and p53-dependent
pathways. Macrophages were exposed to HA (25-200 µg/mL) for 72 h. The protein expression
levels of (A) Fas, caspase-8, and Bid (death receptor pathway); (B) caspase-4 and -12 (ER stress
pathway); (C) p53 and p-p53 were monitored by using specific antibodies. The relative band
intensity is shown just below the gel data. The results are presented as the mean  SD of three
assays. *Significant difference in comparison to the control group (p <0.05).
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Fig. 7. HA induced DNA damage in macrophages. RAW264.7 cells were treated with HA (25-200
g/mL) for 72 h. (A) Cellular DNA was stained with DAPI and photographed under a
fluorescence photomicroscope. (B) The comet-like DNA formations were categorized into five
classes (0, 1, 2, 3 or 4) representing increasing DNA damage in the form of a “tail”. Each comet
was assigned a value according to its class. The overall score for 100 comets ranged from 0 (100%
of comets in class 0) to 400 (100% of comets in class 4). Results are the mean ± SD of three
assays. *Significant difference in comparison to the control group (p <0.05).
Fig. 8. Schematic representation of HA-induced inflammation and apoptosis in macrophages.
27